粘果山羊草(Aegilops kotschyi) puroindoline b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。puroindoline a（Pin a）和puroindoline b（Pin b）是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pin b基因的保守序列，设计合成了一对特异性引物ForB1与RevB1，对粘果山羊草（Aegilops kotschyi，C^uM^k）的三个材料的基因组DNA和胚乳cDNA进行Pin b基因扩增、克隆、序列测定和表达分析，发现了4个新型Pin b等位基因，基因序列与六倍体小麦的同源基因存在较大的差异。与软粒小麦品种Capitole的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％。其ORF长447bp，编码148个氨基酸残基，都具有麦类作物Pinb基因特有的19个氨基酸的信号肽序列和WPTKWWK的色氨酸结构城。等位基因Pin b-11-1含有1个紧邻色氨酸结构城的突变位点（Val66Phe）。RT—PCR证实了Pin b基因在籽粒胚乳中的表达。Southern blot分析结果显示，三种材料均含有两个拷贝的Pin b基因。研究结果表明，粘果山羊草中包含与小麦差异较大的籽粒硬度控制基因，为栽培小麦的品质改良提供了丰富的基因资源。

Cloning and Expression Analysis of Puroindoline b Gene in Aegilops kotschyi

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 controlling grain hardness in wheat.According to the conserved regions of Pin b gene reported,one pair of specific primers ForB_1 and RevB_1 were designed and used to amplify Pin b from genomic DNA of young leaves and developing endosperm RNA of three accessions of Aegilops kotschyi.Through gene cloning and sequencing,four new alleles of Pin b were characterized.There existed large differences of sequences of Pin b between Ae.kotschyi and Triticum aestivum.Compared with Pinb-D1a from T.aestivum cv.Capitole,the Pin b genes in Ae.kotschyi 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.The ORF of them was 447 bp,encoding 148 amino acid residues, containing the signal peptide of 19 amino acid residues and the tryptophan motif of WPTKWWK,which were specific to Pin b gene in cereal crop.Pin b-11-1 contained a mutation adjacent to the tryptophan motif(Val66Phe).The expression of Pin b in grain endosperm was verified by RT-PCR.Southern blot analysis showed that there were two copies of the gene in all the three accessions.The results showed that Aegilops provide an extensive reservoir of genetic variation,which is available for incorporation into cultivated wheat by wide crossing.