Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants wil...Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.展开更多
As one of the important materials in landscaping for flower terrace and border, Petunia hybrida needs high environmental conditions and its growth is seriously influenced by the drought. Silicon is considered to be a ...As one of the important materials in landscaping for flower terrace and border, Petunia hybrida needs high environmental conditions and its growth is seriously influenced by the drought. Silicon is considered to be a necessary element for plant growth, and soluble silicon can improve plant resilience. To improve the drought resilience of Petunia hybrida, the silicon transporter protein OsLsi1 and OsLsi2 genes cloned from rice(Oryza sative) were transferred into Petunia hybrida by Agrobacterium-mediated method, and finally got 26 and 32 positive plants, respectively by PCR and RT-PCR detections. With a control of non-transgenic plants, the obtained transgenic plants were taken by drought treatment stress for 0, 4, 7, 10 and 14 days, then re-watered and measured physiological indexes as malondialdehyde(MDA) content, free proline(Pro) content, superoxide dismutase(SOD) activity and peroxidase(POD) activity to study the effect of Petunia's drought resistance. All the results proved that the silicon transporter protein OsLsi1 and OsLsi2 genes were normally transcripted and expressed in transgenic Petunia hybrida; OsLsi1 gene could improve the abilities of plants' drought resistance and recover after drought stress, while OsLsi2 gene could reduce the above abilities. The order of the drought resistance ability of the three strains from strong to weak was OsLsi1>CK>OsLsi2; and silicon indeed improved the ability of drought resistance as well. All these results provided a new way to improve the drought resistance of Petunia, and laid a foundation to improve the ability of garden plants' drought resistance and water saving.展开更多
EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was...EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.展开更多
Biotechnology plays an important role in mitigation of various pollution in a cost effective manner by using the complex chemistry of living organisms,various cell manipulations and their approaches for environmental ...Biotechnology plays an important role in mitigation of various pollution in a cost effective manner by using the complex chemistry of living organisms,various cell manipulations and their approaches for environmental cleanup along with environmental sustainability.One such technology is phytoremediation technology or green technology which has emerged and evolved as a novel tool for remediation of toxic contaminants from environment.Plants with its diverse range show a remarkable range of their phytoremediation potentiality for establishing a sustainable environment.There is a huge exploitation of natural resources through expanded industrialization,urbanization,modern agricultural development,energy generation to fulfill the never-ending human desires and need.This disturbs the balance in nature where we reside and leads to progressive deterioration of the environment.There are several biotechnological advances which are employed for combating both the biotic and abiotic stress problems caused due to toxic contaminants in the environment.Various biotechnological interventions such as bioinformatics,proteomics,genomics,metallomics and metabolomics play a crucial role and open new avenue in this context.This omics approach is now integrated with bioinformatics to serve as a novel tool in phytoremediation technology.This smart technology provides insights into the complex behavior of enzymes,proteins and metabolites action and their biochemical pathways for degradation of wastes.This leads towards deriving a sustainable solution for environmental pollution.展开更多
We created CRISPR-Cas9 knock-out and overexpressing OsbZIP72 transgenic rice plants to gain a better understanding of the role and molecular mechanism of OsbZIP72 gene in stress tolerance,which has remained largely el...We created CRISPR-Cas9 knock-out and overexpressing OsbZIP72 transgenic rice plants to gain a better understanding of the role and molecular mechanism of OsbZIP72 gene in stress tolerance,which has remained largely elusive.OsbZIP72 was expressed and integrated into rice transgenic plant genomes,and the OsbZIP72 transcript in overexpression lines was elicited by salinity,abscisic acid(ABA)and drought stresses.OsbZIP72 overexpressing plants showed higher tolerance to drought and salinity stresses,while knock-out transgenic lines showed higher sensitivity to these stresses.The differentially expressed genes(DEGs)from RNA-sequencing data encompassed several abiotic stress genes,and the functional classification of these DEGs demonstrated the robust transcriptome diversity in OsbZIP72.Yeast one-hybrid,along with luciferase assay,indicated that OsbZIP72 acted as a transcriptional initiator.Remarkably,electrophoresis mobility assay revealed that OsbZIP72 bound directly to the ABAresponsive element in the OsHKT1;1 promoter region and activated its transcription.Overall,our findings revealed that OsbZIP72 can act as a transcriptional modulator with the ability to induce the expression of OsHKT1;1 in response to environmental stress through an ABA-dependent regulatory pathway,indicating that OsbZIP72 can play a crucial role in the ABA-mediated salt and drought tolerance pathway in rice.展开更多
Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations ...Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.展开更多
Seven in absentia(SINA)family proteins play a central role in plant growth,development and resistance to abiotic stress.However,their biological function in plant response to cold stress is still largely unknown.In th...Seven in absentia(SINA)family proteins play a central role in plant growth,development and resistance to abiotic stress.However,their biological function in plant response to cold stress is still largely unknown.In this work,a seven in absentia gene IbSINA5 was isolated from sweet potato.Quantitative real-time polymerase chain reaction(qRT-PCR)analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato,with a predominant expression in fibrous roots,and was remarkably induced by cold,drought and salt stresses.Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus.Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants,which was associated with the up-regulated expression of IbCOR(cold-regulated)genes,increased proline production,and decreased malondialdehyde(MDA)and H2O2 accumulation in the leaves of transgenic plants.Furthermore,transient expression of IbCBF3,a C-repeat binding factor(CBF)gene,in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes.Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato,and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.展开更多
Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefa...Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg·L<sup>-1</sup> kanamycin and 250 mg·L<sup>-1</sup> timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg·L<sup>-1</sup> kanamycin and 250 mg·L<sup>-1</sup> timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T<sub>0</sub> plants and T<sub>1</sub> seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.展开更多
Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultiva...Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.展开更多
Phytophthora pathogens are a persistent threat to the world's commercially important agricultural crops,including potato and soybean.Current strategies aim at reducing crop losses rely mostly on disease-resistance...Phytophthora pathogens are a persistent threat to the world's commercially important agricultural crops,including potato and soybean.Current strategies aim at reducing crop losses rely mostly on disease-resistance breeding and chemical pesticides,which can be frequently overcome by the rapid adaptive evolution of pathogens.Transgenic crops with intrinsic disease resista nee offer a promising alternative and con tinue to be developed.Here,we explored Phytophthora-derived PI3P(phosphatidylinositol 3-phosphate)as a novel control target,using proteins that bind this lipid to direct secreted anti-microbial peptides and proteins(AMPs)to the surface of Phytophthora pathogens.In transgenic Nicotiana benthamiana,soybean,and potato plants,significantly enhanced resistance to different pathogen isolates was achieved by expression of two AMPs(GAFP1 or GAFP3 from the Chinese medicinal herb Gastrodia elata)fused with a PI3P-specific binding domain(FYVE).Using the soybean pathogen P.sojae as an example,we demonstrated that the FYVE domain could boost the activities of GAFPs in multiple independent assays,including those performed in vitro,in vivo,and in planta.Mutational analysis of P.sojae PI3K1 and PI3K2 genes of this pathogen confirmed that the enhanced activities of the targeted GAFPs were correlated with PI3P levels in the pathogen.Collectively,our study provides a new strategy that could be used to confer resistance not only to Phytophthora pathogens in many plants but also potentially to many other kinds of plant pathogens with unique targets.展开更多
