【作者】 金伟波；

【导师】 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 由多个亚基组成的高分子量（HMW）谷蛋白是小麦种子中一类重要的贮藏蛋白，占种子总蛋白的量的 10%以上。大量研究表明，小麦高分子量谷蛋白亚基（HMW-GS）组成与小麦的加工品质密切相关[1]。因此，利用基因工程将优质亚基导入小麦栽培品种以改良其品质是当前小麦品质改良遗传育种的重要课题。因此得到一个小麦优质亚基基因和能驱动此基因在小麦种子中专一且高效表达的启动子，是实现这一目标的前提。 小麦 HMW-GS 是一类种子贮藏蛋白，它的表达具有很高的胚乳专一性。因此，我们利用 PCR 技术克隆了 HMW-GS 基因的启动子。通过对启动子序列的同源性及聚类分析，结果表明：本实验所克隆的启动子为小麦 HMW-GS 1Dx2 启动子，长度约为 1050bp，GenBank 登陆号为:AJ577815。将此片段与β-葡萄糖苷酸酶(Gus)基因编码序列融合，再用基因枪法将其转入小麦种子及叶片中。组织化学检测表明，GUS 基因只在种子得到表达，并比 CaMV 35S 启动子驱动的 GUS 在种子中表达量更高，因此从功能上证明了此HMW-GS 启动子片段能指导外源基因在小麦种子中高效且专一的表达。 小麦种子特异性启动子的分离，为利用小麦种子作为反应器来生产特殊蛋白奠定基础，也是基因工程技术改良小麦品质的前提。在对小麦进行品质改良分子育种时，除拥有自主知识产权的胚乳专一性启动子外，还需要拥有一些与品质呈正相关的优良基因。研究表明：小麦的加工品质与小麦种子中麦谷蛋白和醇溶蛋白的含量和组成关系极为密切，在不同高分子量麦谷蛋白亚基对加工品质的贡献的研究中发现 5+10 亚基为优质亚基，而 2+12 亚基为劣质亚基。然而我国的一些优质面包小麦品种如小偃 6 号（亚基组成为 1，14+15，2+12）等缺乏 5+10 亚基，而具有认为是劣质的 2+12 亚基。分析结果表明导致其优质的原因在于其所具有的 14+15 亚基，14+15 亚基可能具有 5+10 亚基的功能。因此，研究 14 亚基基因的功能对改良小麦品质具有重要意义。本研究在大量分析HMW-GS14 基因序列及总结以往实验结果的基础上，利用改进的 PCR 缓冲体系及一对高 Tm 值的引物，通过二段法成功的克隆了 HMW-GS14 基因。将 HMW-GS14 基因插入到原核表达载体 pET30a(+)质粒上，并使重组质粒在大肠杆菌中得到表达，为验证HMW-GS14 基因的功能奠定基础，也为利用基因工程改良小麦作准备。更多还原

【Abstract】 High-molecular-weight glutenins which constitute of many subunits is a kind ofimportant storage protein and occupy 10% of whole protein in seed. Plenty of studies showedthat constitution of high-molecular-weight glutenin subunit (HMW-GS)play a signification roleon Triticeae processing quality. It is an important subject that transfer gene into cultivateTriticeae to improve quality utilizing gene engineering. So receiving a quality gene andpromoter which can drive it specially expression in embryo is a precondition to qualitybreeding. Triticeae high-molecular-weight glutenins is a important seed storage protein andspecially expression in embryo. We cloned a promoter utilizing PCR technology. Sequencing,homology and alignment analysis showed that the cloned product is HMW-GS 1Dx2’spromoter, the length is 1041 base pairs and the number of Genbank is AJ577815. Linked itwith Gus gene and transferred it into Triticeae seed and slide with gene gun, only observedinstant expression product in Triticeae seed, which certificated that HMW-GS promoter candirect reporting gene especially expression in Triticeae seed. Isolation specially of Triticeae seed settle foundation to use plant as vector to produceprotein and also is precondition to improve Triticeae quality utilizing gene engineeringtechnology. During improve Triticeae quality must have some quality genes and speciallypromoters. The content and constitutes of glutenins and prolamin in Triticeae seed withprocessing quality have closed relation. During the analysis contribute of different HMW-GSto processing quality find that 1Dx5+1Dy10 is quality subunits but 1Dx2+1Dy12 is badsubunits. Some quality bread Triticeae such as xiaoyan 6 (1Bx14+1By15 and1Dx2+1Dy12)have no 1Dx5+1Dy10 but have 1Dx2+1Dy12 in our country. The analysisshowed that 1Bx14+1By15 have the same function with 1Dx5+1Dy10, so research1Bx14+1By15 have signification to quality breeding. We abundance analysis HMW-GS14gene sequence, summarize anciently experimental result and succeed received HMW-GS14gene making use of ameliorative PCR buffer system and a pair of high melting temperature(Tm)primers. Insert it into prokaryotic expression vector pET30a(+), gained recombinant plasmidpET30a-HMW and transformed pET 30a -HMW into E.coli and were expressed intentionprotein product which establish foundation to function identification HMW-GS14 and improveTriticeae utilizing gene engineering.更多还原