小麦突变体AS208中Glu-B1位点缺失对籽粒中蛋白体形成和储藏蛋白合成与加工相关基因表达的影响

摘要

高分子量谷蛋白亚基(HMW-GS)是小麦籽粒贮藏蛋白的重要成分,其组成、表达和含量决定面团弹性和加工品质.本研究以Glu-B1位点上1Bx20和1By20双亚基沉默的小麦无性系变异体AS208为材料,以AS208的来源亲本轮选987作为对照,对AS208中Glu-B1位点的沉默机制和1Bx20、1By20亚基沉默对种子中蛋白体融合以及合成加工相关基因表达的影响进行了研究.Southern blot分析发现AS208比轮选987少2条特异带,染色体原位杂交(FISH)结果显示轮选987中有6条染色体出现杂交信号,而在AS208中有4条染色体出现杂交信号,表明AS208基因组中缺失Glu-B1位点.透射电镜观察发现,与轮选987相比,AS208的籽粒蛋白体形成过程,以及蛋白体大小和形状基本一致.qRT-PCR检测发现,在12个与籽粒蛋白质合成与加工相关基因中,有7个的表达模式两品种相似,有8个的表达量在AS208中高于在轮选987中,特别是Bip、PDI-1和PDI-5基因(高1.5~2.0倍).本研究结果表明,小麦Glu-B1位点的基因对种子中蛋白体的形成没有明显影响,但一定程度上刺激了多数蛋白质合成和加工相关基因的表达,保证了种子内蛋白含量、蛋白体外形和大小基本不变,表现负反馈调节效应.%High-molecular-weight-glutenin subunits (HMW-GSs) are important components of seed storage proteins in grains of wheat (Triticum aestivum L.), which determine wheat dough elasticity and processing quality. Using AS208, a wheat somatic variation line with silenced 1Bx20 and By20 at Glu-B1 locus, and its originating cultivar Lunxuan 987, we studied the present status of Glu-B1, protein body (PB) formation progress, and expression trends of four kinds of genes (BiP,Dof,PDI, and SPA) related to glutenin synthesis and accumulation at different stages of grain development. Southern blotting analysis revealed that AS208 had two specific bands less than its control parent Lunxuan 987. The fluorescencein situ hybridization (FISH) assay showed six and four chromosomes with signals in Lunxuan 987 and AS208, respectively. These results indicate that the Glu-B1 locus has been deleted from the genome of AS208. Compared to Lunxuan 987, AS208 showed similar size and shape of seed PB during PB formation under a transmission electron microscope (TEM). A total of 12 transcription-factor or molecular-chaperone genes were subjected to qRT-PCR assay. These genes were related to glutenin coding genes expression, storage proteins accumu-lation and processing, PB assembling and transportation. Seven out of 12 genes had simialr expression patterns in AS208 and Lunxuan 987, and eight geneswere expressed more in AS208 than in Lunxuan 987, especially the expression levels of Bip,PDI-1, and PDI-5 were 1.5–2.0 folds higher in AS208 than in Lunxuan 987. These results suggested that Glu-B1 silencing had no sig-nificant effect on the formation of PB during wheat grain development, but partially spurred the expression of some genes regu-lating the synthesis and processing of seed-storage proteins. As a result, via a negative feedback regulation pathway, the protein content in seed and the shape and size of PB remain in similar levels between AS208 and its originating cultivar.