In clinical therapy,the amount of antigen administered to achieve oral tolerance for allergic diseases is large,and the cost is a major consideration.In this study,we used tobacco plants to develop a large-scale prote...In clinical therapy,the amount of antigen administered to achieve oral tolerance for allergic diseases is large,and the cost is a major consideration.In this study,we used tobacco plants to develop a large-scale protein production system for allergen-specific immunotherapy,and we investigated the mechanisms of oral tolerance induced by a transgenic plant-derived antigen.We used plants(tobacco leaves)transgenic for the Dermatophagoides pteronyssinus 2(Der p2)antigen to produce Der p2.Mice received total protein extract from Der p2 orally once per day over 6 days(days 0–2 and days 6–8).Mice were also sensitized and challenged with yeast-derived recombinant Der p2(rDer p2),after which the mice were examined for airway hyper-responsiveness and airway inflammation.After sensitization and challenge with rDer p2,mice that were fed with total protein extracted from transgenic plants showed decreases in serum Der p2-specific IgE and IgG1 titers,decreased IL-5 and eotaxin levels in bronchial alveolar lavage fluid,and eosinophil infiltration in the airway.In addition,hyper-responsiveness was also decreased in mice that were fed with total protein extracted from transgenic plants,and CD4^(+)CD25^(+)Foxp3^(+) regulatory T cells were significantly increased in mediastinal and mesenteric lymph nodes.Furthermore,splenocytes isolated from transgenic plant protein-fed mice exhibited decreased proliferation and increased IL-10 secretion after stimulation with rDer p2.The data here suggest that allergen-expressing transgenic plants could be used for therapeutic purposes for allergic diseases.展开更多
Phytophthora pathogens lead to numerous economically damaging plant diseases worldwide,including potato late blight caused by P.infestans and soybean root rot caused by P.sojae.Our previous work showed that Phytophtho...Phytophthora pathogens lead to numerous economically damaging plant diseases worldwide,including potato late blight caused by P.infestans and soybean root rot caused by P.sojae.Our previous work showed that Phytophthora pathogens may generate abundant phosphatidylinositol 3-phosphate(PI3P)to promote infection via direct association with RxLR effectors.Here,we designed a disease control strategy for metabolizing pathogen-derived PI3P by expressing secreted Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase 1(AtPIP5K1),which can phosphorylate PI3P to PI(3,4)P2.We fused AtPIP5K1 with the soybean PR1a signal peptide(SP-PIP5K1)to enable its secretion into the plant apoplast.Transgenic soybean and potato plants expressing SP-PIP5K1 showed substantially enhanced resistance to various P.sojae and P.infestans isolates,respectively.SP-PIP5K1 significantly reduced PI3P accumulation during P.sojae and soybean interaction.Knockout or inhibition of PI3 kinases(PI3Ks)in P.sojae compromised the resistance mediated by SP-PIP5K1,indicating that SP-PIP5K1 action requires a supply of pathogen-derived PI3P.Furthermore,we revealed that SP-PIP5K1 can interfere with the action of P.sojae mediated by the RxLR effector Avr1k.This novel disease control strategy has the potential to confer durable broad-spectrum Phytophthora resistance in plants through a clear mechanism in which catabolism of PI3P interferes with RxLR effector actions.展开更多
Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in ...Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.展开更多
Cytochrome P450 CYP2E1 is mainly present in hepatocytes in the livers of mammals,where it plays an important role in the metabolism of xenobiotic organic substances. Previous studies showed that transgenic petunia(Pet...Cytochrome P450 CYP2E1 is mainly present in hepatocytes in the livers of mammals,where it plays an important role in the metabolism of xenobiotic organic substances. Previous studies showed that transgenic petunia(Petunia hybrid) plants harboring a mammalian cyp2e1 gene(designated cyp2e1-transgenic petunia) exhibited increased resistance to formaldehyde stress. In this study,we used cyp2e1-transgenic petunia plants to analyze physiological indexes related to formaldehyde stress responses. The results indicated that under formaldehyde stress,the malondialdehyde content in cyp2e1-transgenic petunia plants was lower than in β-glucuronidase gene(gus)-transgenic and wild-type petunia plants. The activities of both superoxide dismutase and peroxidase in the cyp2e1-transgenic plants were higher than in gus-transgenic and wild-type plants. The alcohol dehydrogenase activity was slightly increased and more glutathione was consumed. Additionally,under formaldehyde stress,the levels of plant hormones including indole-3-acetic acid,zeatin and abscisic acid in cyp2e1-transgenic petunia plants displayed decreasing trends,whereas the level of gibberellic acid displayed an increasing trend. In contrast,the indole-3-acetic acid,zeatin and abscisic acid levels in gus-transgenic and wild-type petunia plants displayed increasing trends,whereas the gibberellic acid level displayed a decreasing trend. At 72 h after incubation of 0.5 g of cyp2e1-transgenic petunia plants in 40 mL of treatment solution containing formaldehyde at 50 mg·L^(-1),the formaldehyde content remaining in the treatment solution was close to zero while approximately half of original formaldehyde remained in the treatment solutions containing gus-transgenic and wild-type petunia plants.展开更多
