小麦HMW-GS基因克隆及转基因沉默的HMW-GS

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	小麦HMW-GS基因克隆及转基因沉默的HMW-GS
其他题名	Molecular cloning of the novel HMW-GS genes and inheritance of transgenic silenced HMW-GS genes in wheat
	相微微
学位类型	硕士
导师	张怀刚
	2009-06-02
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	Hmw-gs 基因分离基因沉默转基因沉默遗传和表达
摘要	高分子量麦谷蛋白亚基(High-molecular-weight glutenin subunits)是普通小麦(Triricum aestivum L.)胚乳中的重要贮藏蛋白。高分子量麦谷蛋白亚基的组成和含量影响小麦的加工品质。农家品种中含有丰富的亚基多态性，本试验拟从农家品种中克隆表达的和沉默的亚基基因，研究其组成、序列特征及进化关系；探讨HMW-GS基因沉默的分子机制与亚基转基因沉默后遗传规律，并为小麦育种提供丰富的种质资源。主要结果如下：（1）利用SDS-PAGE分别在小麦农家品种“2004s-47”和“红火穗”中分离和鉴定了一个HMW-GS，其迁移率大小介于1Dx2和1Ax1之间。而在有芒冰麦中只鉴定出一条过表达的亚基，与“中国春”的1Bx7的亚基的迁移率一致。用grain protein content gene 的保守引物在有芒冰麦中只扩增出2B、2A、4A、4B、6A、6B，确定有芒冰麦为四倍体小麦。（2）利用一对小麦高分子量麦谷蛋白亚基基因的兼并性引物，从2004s-47、红火穗和有芒冰麦中分别扩增出了分子量为2700bp，2700bp和2000bp的片段。根据目的片段的酶切位点的差异，将2004s-47和红火穗的目的片段克隆到PMD18-T Vector 上，有芒冰麦的目的片段克隆到PGEMT-easy Vector上后，采用定向缺失亚克隆的方法获得一系列定向缺失体，选择几个带有适当大小片段的克隆，使相邻克隆嵌套200bp以上，用载体的上游引物测序，并拼接成基因全序列。（3）2004s-47和红火穗的该基因具有典型的小麦HMW-GS基因序列结构特征，即包括信号肽、N-端、C-端和中央重复区等4个部分。信号肽是21个氨基酸残基组成的短肽，N-端由89个氨基酸残基构成，其中有3个半胱氨酸残基（Cys）；C-端有42个氨基酸残基，包括一个半胱氨酸残基（Cys）；中央重复区未发现有半胱氨酸残基（Cys），表明从2004s-47和红火穗中克隆的该HMW-GS基因为x-型亚基基因。（4）经NCBI上的相关软件比对，2004s-47和红火穗的1Dx基因与节节麦(Aegilops tauschii)1Dx2.1t（AF480486）同源性最高。2004s-47的Dx亚基仅有两个氨基酸残基与1Dx2.1t有差异，且都出现在重复序列区。而红火穗虽然基因序列与1Dx2.1t的同源性最高，但由于发生了碱基的缺失，导致在中间重复区域发生了移码突变，编码的氨基酸出现了改变。后由于发生了三个碱基的缺失，正好缺失了一个氨基酸，在之后编码的氨基酸又出现很高的同源性。有芒冰麦沉默表达的亚基基因序列，与1By8（AY245797）亚基的同源性最高，推断有芒冰麦的测序基因为一个沉默表达的亚基基因，命名为1ByX，登录号为FJ959383。沉默的原因在于该基因在编码序列开始后第16-262个碱基处发生小片段缺失。导致发生移码突变，出现多个提前终止密码子，最终发生基因沉默现象，这是一种新的沉默机制。（5）采用杂交及回交的方法研究转基因小麦沉默的HMW-GS基因的遗传及表达。选用Bobwhite转百萨偃麦草Glu-1Ebx基因后所有HMW-GS沉默的小麦品系QQ5与育成品种“高原314”和“新春13号”进行正反交，再用育成品种进行回交，采用SDS-PAGE 技术检测并分析各组合亲本、F1、F2、回交BC1F1的HMW-GS 组成。结果表明，HMW-GS基因沉默表现为显性，且能稳定遗传，杂交及回交后代符合3：1和1：1的分离比，遵循孟德尔遗传方式；稳定的HMW-GS沉默株系的获得为进一步获得弱筋小麦纯系奠定了基础。
其他摘要	The high-molecular-weight glutenin subunits (HMW-GSs) are a major class of storage proteins in common wheat (Triticum aestivum L.). The bread-making quality of common wheat flour is directly influenced by the composition and quantity of HMW-GSs. Since local wheat varieties are rich of HMW-GSs, the objiectives of the present study were 1) to clone expressed and silenced HMW-GS genes from three local varieties, 2) to study their compositions, sequence characteristics and evolution, 3) evaluate molecular mechinisim of HMW-GS gene silence and inheritance of transgenic silenced of HMW-GS genes in progenies and 4) to provide new wheat germplasm without HMW-GS or with weak expression of the genes. The main results of the experiments are as follows. (1) A high molecular weight glutenin subunit (HMW-GS) with electrophorctic mobility between1Dx2 subunit and 1Ax1 subunit in 2004s-47 and Honghuosui was found by SDS-PAGE. However, there was only an overexpressed HMW-GS band in Youmangbingmai, which had similar electrophorctic mobility to that of 1Bx7 of Chinese Spring. Grain protein content gene was isolated with a pair of conservative primer following the PCR procedure. The results showed that Youmangbingmai only had 2B, 2A, 4A, 4B, 6A and 6B, so it is conformed that