A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 6...A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.展开更多
The legume species Astragalus sinicus(Chinese milk vetch[CMV])has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preve...The legume species Astragalus sinicus(Chinese milk vetch[CMV])has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration.In this study,we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture(Hi-C)technology.The CMV genome was 595.52 Mb in length,with a contig N50 size of 1.50 Mb.Long terminal repeats(LTRs)had been amplified and contributed to genome size expansion in CMV.CMV has undergone two whole-genome duplication(WGD)events,and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation.The chalcone synthase(CHS)gene family was expanded and was expressed primarily in the roots of CMV.Intriguingly,we found that resistance genes were more highly expressed in roots than in nodules of legume species,suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes.Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.展开更多
基金Supported by Agricultural Science and Technology Innovation Fund of Jiangsu Province.[CX(11)2022]
文摘A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.
基金This research was financially supported by China Agricultural Research System of MOF and MARA(CARS-22)Chinese Outstanding Talents Pro-gram in Agricultural Science,Agricultural Science and Technology Innova-tion Program of CAASChina National Crop Germplasm Resources Platform for Green Manure(NICGR-2021-19).
文摘The legume species Astragalus sinicus(Chinese milk vetch[CMV])has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration.In this study,we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture(Hi-C)technology.The CMV genome was 595.52 Mb in length,with a contig N50 size of 1.50 Mb.Long terminal repeats(LTRs)had been amplified and contributed to genome size expansion in CMV.CMV has undergone two whole-genome duplication(WGD)events,and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation.The chalcone synthase(CHS)gene family was expanded and was expressed primarily in the roots of CMV.Intriguingly,we found that resistance genes were more highly expressed in roots than in nodules of legume species,suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes.Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.
