【作者】 郑继刚；

【导师】 晏月明；

【作者基本信息】 首都师范大学 ， 遗传学， 2004， 硕士

【摘要】 小麦高分子量麦谷蛋白亚基(HMW-GS)是由1A、1B、1D染色体长臂上Glu-1位点编码的一组种子贮藏蛋白，按分子量大小分为x-型和y-型亚基，其组成与面包烘烤品质密切相关。研究表明，D基因组对面包品质具有十分重要的作用，然而普通小麦Glu-D1位点等位基因变异较小，HMW谷蛋白基因资源十分有限。因此，从小麦近缘种中寻找新的候选优质基因对小麦品质改良具有特别重要的意义。本研究以小麦D基因组的祖先——粗山羊草(Aegilops squarrosa或Triticum tauschii，又叫节节麦，DD，2n=2x=14)为材料，利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、酸性聚丙烯酰胺凝胶电泳(A-PAGE)、双向聚丙烯酰胺凝胶电泳(2-DE：A-PAGE×SDS-PAGE)以及高效毛细管电泳(HPCE)等方法对特异HMW-GS进行分离和鉴定，然后通过设计等位基因特异PCR(AS-PCR)引物对其编码基因进行分子克隆、序列测定和比较分析，并将克隆的Dy12.1~t基因连接到表达载体，在大肠杆菌中进行了表达分析与鉴定。主要研究结果如下： ·粗山羊草特异HMW谷蛋白亚基的分离与鉴定 应用单、双向凝胶电泳方法，在粗山羊草中鉴定了6个新的高分子量谷蛋白亚基，分别命名为1Dx1.5~t、1Dx5.2~t、1Dy10.1~t、1Dy12.1~t、1Dy12.2~t和1Dy12.5~t。同时，用高效毛细管电泳技术对这些新亚基进行了表征和鉴定，确定了这些亚基的毛细管电泳图谱，证实它们都为单一蛋白组分，由各自特定的基因编码。从而为特异谷蛋白亚基基因的分子克隆奠定了基础。在磷酸-甘氨酸酸性缓冲液(含20％乙氰和0.05％羟丙基甲基纤维素)条件下，用内径50μm、长度25.5cm(检测长度20cm)的毛细管分离HMW-GS可获得较高的分辨度。在这些条件下，多数x-型高分子量谷蛋白亚基表现为2-3个多态峰，其原因可能与蛋白质翻译后的修饰有关。从本研究结果可以看出，高效毛细管电泳技术具有操作简便、成本低、快速、样品用量少、分辨率和重复性较高以及高度自动化分离等优点，适于对HMW-GS进行高效的分离和和表征。 ·特异HMW-GS编码基因的克隆、序列比较与分子进化分析 根据高分子量谷蛋白亚基基因5’端和3’端的保守性，设计了两对引物分别扩增y-型高分子量谷蛋白亚基基因(1Dy12.1~t和1Dy10.1~t)编码区，以及1Dy12.1~t亚基编码基因的上游DNA序列。对含有目的基因的粗山羊草TD81和TD159进行了AS-PER扩增，均获得了约1950bp的单一强扩增带，分别回收克隆首都师范大学硕士论文后进行DNA序列测定，获得了IDy12.1‘和IDy10.1‘基因的完整编码区序列。同时，对TD159中y一型亚基基因上游序列的PCR也得到12O0bp的强扩增带，克隆测序后获得了IDyl2.1‘的上游启动子序列。IDy12.1‘基因DNA序列全长为2807bp，其中857bp的上游序列具有H姗一GS或真核生物基因的特征序列，如TATA框、类CCAAT序列、“一300元件”保守序列以及增强子序列等。该基因编码区与己知y一型HMW一GS基因结构相同。推导出的氨基酸序列由648个氨基酸残基组成，其蛋白质结构与面包小麦中的IDy10亚基具有很高的同源性，只有5个氨基酸残基的替换。因此，IDy12.l‘基因很有可能对面包品质具有重要作用(该基因已登陆GeneBank，序列号为AY248704)。从粗山羊草TD81中得到的DNA序列全长1980bp，以ATG起始密码子开始，结尾为两个连续的终止密码子 TGATAG，5’端和3’端均为非重复序列，中部为有规律的六肤和九肤重复区，具有y一型HMW一GS基因典型的编码区结构。通过DNA序列推导出的蛋白序列含有638个氨基酸残基，与面包小麦中的IDy12亚基同源性很高。分子进化分析表明，来自粗山羊草的y一型亚基与面包小麦和其它近缘种中的y一型亚基具有很高的相似性，y一型和x一型两个紧密连锁基因在由一个位点分化后沿各自的方向独立进化。.IDy12.1‘基因的原核表达与鉴定 因Tm 59中的Dy12.1‘亚基与普通面包小麦中的的优质亚基Dy1O具有很高的同源性，推测其为一候选优质亚基。为进一步鉴定并研究其功能，对该亚基编码基因进行了原核表达。结果证明该序列能在大肠杆菌中表达，且表达产物大小与TD159种子中的Dy12.l‘亚基相同。对该基因进一步的遗传转化与功能鉴定等工作正在进行之中。更多还原