Plant natural products(PNPs)are the main sources of drugs,food additives,and new biofuels and have become a hotspot in synthetic biology.In the past two decades,the engineered biosynthesis of many PNPs has been achiev...Plant natural products(PNPs)are the main sources of drugs,food additives,and new biofuels and have become a hotspot in synthetic biology.In the past two decades,the engineered biosynthesis of many PNPs has been achieved through the construction of microbial cell factories.Alongside the rapid development of plant physiology,genetics,and plant genetic modification techniques,hosts have now expanded from single-celled microbes to complex plant systems.Plant synthetic biology is an emerging field that combines engineering principles with plant biology.In this review,we introduce recent advances in the biosynthetic pathway elucidation of PNPs and summarize the progress of engineered PNP biosynthesis in plant cells.Furthermore,a future vision of plant synthetic biology is proposed.Although we are still a long way from overcoming all the bottlenecks in plant synthetic biology,the ascent of this field is expected to provide a huge opportunity for future agriculture and industry.展开更多
Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolat...Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.展开更多
基金the Ministry of Agriculture of China for the National Transgenic Research Program (2016ZX08010004)the Chinese Academy of Agricultural Sciences for the Agricultural Science and Technology Innovation Program (ASTIP-2060302-2-19)
文摘Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.
基金Supported by the Fund of Science and Technology Research Project of Education Department in Heilongjiang Province(12531014)
文摘As one of the important materials in landscaping for flower terrace and border, Petunia hybrida needs high environmental conditions and its growth is seriously influenced by the drought. Silicon is considered to be a necessary element for plant growth, and soluble silicon can improve plant resilience. To improve the drought resilience of Petunia hybrida, the silicon transporter protein OsLsi1 and OsLsi2 genes cloned from rice(Oryza sative) were transferred into Petunia hybrida by Agrobacterium-mediated method, and finally got 26 and 32 positive plants, respectively by PCR and RT-PCR detections. With a control of non-transgenic plants, the obtained transgenic plants were taken by drought treatment stress for 0, 4, 7, 10 and 14 days, then re-watered and measured physiological indexes as malondialdehyde(MDA) content, free proline(Pro) content, superoxide dismutase(SOD) activity and peroxidase(POD) activity to study the effect of Petunia's drought resistance. All the results proved that the silicon transporter protein OsLsi1 and OsLsi2 genes were normally transcripted and expressed in transgenic Petunia hybrida; OsLsi1 gene could improve the abilities of plants' drought resistance and recover after drought stress, while OsLsi2 gene could reduce the above abilities. The order of the drought resistance ability of the three strains from strong to weak was OsLsi1>CK>OsLsi2; and silicon indeed improved the ability of drought resistance as well. All these results provided a new way to improve the drought resistance of Petunia, and laid a foundation to improve the ability of garden plants' drought resistance and water saving.
基金Supported by the Natural Science Foundation of Heilongjiang Province(LH2021C032)。
文摘EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.
文摘Biotechnology plays an important role in mitigation of various pollution in a cost effective manner by using the complex chemistry of living organisms,various cell manipulations and their approaches for environmental cleanup along with environmental sustainability.One such technology is phytoremediation technology or green technology which has emerged and evolved as a novel tool for remediation of toxic contaminants from environment.Plants with its diverse range show a remarkable range of their phytoremediation potentiality for establishing a sustainable environment.There is a huge exploitation of natural resources through expanded industrialization,urbanization,modern agricultural development,energy generation to fulfill the never-ending human desires and need.This disturbs the balance in nature where we reside and leads to progressive deterioration of the environment.There are several biotechnological advances which are employed for combating both the biotic and abiotic stress problems caused due to toxic contaminants in the environment.Various biotechnological interventions such as bioinformatics,proteomics,genomics,metallomics and metabolomics play a crucial role and open new avenue in this context.This omics approach is now integrated with bioinformatics to serve as a novel tool in phytoremediation technology.This smart technology provides insights into the complex behavior of enzymes,proteins and metabolites action and their biochemical pathways for degradation of wastes.This leads towards deriving a sustainable solution for environmental pollution.
