α-醇溶蛋白与小麦育种

【作者】 陈梦竹；

【导师】 夏光敏； 陈凡国；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 硕士

【摘要】 为了研究α-醇溶蛋白基因组成及其与小麦育种的关系,我们利用基因组PCR技术分离了158个α-醇溶蛋白基因的编码序列。其中,从二倍体长穗偃麦草（diploid Agropyron elongatum）和老芒麦（Elymus sibiricus）中获得63个α-醇溶蛋白基因的开放阅读框（ORFs）,这些序列中90%存在内部终止密码子或移码,前者主要是由于编码谷氨酰胺的密码子（CAG或CAA）发生了碱基C到T的颠换变为终止密码子（TAG或TAA）。通过分析α-醇溶蛋白基因推导的氨基酸序列发现α-醇溶蛋白是由N-端信号肽,N-端重复区,第一个多聚谷氨酰胺区,N-端非重复区,第二个多聚谷氨酰胺区和C-端非重复区六部分组成,其中除了两个变异较大的多聚谷氨酰胺区外,其他区域比较保守且序列间相似性较大:其差异主要归因于单核昔酸多态性（SNPs）、序列中的密码子的改变或移码突变。在重复区的前三个密码子中,我们发现了一些新的基序,例如GRV和RV;在第一个多聚谷氨酰胺区出现了（Q）₃AR（Q）₅,（Q）₂ AR（Q）₅和（Q）₃A新基序;我们也发现一些具有奇数个半胱氨酸残基的α-醇溶蛋白,并且非保守的半胱氨酸残基在N-和C-末端非重复区都存在。在二倍体长穗偃麦草的假基因中发现了一种已知的乳糜泻疾病（CD）抗原决定簇;在老芒麦的真基因中发现了两种CD抗原决定簇。系统进化树的分析表明,来自于二倍体长穗偃麦草的α-醇溶蛋白基因由若干个亚家族组成,与十倍体来源的基因聚在一起;而来自于老芒麦的α-醇溶蛋白基因则与小麦属的α-醇溶蛋白基因彼此之间具有更高的序列相似性。同时,我们从小麦体细胞杂种渐渗系Ⅱ-12及其亲本普通小麦JN177和十倍体长穗偃麦草中分离了95个α-醇溶蛋白基因的ORFs。序列比对的结果表明杂种Ⅱ-12中α-醇溶蛋白基因的组成和来源如下:（1）杂种中大多数α-醇溶蛋白基因和IN177α-醇溶蛋白基因相似;（2）小部分来自于十倍体长穗偃麦草的渐渗;（3）一些新的基因是由点突变、不等交换或者亲本基因的复制滑动所造成的。因此,我们证实了新的α-醇溶蛋白基因可能通过体细胞杂交而更快的产生,这种产生的方式与HMW-GS自然进化的机制是一致的。进一步氨基酸序列分析表明,在亲本小麦JN177和Ⅱ-12的α-醇溶蛋白基因中鉴定出了四种已知类型的CD抗原决定簇,在十倍体长穗偃麦草中只发现一种。值得注意的是虽然有相同的四种类型的CD表面抗原决定簇,但是杂种Ⅱ-12中编码CD表面抗原决定簇序列的基因数量要低于JN177,特别是在假基因中则更低。文章讨论了体细胞杂种α-醇溶蛋白与小麦育种的关系。α-醇溶蛋白中半胱氨酸的数目和位置影响面粉的品质,因此,我们选择了两个与面粉品质具有潜在相关性的基因,一个来自于老芒麦,另一个来自于Ⅱ-12,对其进行原核表达,然后构建了基于ubiquitin启动子的真核表达载体pCAMBIA3301-E42和pCAMBIA3301-Ⅱ-93,接着运用农杆菌介导的转化方法分别转化小麦株系,通过双引物PCR筛选后,初步获得了19株阳性株系,为进一步的分子鉴定、遗传分析、功能鉴定和育种应用奠定基础。从Ⅱ-12中克隆的H1Dx5基因是一个潜在的优质HMW-GS基因,利用胚乳特异的Bxl7启动子构建了该基因的真核表达载体pBIN20-H1Dx5,为今后该基因功能研究和应用于小麦育种打下了基础。更多还原

