高大山羊草Aegilops longissima puroindoline b基因的克隆与表达分析

Cloning and Expression Analysis of Puroindoline b Gene in Aegilops longissima

籽粒硬度是小麦加工品质的重要影响因素。puroindoline a(Pina)和puroindoline b(Pinb)是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对高大山羊草Aegilops longissima(SS)的基因组DNA和胚乳RNA进行Pinb基因扩增、克隆、序列测定和表达分析,发现了一个新型Pinb等位基因。基因长360bp,编码119个氨基酸残基,对应于麦类作物Puroindoline B(PinB)成熟蛋白的结构区域,具有麦类作物Pinb基因特有的WPTKWWK的色氨酸结构域基因序列和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,其核苷酸和氨基酸同源性分别为93.3%和92.4%。RT-PCR证实了Pinb基因在籽粒胚乳中的表达。研究结果表明,高大山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为栽培小麦品质改良提供了丰富的遗传资源。

Grain hardness is one of the main characters that determine the processing properties of wheat. Puroindolines, including purnindoline a （Pina） and puroindoline b （Pinb） are major genes controlling grain hardness in wheat. According to the reported conserved regions of Pinb gene, one pair of specific primers were designed and used to amplify Pinb from young leaves genomic DNA and developing endosperm RNA of Aegilops longissima（SS）. Through gene cloning and sequencing, one new allele of Pinb was characterized. It was 360bp in size, encoding 119 amino acid residues which corresponded to the structure of mature Puroindoline B （PinB） protein. It contained the tryptophan motif of WFrKWWK and five disulphide bonds formed by ten cysteines, which were specific to Pinb gene in cereal crop. Compared with Pinb - Dla from T. aestivum cv. Capitole, the Pinb genes in Ae.longissima shared 93.3% homologies in nucleotide sequence and 92.4% homologies in amino acid sequence respectively. The expression of Pinb in grain endosperm was verified by RT- PCR. The results showed that Ae. Iongissima contained grain hardnesscontrolling gene quite different from that of T. aestivum. It＇s possible that Aegilops provides an extensive reservoir of genetic resources for cultirated wheat quality improvement by wide crossing.