粘果山羊草(Aegilops kotschyi)puroindoline基因的克隆与表达分析

籽粒硬度是小麦加工品质的重要影响因素.籽粒硬度生化标记Friabilin蛋白主要由Puroindoline A（PinA）和Puroindoline B（PinB）构成,它们的编码基因puroindoline a（Pina）和puroindoline b（Pinb）紧密连锁,位于Ha（hardness）位点,是控制籽粒硬度的主效基因.根据小麦Pina、Pinb的保守序列,设计合成了2对特异性引物,对粘果山羊草Aegilops kotschyi（UUSS）3个材料的基因组DNA和胚乳RNA进行Pina、Pinb基因克隆、序列测定和表达分析,发现了3个新型Pina等位基因和4个新型Pinb等位基因.新型puroindoline等位基因全长447bp,编码148个氨基酸残基,具有麦类作物puroindoline基因特有的信号肽序列和色氨酸结构域.与Pina-D1a相比较,新型Pina等位基因核苷酸同源性分别为98.7%、98.4%、97.5%,氨基酸同源性分别为96.6%、95.9%、93.9%.等位基因Pina-allele-2包含一个位于色氨酸结构域内的突变位点（Lys70Arg）.新型Pinb等位基因与Pinb-D1a相比较,其核苷酸同源性分别为93.3%、94.6%、94.6%、94.4%,氨基酸同源性分别为90.5%、93.2%、93.2%、92.6%.等位基因Pinb-allele-1含有一个紧邻色氨酸结构域的突变位点（Val66Phe）.Southern blot分析结果表明,3个材料中Pina和Pinb基因都含有2个拷贝.RT-PCR和Western blot都证实了Pina、Pinb基因在籽粒胚乳中的表达.研究结果显示山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为小麦分子育种提供了丰富的的遗传资源.

Cloning and Expression Analysis of Puroindoline Genes in Aegilops Kotschyi

Grain hardness is one of the main characters that determine the processing properties of wheat. Friabilin, the biochemical marker of wheat grain hardness, is mainly composed of Puroindoline A (PinA) and Puroindoline B (PinB), whose encoding genes puroindoline a (Pina) and puroindoline b (Pinb) are tightly linked at the Ha (hardness) locus. Pina and Pinb are major genes controlling grain hardness in wheat. According to the conserved regions of Pina and Pinb genes reported, two pairs of specific primers were designed and used to amplify puroindolines from young leaves genomic DNA and developing endosperm RNA of three accessions of Aegilops kotschyi (UUSS). Through gene cloning and sequencing, three new alleles of Pina and four new alleles of Pinb were characterized. The whole length of new puroindoline alleles was 447bp, encoding 148 amino acid residues, containing the signal peptide and the tryptophan motif, which were specific to puroindoline genes in cereal crop. Compared with Pina-D1a, the new Pina alleles shared 98.7%, 98.4% and 97.5% homologies in nucleotide sequence and 96.6%, 95.9% and 93.9% homologies in amino acid sequence respectively. Pina-allele-2 contained one mutation inside the tryptophan motif (Lys70Arg). Compared with Pinb-D1a, the new Pinb alleles shared 93.3%, 94.6%, 94.6% and 94.4% homologies in nucleotide sequence and 90.5%, 93.2%, 93.2% and 92.6% homologies in amino acid sequence respectively. Pinb-allele-1 contained a mutation adjacent to the tryptophan motif (Val66Phe). Southern blot analysis showed that there were two copies of Pina and Pinb genes in the three accessions. The expression of puroindolines in grain endosperm was verified by RT-PCR and Western blot analysis.The results showed that large differences of sequences of puroindolines existed between Aegilops and Triticum aestivum, which implied that Aegilops provides an extensive genetic resource in wheat molecular breeding.