牡山羊草puroindoline a基因的克隆与表达分析  

Cloning and Expression Analysis of Puroindoline a Gene in Aegilops juvenalis

作　　者：陈明洁[1] 罗立廷[1] 刘勇[1] 何勇刚[1] 何光源[1] 

机构地区：[1]华中科技大学中英HUST-RRes基因工程和基因组学联合实验室,武汉430074

出　　处：《华中农业大学学报》2005年第6期544-548,共5页Journal of Huazhong Agricultural University

基　　金：国家"973"计划项目(2002CB111302)资助

摘　　要：puroindoline a(Pin a)和puroindoline b(Pin b)是控制小麦籽粒硬度的主效基因。根据已报道的小麦Pin a基因的保守序列,设计合成了1对特异性引物ForA1和RevA1,对六倍体牡山羊草Aegilops juvenalis(UUMMDD)的基因组DNA和胚乳cDNA进行Pin a基因扩增、克隆、序列测定和表达分析,发现了1个新型Pin a(Pin a-allele),其ORF长447 bp,编码148个氨基酸残基,具有麦类作物Pin a基因特有的28个氨基酸的信号肽序列和WRWWKWWK的色氨酸结构域基因序列,与软粒小麦cv.capitole的Pina-D1a相比较,其核苷酸同源性和氨基酸同源性分别为98.7%与96.6%。Pin a-allele含有1个紧邻色氨酸结构域的突变位点(Gln77Leu)。RT-PCR证实了Pin a在籽粒胚乳中的表达。Southern Blot分析结果表明,牡山羊草中Pin a基因含有1个拷贝。研究结果表明,山羊草中包含着与小麦差异较大的籽粒硬度控制基因。Grain hardness is one of the main characters that determine the processing properties of wheat. Puroindolines,including puroindoline a （Pin a ） and puroindoline b （Pin b） are major genes to control grain hardness in wheat. According to the conserved regions of Pin a gene reported,one pair of - specific primers ForA1 and RevAl were designed and used to amplify Pin a from young leaves genomic DNA and developing endosperm RNA of hexaploid Aegilops juvenalis（UUMMDD）. Through gene cloning and sequencing, one new allele of Pin a gene was characterized. Great difference of sequences of Pin a gene existed between Ae. juvenalis and Triticum aestivum. Compared with Pina-Dla from T. aestivum cv. Capitole,the Pin a gene in Ae. juvenalis shared 98. 7% and 96.6% homologies in nucleotide sequence and amino acid sequence respectively. Its ORF was 447 bp, encoding 148 amino acid residues,containing the signal peptide of 28 amino acid residues and the tryptophan motif of WRWWKWWK, which were specific to Pin a gene in cereal crop. The new allele contained a mutation adjacent to the tryptophan motif （Gln77Leu）. The expression of Pin a in grain endosperm was verified by RT-PCR. Southern blot analysis showed that there was one copy of Pin a gene in Ae. juvenalis. The results showed the possibility that Aegilops provide an extensive reservoir of genetic variation which is available for incorporation into cultivated wheat by wide crossing.

关 键 词：牡山羊草 PUROINDOLINE a 籽粒硬度 克隆 

分 类 号：Q949.714.2[生物学—植物学] Q78