乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列.表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似.在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区.但是,本研究所克隆的沉默型1Av基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白.讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制.

Cloning and Characterization of the Coding Sequences of the 1Ay High Molecular Weight Glutenin Subunit Genes from Triticum urartu

Using degenerate oligonucleotide primers and genomic PCR reactions, the complete coding region sequences of two 1Ay high molecular weight (HMW) glutenin subunit genes were amplified from Triticum urartu accessions that showed differential expression of the 1Ay HMW glutenin subunits in their seeds. The coding sequence amplified from the accession that expressed the 1Ay gene (Tu1Ay-e) was highly homologous to that of known y type HMW glutenin subunit genes. Consequently, the primary structure of the protein translated from the coding sequence of Tu1Ay-e was identical to that of previously published y type subunits. Bacterial expression of the coding sequence of Tu1Ay-e produced a polypeptide identical to the 1Ay subunit extracted from seeds, indicating that the cloned sequence was an accurate representation of the original coding region of Tu1Ay-e. In contrast, the coding region sequence amplified from the accession that did not express 1Ay subunit contained three in-frame premature stop codons. Based on past findings on silenced HMW glutenin subunit genes, we conclude that the presence of in-frame premature stop codon(s) is an important feature of the silenced 1Ay gene (Tu1Ay-s) in T. urartu. The potential value of the active 1Ay gene in improving the end use quality of common wheat and the mechanism underlying 1Ay gene silencing are discussed.