基金supported by the earmarked funds for China Agriculture Research System(Grant No.CARS-01-61)National Science and Technology Support Program of China(Grant No.2015BAD01B01)+3 种基金Science and Technology Support Program of Jiangsu Province,China(Grant Nos.BE2016370-3 and BE2017323)the Natural Science Foundation of Jiangsu Province,China(Grant No.BK20161299)the Financial Grant Support Program of Lianyungang City,Jiangsu Province,China(Grant Nos.QNJJ1704 and QNJJ1912)National Natural Science Foundation of China(Grant No.31701395).
文摘We created CRISPR-Cas9 knock-out and overexpressing OsbZIP72 transgenic rice plants to gain a better understanding of the role and molecular mechanism of OsbZIP72 gene in stress tolerance,which has remained largely elusive.OsbZIP72 was expressed and integrated into rice transgenic plant genomes,and the OsbZIP72 transcript in overexpression lines was elicited by salinity,abscisic acid(ABA)and drought stresses.OsbZIP72 overexpressing plants showed higher tolerance to drought and salinity stresses,while knock-out transgenic lines showed higher sensitivity to these stresses.The differentially expressed genes(DEGs)from RNA-sequencing data encompassed several abiotic stress genes,and the functional classification of these DEGs demonstrated the robust transcriptome diversity in OsbZIP72.Yeast one-hybrid,along with luciferase assay,indicated that OsbZIP72 acted as a transcriptional initiator.Remarkably,electrophoresis mobility assay revealed that OsbZIP72 bound directly to the ABAresponsive element in the OsHKT1;1 promoter region and activated its transcription.Overall,our findings revealed that OsbZIP72 can act as a transcriptional modulator with the ability to induce the expression of OsHKT1;1 in response to environmental stress through an ABA-dependent regulatory pathway,indicating that OsbZIP72 can play a crucial role in the ABA-mediated salt and drought tolerance pathway in rice.
基金supported by the National"863"High-Tech Program,the Special Foundation of the Ministry of Agriculture for"Developing Cotton Production"and the Chinese Foundation for Agriculture Science and Education.
文摘Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.
基金This work was jointly supported by the following grants:Agricultural Variety Improvement Project of Shandong Province[Grant Nos.2019LZGC009,2019LZGC010,2020LZGC007]the National Key R&D Program of China[Grant Nos.2018YFD1000500,2019YFD1000500]+4 种基金the National Natural Science Foundation of China[Grant Nos.31870576,31901572,32071733]the Natural Science Foundation of Shandong Province[Grant Nos.ZR2018PH041,ZR2019PC015,ZR2020MC138]the Modern Agricultural Industry Technology System Innovation Team of Shandong Province of China[Grant No.SDAIT-02-05]the Key R&D Program of Shandong Province of China[2019GSF108154]the Science and Technology Development Project in Yantai[Grant No.2018XSCC041].
文摘Seven in absentia(SINA)family proteins play a central role in plant growth,development and resistance to abiotic stress.However,their biological function in plant response to cold stress is still largely unknown.In this work,a seven in absentia gene IbSINA5 was isolated from sweet potato.Quantitative real-time polymerase chain reaction(qRT-PCR)analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato,with a predominant expression in fibrous roots,and was remarkably induced by cold,drought and salt stresses.Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus.Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants,which was associated with the up-regulated expression of IbCOR(cold-regulated)genes,increased proline production,and decreased malondialdehyde(MDA)and H2O2 accumulation in the leaves of transgenic plants.Furthermore,transient expression of IbCBF3,a C-repeat binding factor(CBF)gene,in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes.Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato,and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.
