摘要
根据捕光叶绿素a／b结合蛋白基因（cab）家族中的保守序列设计PCR引物，扩增出的约310bpcDNA小片段为分子杂交探针，对构建的水稻cDNA文库进行杂交筛选，并结合PCR分析确定阳性克隆中cDNA片段的大小。通过对插入的cDNA片段最长的阳性克隆进行测序分析验证，克隆了水稻中1个cab基因全长cDNA，命名为cab-n8（GenBank登记号：AY445626）。cab-n8长为1128bp，从第55bp开始至789bp含有1个开放阅读框和1个终止密码子，编码244个氨基酸（GenBank登记号为AAR19267．1）；在3’端含有330bp的非编码区和Poly（A）18；在5’端有45bp的非编码区，在转录起始位点附近有TCA序列。通过序列分析，cab-n8编码的蛋白质在第54—216位包括典型的捕光叶绿素a／b结合蛋白功能域（chlorophll a／b binding domain）；在第141—158位含有无名指结构功能域（ring finger structure domain），该位点可能与捕光叶绿素结合蛋白与叶绿素a／b的结合有关；在第194—231位含有甘油醛-3-磷酸脱氢酶C端功能域（C-terminal domain of glyceraldehyde-3-phosphate dehydrogenase-like）。cab-n8编码的蛋白质预测的等电点和分子量分别为6．52和26955．80 Da。通过比较分析，cab-n8DNA序列（AY445626）和编码的氨基酸序列（AAR19267．1）与cab27DNA序列（AF094775．1）和编码的氨基酸序列（AAC67557．1）相似性最高，均为97％，显示cab家族基因在进化过程中是相当保守的。Northern blot分析表明，该基因在水稻叶和茎中表达没有差异，但光对其表达有明显的诱导促进作用。
A pair of PCR primers was designed according to conserved sequences of cab family gene of different varieties. A fragment with about 350 bp of cab gene was amplified from the first strand of rice cDNA and PCR product employed for probe to screen cDNA library of rice. The inserted cDNA lengths of positive clones were analysed by PCR method. While the largest inserted cDNA was sequenced, a full-length cDNA of cab, named as cab-n8 （ cab gene from Norin 8 ） was obtained （GenBank accession number： AY445626） in rice. The length of cab-n8 is 1 128 bp, which contains an open reading frame encoding 244 amino acids （ GenBank accession number： AAR19267.1 ） and one stop coden from 55th to 789th position, and habours a 330 bp UTR with Poly（A） 18 at 3＇ end and 45 bp UTR with TCA sequences near transcription initiation site at 5＇ end. The bioinformatics characterization indicated that the protein encoded by cab-n8 has a chlorophll a/ b binding domain （54th - 216th position）, a ring finger structure domain relative to binding to chlorophll a/b （141th - 158th position） and a C-terminal domain of glyceraldehyde-3- phosphate dehydrogenase-like （ 194th - 231th position）. The pl and Mw of protein encoded by cab-n8 were predicted to be 6.53 and 26 955.80 Da respectively. The similarity of cab- n8 DNA sequence （AY445626） and encoding amino acid sequence （AAR19267.1） blast with cab-27 DNA sequence （ AF094775.1 ） and encoding amino acid sequence （ AAC67557.1 ） was the highest at rate of 97 %. The results gave a clue that cDNA of cab family genes are highly homology and conserved. Northern blot analysis showed that the expression of cab-n8 was not different between leaf and stem, while the expression of cab-n8 in leaf and stem was significantly induced by light.
出处
《作物学报》
CAS
CSCD
北大核心
2005年第9期1227-1232,共6页
Acta Agronomica Sinica
基金
浙江省自然科学基金项目(No.Y304083)
浙江省教育厅科研计划项目(No.20040069)
杭州师范学院科研启动基金项目(No.87)资助。
