幼叶黄化油菜突变体Cr3529中Toc33 cDNA的克隆和序列分析  

Molecular Cloning and Sequence Analysis of the Toc33cDNA in Brassica napus Line Cr3529

作　　者：胡远辉[1] 王茂林[1] 张年辉[1] 廖问陶[1] 赵云[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《中国农业科学》2004年第8期1198-1202,共5页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30170500)

摘　　要：分别提取甘蓝型油菜幼叶黄化突变体Cr3529和野生型近等基因系3529幼叶期总RNA,反转录合成cDNA,根据拟南芥Toc33基因的编码区序列设计引物,RT-PCR扩增出叶绿体外膜转运机器构件蛋白基因Toc33。扩增产物与T载体连接,转化E.coli。获得cDNA克隆,分别命名为BnToc33-c和BnToc33,长度均为894 bp,与拟南芥Toc33cDNA有着较高的同源性。突变油菜Cr3529与野生型3529在Toc33的基因序列上有3处碱基存在差异,均为错义突变,其中一处位于TOC33蛋白的跨膜区域内,推测这一变化可能与幼叶黄化突变相关。Two primers, designed according to the Arabidoposis thaliana Toc33 sequence, were used to amplify the full coding region of the Toc33 cDNAs in leaves of a chlorophyll-reduced (Cr) mutant Cr3529 and its wild type 3529, Brassica napus, by RT-PCR technique. The RT-PCR results showed that the fragment homologous to Toc33 was expressed in Cr3529 as well as 3529 seedlings. PCR fragments were inserted into the pMD18-T vector and transferred into E. coli, then two cDNA clones, BnToc33-c and BnToc33, were obtained. Sequence analysis showed that the two sequences were 894 bp and the nucleotide and the deduced amino acid sequences were highly homologous to those of A. thaliana. There were three diverged nucleotides between the Cr3529 and the 3529 Toc33 cDNAs, i.e, GGT/AGT, TTG/TTT, AGG/AGT, all which belonged to missense mutation. The amino acid replacement (Leu/Phe) caused by TTG/TTT mutation located in the membrane anchor domain may result in chlorophyll-reduced character in Cr3529.

关 键 词：幼叶 黄化油菜 突变体 Cr3529 Toc33cDNA 克隆 序列分析 

分 类 号：S565.4[农业科学—作物学]