文摘Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg·L<sup>-1</sup> kanamycin and 250 mg·L<sup>-1</sup> timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg·L<sup>-1</sup> kanamycin and 250 mg·L<sup>-1</sup> timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T<sub>0</sub> plants and T<sub>1</sub> seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.
文摘Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.
基金supported by the National Natural Science Foundation,China(31625023,31721004,and 31801715)the National Key R&D Program,China(2018YFD0201000)supported by Oregon State University,United States.
文摘Phytophthora pathogens are a persistent threat to the world's commercially important agricultural crops,including potato and soybean.Current strategies aim at reducing crop losses rely mostly on disease-resistance breeding and chemical pesticides,which can be frequently overcome by the rapid adaptive evolution of pathogens.Transgenic crops with intrinsic disease resista nee offer a promising alternative and con tinue to be developed.Here,we explored Phytophthora-derived PI3P(phosphatidylinositol 3-phosphate)as a novel control target,using proteins that bind this lipid to direct secreted anti-microbial peptides and proteins(AMPs)to the surface of Phytophthora pathogens.In transgenic Nicotiana benthamiana,soybean,and potato plants,significantly enhanced resistance to different pathogen isolates was achieved by expression of two AMPs(GAFP1 or GAFP3 from the Chinese medicinal herb Gastrodia elata)fused with a PI3P-specific binding domain(FYVE).Using the soybean pathogen P.sojae as an example,we demonstrated that the FYVE domain could boost the activities of GAFPs in multiple independent assays,including those performed in vitro,in vivo,and in planta.Mutational analysis of P.sojae PI3K1 and PI3K2 genes of this pathogen confirmed that the enhanced activities of the targeted GAFPs were correlated with PI3P levels in the pathogen.Collectively,our study provides a new strategy that could be used to confer resistance not only to Phytophthora pathogens in many plants but also potentially to many other kinds of plant pathogens with unique targets.
文摘In clinical therapy,the amount of antigen administered to achieve oral tolerance for allergic diseases is large,and the cost is a major consideration.In this study,we used tobacco plants to develop a large-scale protein production system for allergen-specific immunotherapy,and we investigated the mechanisms of oral tolerance induced by a transgenic plant-derived antigen.We used plants(tobacco leaves)transgenic for the Dermatophagoides pteronyssinus 2(Der p2)antigen to produce Der p2.Mice received total protein extract from Der p2 orally once per day over 6 days(days 0–2 and days 6–8).Mice were also sensitized and challenged with yeast-derived recombinant Der p2(rDer p2),after which the mice were examined for airway hyper-responsiveness and airway inflammation.After sensitization and challenge with rDer p2,mice that were fed with total protein extracted from transgenic plants showed decreases in serum Der p2-specific IgE and IgG1 titers,decreased IL-5 and eotaxin levels in bronchial alveolar lavage fluid,and eosinophil infiltration in the airway.In addition,hyper-responsiveness was also decreased in mice that were fed with total protein extracted from transgenic plants,and CD4^(+)CD25^(+)Foxp3^(+) regulatory T cells were significantly increased in mediastinal and mesenteric lymph nodes.Furthermore,splenocytes isolated from transgenic plant protein-fed mice exhibited decreased proliferation and increased IL-10 secretion after stimulation with rDer p2.The data here suggest that allergen-expressing transgenic plants could be used for therapeutic purposes for allergic diseases.
基金supported by the National Natural Science Foundation of China,China(32072507,32272495,and 31721004)the Natural Science Foundation of Jiangsu Province,China(BK20220147).