Youmangbingmai is a tetraploid wheat. (2) Gene was isolated with a pair of degenerate primers for wheat HMW-GS following the PCR procedure. The amplified products of 2700bp from 2004s-47 and Honghuosui were cloned into PMD18-T Vector and the amplified product of 2000bp from Youmangbingmai was cloned into T-easy Vector. The complete sequences of the target HMW-GS genes were obtained by assembling sequences of several clones developed by nested deletion. (3) The genes from 2004s-47 and Honghuosui showed typical structure of wheat HMW-GS, comprising four parts including signal peptide, N-terminal sequence, C- terminal sequence and central repetitive domain. The deduced amino acid sequences showed they belonged to x-type subunits containing three cysteine residues in N-terminal and only one cysteine residue in C-terminal sequence. There was no cysteine present in central repetitive domain. (4) Through comparision analysis by using BLASTn in NCBI, the DNA sequence of the 1Dx gene from 2004s-47 and Honghuosui showed the highest homology with that of previously published 1Dx2.1t（AF480486）in Aegilops tauschii. The 1Dx gene from 2004s-47 had only two amino acid residues in central repetitive domain different from 1Dx2.1t. However, because of the deletion of nucleotides A, G and C in DNA sequence of 1Dx gene had led to frameshift mutation in central repetitive domain and the amino acid sequences were changed between 290th and 485th amino acid locus in Honghuosui. The DNA sequence of the silent 1By gene from Youmangbingmai showed the highest homology with that of previously published 1By8 in durum wheat (AY245797). Therefore, it was concluded that the inactive HMW-GS gene were mutant 1By8 named 1ByX. The silence of 1ByX (FJ959383) was caused by premature stop codons via the deletion of bases between 16th and 262th, which resulted in framshift mutation and indirectly produced several premature stop codons(TAA,TAG) in the coding sequence. The amino acid sequences were early stopped by premature stop codons. This is a new molecular mechanism for the silence of HMW-GS gene in wheat. (5) Crosses and backcrosses between transgene induced HMW-GS gene silencing line QQ5 and two elite wheat varieties have been made to study the inheritance and stability of silenced HMW-GS genes. The two wheat (Triticum aestivum L.) varieties were Plateau 314 and Xinchun 13 with high yield and good adaptability in northwestern China. Different crosses and generation populations were obtained. The grains of the parents, F1, F2 and backcross progenies BC1F1 were analyzed by SDS-PAGE method for HMW-GS genes expression. The results showed that the phenomenon of HMW-GS gene silencing was dominantly controlled by one functional gene and inherited stably in progenies according to the Mendelian pattern. The stable HMW-GS silenced lines would be beneficial to weak wheat breeding.
页数	67
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3242
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	相微微. 小麦HMW-GS基因克隆及转基因沉默的HMW-GS[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.

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