【Abstract】 The high-molecular-weight glutenin subunits (HMW-GS) are main seed storage proteins encoded by multi-alleles at Glu-1 loci in wheat. There are two subfamilies called x-type and y-type subunits according to their molecular weight. It is well known that bread-making quality is highly associated with the composition of HMW glutenin subunits. Studies showed that the Gli-D1 loci play an important role in bread-making quality. The allelic variations at the Gli-Dl loci in bread wheat, however, are rather limited. Therefore, it is important to widen the genetic basis of bread wheat by using useful genes of related Triticum species. The D genome donor of bread wheat - diploid Asian goatgrass, Aegilops tauschii Coss (syn. Triticum tauschii, 2n = 2x = 14, DD) was found to possess extensive variations at the Gli-Dl loci, and therefore it is expected to serves as an important gene resource for bread wheat quality improvement. In this study, some specific HMW glutenin subunits from Aegilops tauschii were separated and identified by SDS-PAGE, A-PAGE., 2-DE (A-PAGE x SDS-PAGE) and High Performance Capillary Electrophoresis (HPCE): Allele specific PCR primers were designed to amplify and clone some specific HMW-GS genes. The main results were as the followings:Separation and identification of specific HMW-GS in Aegilops tauschii by PAGE and HPCESome novel HMW-GS in Aegilops tauschii were detected by one- and two-dimensional gel electrophoresis and they were named as 1Dx1.5t, 1Dx5.2t, IDy10.1t, lDy12.1t, 1Dy12.2t and lDy12.5t, respectively. They were further characterized by high performance capillary electrophoresis (HPCE). The high resolution CE separations of HMW-GS could be obtained by 0.1 M phosphate-glycine (pH2.50) buffer, containing 20% acetonitrile (ACN) and 0.05% Hydroxypropyl-methylcellulose (HPMC) with a 25.5 cm length capillary (50um i.d.). In addition, the multiple peaks of single HMW-GS under above acidic CE conditions were found, which may relate to post-translational modification of glutenin proteins. Our results showed that HPCE is a powerful tool for HMW-GS separation and characterization with low cost, minor sample requirement, good resolution, and rapidly automatic separation.Cloning, characterization and molecular evolutional analysis of HMW-GS genes at Glu-Dt 1 locusAS-PCR primers were designed to amplify the upstream and coding region of HMW-GS genes from TD159 with IDy12.1t gene and TD81 with 1Dy10.1t gene. Single strongly amplified band with about 1850bp from both accessions were obtained, and then the amplified products were cloned and sequenced. The complete nucleotide sequence of 1Dy12.1t gene was 2807bp, containing an open reading frame of 1950bp and 857bp of upstream sequence. A perfectly conserved enhancer sequence and the -300 element were present at positions of 209-246bp and 424-447bp upstream of the ATG start codon, respectively. The deduced mature protein of 1Dy12.1t subunit comprised 648 amino acid residues and had great similarity to 1Dy10 subunit from bread wheat with only seven amino acid substitutions, suggesting that 1Dy12.1t gene could have positive effect on bread-making quality. The complete nucleotide sequence of 1Dy10.1t gene was 1980bp. Sequence analysis showed that it is the coding sequence of typical y-type HMW-GS genes. Its deduced mature protein had 638 amino acid residues and was highly homologous with 1Dy12 subunit of bread wheat. The phenetic trees produced by nucleotide and protein sequences showed that the x-and y-type subunit genes were clustered together, respectively, and the 1Dy12.1t and 1Dy10.1t genes are closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat. Expression and identification of 1Dy12.1t gene in E. coliBecause of high similarity to 1Dy10 good-quality subunit, it could be concluded that 1Dy12.1t gene have positive effect on bread-making quality. Thus, in order to further understand its function, the expression characters of the 1Dy 12.1t gene were primarily investigated. Cloned 1Dy12.1t gene was connected更多还原