水稻RuBPCase小亚基基因(rbcS)全长cDNA的克隆和分析

Cloning and characterization of a full-length cDNA encoding the RuBPCase small subunit (rbcS) in rice (Oryza sativa L).

根据国际核酸数据库中不同植物的Ru BPCase小亚基基因(rbc S)保守序列设计PCR引物,首先扩增出约35 0 bp的c DNA短片段,以此作为分子杂交探针对构建的水稻c DNA文库进行杂交筛选,同时结合PCR分析确定阳性克隆中c DNA片段的大小,选取插入的c DNA片段最长的阳性克隆进行测序分析验证,从而克隆了水稻中编码Ru BPCase小亚基的全长c DNA(Gen Bank登记号:AY4 4 5 6 2 7) .该c DNA片段从第5 2 bp开始至5 79bp含有编码175个氨基酸的开放阅读框(ORF,Gen Bank登记号:AAR192 6 8.1)和一个终止密码子.AY 4 4 5 6 2 7在3′端有一个长2 6 4 bp的非编码区,而在3′末段有poly(T) 1 7.经Compute p I /Mw软件预测,AAR192 6 8.1的等电点p I和分子量Mw 分别为9.0 3和196 30 .6 6 Da.通过与国际核酸和蛋白质数据库的blast比较分析,AY4 4 5 6 2 7序列与Gen Bank登记号D0 0 6 4 4 .1(水稻rbc S)、U 4 3493.1(大麦rbc S)、AF10 4 2 5 0 .1(燕麦rbc S)和M374 77.1(小麦rbc S)的序列相似性分别为99% (6 5 0 /6 5 5 )、84 % (436 /5 15 )、84 % (42 7/5 0 6 )和84 % (386 /4 5 6 ) .而AAR192 6 8.1氨基酸序列与Gen Bank登记号P185 6 6 (水稻rbc S)、AAF0 794 7.1(燕麦rbc S)、BAA35 16 2 .1(大麦rbc S)和BAB19812 .1(小麦rbc S)的氨基酸序列相似性分别为98% (173/175 )、84 % (14 3/16 9)、84 % (14 0 /16 6 )和83% (14 4 /173) .生物信息学的分析还表明rbc S基因不仅在水稻不同品种基因型之间非常保守,而且在水稻、小麦、大麦和燕麦等禾本科不同物种之间也高度同源.

Based on conserved sequences of RuBPCase small subunit gene (rbcS) of different species in nucleotide databases, PCR primers were designed for amplifying rice rbcS. About 350bp amplified fragment of cDNA was first employed for probe to screen cDNA library of rice, and then the inserted cDNA lengths of positive clones analysed by PCR. While the longest inserted cDNA was sequenced, a full-length cDNA encoding the RuBPCase small subunit was obtained (GenBank accession number AY445627) in rice. The cDNA sequence of AY445627 harbors an open reading frame (GenBank accession number AAR19268.1) and one stop code from 52bp to 579 bp with the 3′end including a 264bp non-encoding region and poly(T)_ 17 . The pI and M_w of AAF07947.1 were estimated to be 9.03 and 19630.66 Da respectively. AY445627 sequences are similar to GenBank accession D00644.1 (Oryza sativa rbcS), U43493.1 (Hordeum vulgar rbcS), AF104250.1(Avena maroccana rbcS)and M37477.1(Triticum aestivum rbcS)with homology of 99%(650/655), 84% (436/515), 84% (427/506) and 84% (386/456) respectively. AAR19268.1 amino acid sequences are similar to P18566 (Oryza sativa rbcS), AAF07947.1 (Avena sterilis rbcS), BAA35162.1 (Hordeum vulgare rbcS) and BAB19812.1(Triticum aestivum rbcS)at rates of 98% (173/175), 84%(143/169), 84%(140/166)and 83%(144/173)respectively. The bioinformatics analysis shows that rbcS gene is not only very conserved in different genotypes of rice, but also highly homology in different species including rice, wheat, barely and oat.