Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associat...Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated endonuclease 9(CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T_0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1(type 1, with an adenine insertion) and cr-isa1-2(type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.展开更多
Semi-dwarfing improves the lodging resistance and yield of rice,and the vast majority of modern rice varieties harbor the sd1 allele to decrease plant height,resulting in reduced genetic diversity and negative agronom...Semi-dwarfing improves the lodging resistance and yield of rice,and the vast majority of modern rice varieties harbor the sd1 allele to decrease plant height,resulting in reduced genetic diversity and negative agronomic traits.Thus,exploring alternative sources of dwarfism is imperative for rice breeding.Here,we identified a novel RGA1 allele,d1-w,from a local indica variety Xiaolixiang(XLX)using a map-based cloning approach.Compared with other rice varieties,RGA1 in XLX contained a unique single nucleotide polymorphism that resulted in an additional transcript and reduced functional RGA1 transcript level.The RGA1 from Nipponbare was introduced into XLX to estimate the value of d1-w in rice breeding.Compared with transgenic XLX plants(XLX^(D1)),XLX exhibited reduced plant height,increased stem strength,lower reactive oxygen species accumulation,delayed senescence,stronger photosynthesis,higher grain yield and quality(including external,milling and nutritional qualities),and enhanced resistance to drought and Rhizoctonia solani.Therefore,we proposed that the d1-w allele has potential as an excellent dwarfism resource for rice breeding.展开更多
NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-h...NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of Os K4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of Os K4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that Os K4 functions downstream of NRL3.Moreover,both NRL3 and Os K4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.展开更多
基金supported by the National Nature Science Foundation of China (Grant Nos. 31471472 and 31521064)the National S&T Major Project of China (Grant No. 2016ZX08001006)the Central Level, Non-Profit, Scientific Research Institutes Basic R and D Operations Special Fund (Grant Nos.Y2017PT46 and 2017RG002-1)
文摘Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated endonuclease 9(CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T_0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1(type 1, with an adenine insertion) and cr-isa1-2(type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.
基金supported by grants from the National Natural Science Foundation of China (Grant Nos. 31960403 and 31501286)Jiangxi Natural Science Foundation-Outstanding Youth Science Fund Project,China (Grant No. 20212ACB215003)
文摘Semi-dwarfing improves the lodging resistance and yield of rice,and the vast majority of modern rice varieties harbor the sd1 allele to decrease plant height,resulting in reduced genetic diversity and negative agronomic traits.Thus,exploring alternative sources of dwarfism is imperative for rice breeding.Here,we identified a novel RGA1 allele,d1-w,from a local indica variety Xiaolixiang(XLX)using a map-based cloning approach.Compared with other rice varieties,RGA1 in XLX contained a unique single nucleotide polymorphism that resulted in an additional transcript and reduced functional RGA1 transcript level.The RGA1 from Nipponbare was introduced into XLX to estimate the value of d1-w in rice breeding.Compared with transgenic XLX plants(XLX^(D1)),XLX exhibited reduced plant height,increased stem strength,lower reactive oxygen species accumulation,delayed senescence,stronger photosynthesis,higher grain yield and quality(including external,milling and nutritional qualities),and enhanced resistance to drought and Rhizoctonia solani.Therefore,we proposed that the d1-w allele has potential as an excellent dwarfism resource for rice breeding.
基金supported by the China National Natural Science Foundation(Grant No.31871597)the Key Research and Development Program of Zhejiang Province,China(Grant No.2021C02063-2)+2 种基金the Key Research and Development Program of China National Rice Research Institute(Grant No.CNRRI-2020-02)the Science and Technology Project of Jiangxi Provincial Department of Education,China(Grant No.GJJ180217)the Project supported by Jiangxi Youth Science Foundation,China(Grant No.20202BAB215001)。
文摘NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of Os K4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of Os K4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that Os K4 functions downstream of NRL3.Moreover,both NRL3 and Os K4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.