文摘Phytophthora pathogens lead to numerous economically damaging plant diseases worldwide,including potato late blight caused by P.infestans and soybean root rot caused by P.sojae.Our previous work showed that Phytophthora pathogens may generate abundant phosphatidylinositol 3-phosphate(PI3P)to promote infection via direct association with RxLR effectors.Here,we designed a disease control strategy for metabolizing pathogen-derived PI3P by expressing secreted Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase 1(AtPIP5K1),which can phosphorylate PI3P to PI(3,4)P2.We fused AtPIP5K1 with the soybean PR1a signal peptide(SP-PIP5K1)to enable its secretion into the plant apoplast.Transgenic soybean and potato plants expressing SP-PIP5K1 showed substantially enhanced resistance to various P.sojae and P.infestans isolates,respectively.SP-PIP5K1 significantly reduced PI3P accumulation during P.sojae and soybean interaction.Knockout or inhibition of PI3 kinases(PI3Ks)in P.sojae compromised the resistance mediated by SP-PIP5K1,indicating that SP-PIP5K1 action requires a supply of pathogen-derived PI3P.Furthermore,we revealed that SP-PIP5K1 can interfere with the action of P.sojae mediated by the RxLR effector Avr1k.This novel disease control strategy has the potential to confer durable broad-spectrum Phytophthora resistance in plants through a clear mechanism in which catabolism of PI3P interferes with RxLR effector actions.
文摘Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.
基金supported by the Hangzhou Science and Technology Development Plan(Grant No.20140432B05)the New-shoot Talents Program of Zhejiang Province(Grant No.2013R421008)
文摘Cytochrome P450 CYP2E1 is mainly present in hepatocytes in the livers of mammals,where it plays an important role in the metabolism of xenobiotic organic substances. Previous studies showed that transgenic petunia(Petunia hybrid) plants harboring a mammalian cyp2e1 gene(designated cyp2e1-transgenic petunia) exhibited increased resistance to formaldehyde stress. In this study,we used cyp2e1-transgenic petunia plants to analyze physiological indexes related to formaldehyde stress responses. The results indicated that under formaldehyde stress,the malondialdehyde content in cyp2e1-transgenic petunia plants was lower than in β-glucuronidase gene(gus)-transgenic and wild-type petunia plants. The activities of both superoxide dismutase and peroxidase in the cyp2e1-transgenic plants were higher than in gus-transgenic and wild-type plants. The alcohol dehydrogenase activity was slightly increased and more glutathione was consumed. Additionally,under formaldehyde stress,the levels of plant hormones including indole-3-acetic acid,zeatin and abscisic acid in cyp2e1-transgenic petunia plants displayed decreasing trends,whereas the level of gibberellic acid displayed an increasing trend. In contrast,the indole-3-acetic acid,zeatin and abscisic acid levels in gus-transgenic and wild-type petunia plants displayed increasing trends,whereas the gibberellic acid level displayed a decreasing trend. At 72 h after incubation of 0.5 g of cyp2e1-transgenic petunia plants in 40 mL of treatment solution containing formaldehyde at 50 mg·L^(-1),the formaldehyde content remaining in the treatment solution was close to zero while approximately half of original formaldehyde remained in the treatment solutions containing gus-transgenic and wild-type petunia plants.
基金supported by grants from the National Natural Science Foundation of China(grant no.31901026)the China Postdoctoral Science Foundation(grant no.2019M661032)Tianjin Science and technology plan project(grant no.19PTZWHZ00060).
文摘Plant natural products(PNPs)are the main sources of drugs,food additives,and new biofuels and have become a hotspot in synthetic biology.In the past two decades,the engineered biosynthesis of many PNPs has been achieved through the construction of microbial cell factories.Alongside the rapid development of plant physiology,genetics,and plant genetic modification techniques,hosts have now expanded from single-celled microbes to complex plant systems.Plant synthetic biology is an emerging field that combines engineering principles with plant biology.In this review,we introduce recent advances in the biosynthetic pathway elucidation of PNPs and summarize the progress of engineered PNP biosynthesis in plant cells.Furthermore,a future vision of plant synthetic biology is proposed.Although we are still a long way from overcoming all the bottlenecks in plant synthetic biology,the ascent of this field is expected to provide a huge opportunity for future agriculture and industry.
基金supported by the "100-Talent Program of the Chinese Academy of Sciences" foundationthe National Natural Science Foundation of China (31470390)the National Key Technologies Research & Development Program-Seven Major Crop Breeding Project (2016YFD0101803)
文摘Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.