【Abstract】 In order to exploitα-gliadin composition and possible relationship with wheat breeding,one hundred and fifty-eightα-gliadin open reading frames were cloned and sequenced from wheat relative grasses,the somatic hybrid wheat introgression lineⅡ-12 and its biparents.Out of them,sixty-threeα-gliadin open reading frames were characterized from diploid Agropyron elongatum and Elymus sibiricus.About 90%of these sequences contained an internal stop codon（TAG or TAA）or frameshift,the former maily due to the change from C to T in glutamine（CAG or CAA）.Aligned with reportedα-gliadin sequences,signal peptide domain,repetitive domain and C-terminal region were significantly identical,but both polyglumatine domains different.Most of the amino acid differences could be attributed to varieties of SNPs and sequences involving complete codons,or frameshift changes.Two new types of motifs GRV and RV were found in repetitive domains of the first three codons.Novel types of motifs,including（Q）₃AR（Q）₅,（Q）₂AR（Q）₅,（Q）₃A,were characterized in the first polyglumatine region.Some ghadins with odd number of cysteine residues were discovered in both N- and C- terminal unique domain,α-gliadin of E.sibiricus contained two types of known CD epitode in potential real gene,whereas,those of diploid Agropyron elongatum has only one type in pseudogenes,no in potential real ones.Phylogenetic analysis showed thatα-gliadins from diploid Agropyron elongatum were comprised of several subfamilies,which were closely related to those from decaploid Agropyron elongatum;while that from E.sibiricus were more homologous to those from other Triticum species.Ninety-fiveα-gliadin open reading frames were cloned and sequenced from somatic hybrid wheat introgression lineⅡ-12 and its parents Triticum aestivum cv. Jinan177（JN177）and decaploid Agropyron elongatum.The data showed the composition and origin of theα-gliadin gene inⅡ-12 included:（1）most that were homologous to those of JN177;（2）a few derived direct from decaploid A.elongatum; （3）some new genes that were created via point mutation,unequal,crossover or slippage of a parental gene.Therefore,Novelα-gliadin genes were found to originate via point mutation,unequal crossover or slippage of a parental gene,demonstrating new genes could be rapidly created through somatic hybridization in a manner similar to that previously shown for HMW-GS genes.A particular quality attribute of interest was the presence or absence of celiac disease（CD）epitopes.These were found to be 4 times more common amongα-gliadin genes from the parent wheat JN177 than from decaploid A.elongatum.Although four types of CD-epitopes were found in introgression lineⅡ-12,the number of the genes encoded CD-epitopes was lower than in JN177,due to the occurrence of pseudogenes.We discussed the benefit of theseα-gliadins to wheat breeding.The number and the locus of cysteine residues inα- gliadin gene play very important role in flour quality.In order to investigate their function,Two genes with odd number cysteine residues,one is from Elymus sibiricus,and the other fromⅡ-12, were conducted prokaryotic expression and then constructed the eukaryotic expression vectors pCAMBIA3301-E42,pCAMBIA3301-Ⅱ-93 controlled by the promoter ubiquitin.After that these two genes were transformed into the wheat strain respectively by using Agrobacterium-mediated transformation method.Nineteen positive transgenic plants were obtained by PCR screening.It can greatly promote the further study of these genes concerning molecular characteristics,genetic analysis, functional detection and the application for wheat breeding.H1Dx5,which is cloned from the somatic hybrid wheat introgression lineⅡ-12, is a potential quality-associated HWM-GS subunit according to its primary structure of deduced amino acid.We have constructed the eukaryotic expression vector pBIN20-H1Dx5 successfully as to pave a way for studying its function and potential utility,in plant breeding.更多还原