Function Analysis Of A Glycine-rich Protein BrGRP1 Of Chinese Cabbage

GRPs(Glycine-rich proteins),a large family protein consisting of highly repetitive sequences that are glycine rich and simple,plays an important role in plant growth,biological defense and response to abiotic stresses.In this study,we found differences a gene-BrGRP1,which expressed in the sterile and fertile flower buds,highly expressed in the former.The experiment preliminarily reveal the biological function of the gene though construction and screening of cDNA library,construction and genetic transformation of BrGRP1 overexpression vector,and identification of resistance of transgenic Arabidopsis.The main results of this experiment are described as follows:(1)To obtain normalized cDNA library of Chinese cabbage buds with pol cytoplasmic male sterile,the library was constructed by DSN(duplex-specific nuclease)normalization method in combination with SMART(switching mechanism at 5' end of RNA transcript)technology.Then the purified cDNA was recombined with pDONRTM/Zeo vector and transformed into E.coli DH10B.The titer of the library was about 3.2×106 cfu/mL,the total CFU was about 1.28×107 cfu,and the average size of the inserted fragments was about 1.2 kb with a recombinant about 97%.The redundancy of the library which tested by 100 randomly sequenced clones was only 3.7%.(2)The cDNA sequence of BrGRP1 gene was cloned using the specific primer GRP-F/R.BrGRP1 contained a complete open reading frame(ORF)of 804 bp,which encoded a predicted protein containing 267 amino acid residues.The molecular weight of BrGRP1 is 29.7 kDa,and the isoelectric point is 7.3.It is concluded that BrGRP1 protein is not transmembrane protein or secreted protein.The results of BLAST alignment showed that the similarity with Bra004066 sequence was 93%,and the similarity of At1G67870 gene of Arabidopsis thaliana was 65%.(3)The overexpression vector Cam35S-BrGRP1 was successfully constructed by seamless cloning technology,and the genetic transformation of Arabidopsis thaliana was transformed by floral-dip.Five homozygous transgenic lines were obtained by antibiotic resistance screening and PCR confirmation.Three independent T3 homozygous transgenic lines(GRP1,GRP4 and GRP5)were randomly selected for further analysis.Compared with WT plants,no obvious morphological or developmental abnormalities were observed in transgenic plants when they were grown in same artificial soil at normal temperatures in the greenhouse.qRT-PCR analysis showed that BrGRP1 was highly expressed in transgenic Arabidopsis thaliana.Stress response research shows:0.3 ?mmol/LABA?100 mmol/LNaC1 treatment could inhibit the seed germination and seedling growth of transgenic Arabidopsis.These results suggest that the BrGRPl gene is involved in ABA signal transduction and NaCl stress response.