Cloning, Expression And Function Of C-8, 7 Sterol Isomerase Genes Of Barley (Hordeum Vulgare)

Using arabidopsis C-8,7 sterol isomerase(GenBank accession No.AAD03489)) amino acid sequence as a querying probe,many highly homologous EST sequences were obtained from GenBank and a putative cDNA sequence of barley C-8,7 sterol isomerase was assembled. Futhermore,a barley C-8,7 sterol isomerase gene cDNA,named HvSI(GenBank accession number EF190989) was cloned by RT-PCR along with 3' RACE amplification from barley (Hordeum vulgare) seedlings.Then,the characterization structure and expression pattern of HvSI were analysized.To elucidate the function of HvSI,two prokaryotic expression vector and four plant expression vector,pUbi::HvSI,pUbi::HvSI-RNAi,pUbi::HvSI::GFP and pGluB7::HvSI were constructed respectively.The results were as followings:1.Cloning,expression,and characterization of one cDNA encoding C-8,7 sterol isomerase from barley.After RT-PCR amplification along with 3' RACE,a full-length C-8,7 sterol isomerase gene cDNA,named HvSI(GenBank accession number EF190989) was cloned.HvSIwas 972 bp in full length,including 5' untranslated region of 186 bp,3' untranslated region of 135 bp with Poly A, and an open reading frame(ORF) of 651 bp encoding 216 amino acid with the molecular weight about 24.2 kD,and the isoelectric point 8.75.Homology analysis showed that HvSI protein shared 35.2%-79.6%identity on the amino acid level with the C-8,7 sterol isomerase family reported previous.Quantitative RT-PCR analysis revealed that HvSI was expressed at high level both in shoots and roots,moderate level in embryos,but not in endosperm.Furthermore reseach showed that the trascripts accumulated rapidly after seeds germination,peaked at 8 DAG,and remained high level until 16 DAG with a little decrease.2.Construction and expression of HvSI recombinant protein in prokaryotic expression systemTo construct recombinant plasmid and detect its expression in E.coli BL21 cell,the HvSI cDNA was subcloned into prokaryotic expressed vector pGEX-4T with the correct ORF.The recombinant plasmids,named pGEX-4T-HvSI1 and pGEX-4T-HvSIs,were identified by restriction analysis,PCR and DNA sequencing analysis.And then,both the two recombinant plasmids were transfected into BL21 and the recombinant proteins were detected by SDS-PAGE. The results showed that both the two recombinant C-8,7 sterol isomerase proteins,with signal peptide and no signal peptide can be found in SDS-PAGE clearly,and the MW were 44 kDa and 41 kDa respectively,confirming the putative amino acids sequences ofHvSI cDNA was correct.3.Construction of various eukaryotic expression vectors and foreign genes transformation in riceTo elucidate the function of HvSI,four plant expression vector,pUbi::HvSI, pUbi::HvSI-RNAi,pUbi::HvSI::GFP and pGluB7::HvSI were constructed respectively,and the four constructs along with the empty vector were transformed into rice(Oriza sativa,japannica) variety Kitaake by Agrobacterium-mediated,respectively.After identified with special PCR,13, 12,13,25 and 3 independent transgenic lines were obtained,and the total positive percents was about 95%.To detect the expression pattern of exogenous gene in various transgenic rice lines, quantitative RT-PCR were used.The results showed that the the foreign gene can expressed in many transgenic lines with different level.The results also showed that the expression level of endogenetic C-8,7 sterol isomerase gene,OsSI1 were decreased greatly in some of lines,while the other have no or weak influence.