【作者】 余泓漾；

【导师】 郑有良； 贾继增；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 小麦高分子量谷蛋白是主要种子储藏蛋白,对小麦加工品质有重要影响。已有研究发现位于Glu-1B位点的1Bx13+1B16组合(即Glu-B1f)所在的导入系,在不同高分子量谷蛋白亚基构建的导入系群体中对应着最高的蛋白含量,但1Bx13+1By16组合对应高蛋白含量的机理却并不清楚。1Bx13+1By16组合(即Glu-B1f是公认优质亚基,我国只有济麦20等极少数的品种含有,1Bx13+1By16组合在改良我国小麦加工品质上具有重要应用价值,但一些基础研究工作仍然需要完成。针对以上问题,本研究从济麦20中克隆得到1Bx130RF和1By16ORF,构建过表达载体pCUbi1390-1Bx13和pCUbi 1390-1By16,利用农杆菌介导的小麦成熟胚愈伤组织转化体系,对这两个基因进行小麦转基因功能验证。利用济麦20/偃展1号重组自交系群体,分析了1Bx13+1By16组合对小麦主要产量相关性状的影响。主要研究结果如下：1.通过对获得的基因ORF区的镶嵌缺失测序,与NCBI上已发表序列相比,本研究获得的1Bx13 ORF和1By16ORF存在一些序列差异,其中一些差异导致了推导出的蛋白质一级结构上氨基酸序列变化。1Bx13 ORF中氨基酸发生第646位Arg-Gln、702位Leu-Pro、714位Pro-Ser、777位Ser-Gln和792位Thr-Ala的变化。这些氨基酸位点变化,一个位于中间重复区,其余五个位点位于C-末端区域。1By16ORF氨基酸序列中第247位氨基酸由Glu变为Gly,位于中间重复区域。氨基酸的改变导致了1Bx13 ORF具有更高的Gln含量,更加有利于谷蛋白大分子聚合体的形成和稳定。2.利用用于小麦农杆菌转化的过表达载体pCUbi1390,构建了高分子量谷蛋白亚基过表达载体pCUbi1390-1Bx13和pCUbi1390-1By16,用于遗传转化。3.利用农杆菌C58Cl介导的小麦成熟胚转化体系,获得了一批转基因抗性再生植株。小麦转基因实验依然面临愈伤组织褐化现象严重,抗性愈伤组织再生能力不强等问题。由于时间关系,目前正在壮苗培养阶段,待移栽后可以进行检测获得TO代转基因植株。4.利用济麦20/偃展1号重组自交系对1Bx13+1By16亚基组合(即Glu-Blf)与重要产量性状之间的关系作了分析,结果表明,重组自交系群体中含来自亲本济麦20的1Bx13+1By16组合的群体与来自亲本偃展1号的含1Bx14+1By15组合的群体相比,在穗数、株高、穗长、结实小穗数、穗粒数、千粒重这些重要产量性状上,都没有显著性差异。因此,来自济麦20的这条含有优质高分子量谷蛋白亚基组合1Bx13+1By16的染色体区段,能够在不影响产量性状的前提下,提高小麦的蛋白含量。1Bx13+1By16亚基组具有重大的应用潜力。更多还原

【Abstract】 High-molecular-weight glutenin subunits were major parts of wheat storage proteins, and played a significant role in determing wheat processing quality. An early research based on introgression lines with varied HMW-GS alleles showed that the introgression line with 1Bx13+1By16 had the highest grain protein content in all of the investigated introgression lines. However, the mechanism of 1Bx13 and 1By16 correlated with high grain protein content was still unknown.Few of the wheat varieties in China contained those good quality alleles except Jimai20 and several other cultivars,therefore,it had a great potential to use 1Bx13 and 1By16 to improve wheat processing quality in China. In this research 1Bx13ORF and 1By16ORF were cloned from Jimai20,constructed into expressing vectors and transferred into bread wheat through agrobacterium-mediated wheat mature embryo calli transformation system to do functional identification.We also used recombination inbred lines derived from crossing of Jimai20 and Yanzhan No.1 to analysis correlations between yield related traits and alleles 1Bx13+1Byl6. The results were outlined as following.1.We sequenced the full length of 1Bx13ORF and 1By16ORF based on the deletion subclones and BLAST them with 1Bx13ORF and 1By16ORF from NCB1.The results showed that there are several single nucleotide diversities between the two sequences which came from this research and came from NCBI, and some single nucleotide changes leaded to alteration of protein primary structure.They were AA 646 Arg-Gln, AA 702 Leu-Pro,AA 714 Pro-Ser,AA 777 Ser-Gln,and AA 792 Thr-Ala changes in 1Bx130RF, in which AA 646 change was located on repetitive domain while others located on C-terminal domain. In terms of 1By16ORF only one amino acid changed on AA 247 in repetitive domain from Glu on NCBI to Gly in this research. These protein primary structure changes resulted in a higher Gln content in 1Bx13ORF, which were advantageous to the aggregation and stability of the glutenin macropolymer.2.Over expressing vectors pCUbi1390-1Bx13 and pCUbi1390-1By16 were constructed to do agrobacterium-mediated wheat transformation.Resistant plantlets were obtained by using Agrobacterium C58C1 through agrobacterium-mediated wheat mature embryo calli transformation system, and at this moment they were cultured on plantlets growth medium.PCR analysis for the positive TO transgenic plants would be performed when they were transplanted in fields and grain protein content would be measured in T3 or T4 generation according to experimental schedule. The lower regeneration rate and higher browning rate during tissue culture were still a block to improve the efficiency wheat transgenic system.3. The correlations between 1Bx13+1By16 and some yield related traits were analysed using a recombination inbred lines derived from crossing of Jimai20 and Yanzhan No.1, and the result showed that there were no significant difference in spikes per plant, plant height, spike height, number of fertile spikelet, grain number per spike, thousand-grain weight between lines contained 1Bx13+1By16 from Jimai20 and lines contained 1Bx14+1Bx15 from Yanzhan No.1.So we inferred that 1Bx13+1By16 from Jimai20 or the chromosomal segment contained 1Bx13+1By16 from Jimai20 could improve wheat grain protein content while were with little influence on yield related traits, therefore, it was with a great productive potentialities.更多还原