粗山羊草(Aegilops tauschii)中Pinb基因的克隆和表达分析  

Cloning and Expression of Pinb Gene in Aegilops tauschii

作　　者：吴昊[1] 何勇刚[1] 陈鹏[1] 张欣鑫[1] 黄健忠[1] 陈明洁[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：《武汉植物学研究》2007年第6期535-538,共4页Journal of Wuhan Botanical Research

基　　金：国家自然科学基金资助项目(30640043);华中科技大学校科学研究基金(2006M033B)

摘　　要：puroindoline a(Pina)和puroindoline b(Pinb)是控制小麦籽粒硬度的主效基因。根据已报道的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对粗山羊草Aegilops tauschii(DD)的基因组DNA进行Pinb基因扩增、克隆和序列分析,发现了一个新型Pinb等位基因。该基因长447 bp,编码148个氨基酸残基,具有麦类作物PinB蛋白所特有的WPTKWWK色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv.Capitole的Pinb-D1a相比较,该基因含有14个氨基酸变异位点,其中包括一个紧邻色氨酸结构域的变异位点(Val66Phe),其核苷酸和氨基酸同源性分别为93.3%和90.5%。RT-PCR和Western Blot证实了Pinb基因在籽粒胚乳中的表达。Southern Blot分析结果表明,粗山羊草中Pinb基因为单拷贝。研究结果表明,粗山羊草中包含着与小麦差异较大的籽粒硬度控制基因,对此基因的进一步研究将加深对小麦籽粒硬度形成分子机制的了解。Puroindoline a （ Pina ） and puroindoline b （ Pinb ） are the major genes controlling grain hardness in wheat. According to the conserved regions of Pinb gene reported, one pair of specific primers was designed and used to amplify one new Pinb allele from genomic DNA of young leaves of Aegilops tauschii （DD）. Sequence analysis indicated that it was 447 bp in size, encoding 148 amino acid residues which corresponded to the structure of PinB protein, with the tryptophan motif of WPTKWWK and five disulphide bonds formed by ten cysteines ,which were specific to PinB in cereal crop. Compared with Pinb-Dla from Triticum aestivum cv. Capitole, this new Pinb allele in Ae. tauschii contained 14 amino acid mutations, among which there was one mutation adjacent to the tryptophan motif （Val66Phe）, sharing 93.3 % homologies in nucleotide sequence and 90. 5% homologies in amino acid sequence, respectively. The expression of Pinb in grain endosperm was verified by RT-PCR and Western Blot analysis. The results showed that Ae. tauschii contained grain hardness-controlling gene quite different from that of T. aestivum and further study on this gene would benefit our understanding of the mechanism underlying the formation of wheat kernel texture.

关 键 词：粗山羊草 Pinb6基因 籽粒硬度 克隆 

分 类 号：Q78[生物学—分子生物学] S512[农业科学—作物学]