Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd. tauri and Pd. strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminal domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species.     

小麦资源胚乳蛋白Glu-1、Glu-3、Gli-1基因位点变异特点

141个普通小麦品种及农家种中，由Glu-1位点控制的高分子量谷蛋白亚基共27种图谱，最常见的图谱是（N，7+8，2+12）占22%和（N，7+9，2+12）占19.9%，Glu-A1、Glu-B1、Glu-D1位点控制的均为正效应亚基，其图谱（1，7+8，5+10），（1，14+15，5+10），（1，13+16，5+10），（1，17+18，5+10），（2*，7+8，5+10），（2*，13+16，5+10）占13.4%; 由Glu-3位点控制的低分子量谷蛋白亚基共48种以上的图谱，最常见的图谱是（a, j, c）, Glu-A3位点存在6个以上等位基因，新发现的占5.7%, Glu-B3位点存在10个以上等位基因，新发现的占2.8%, Glu-D3位点存在3个等位基因；由Gli-1位点控制的醇溶蛋白共81种以上图谱，Gli-1A1位点存在7个以上等位基因，新发现的占7.1%, Gli-B1位点存在12个以上等位基因，新发现的等位基因占3.5%, Gli-D1位点存在10个等位基因，Gli-B1位点的l为1B/1R易位系，占总数的33.6%; 由Gli-1位点控制的醇溶蛋白和由Glu-3位点控制的低分子量谷蛋白亚基基因变异远比由G1u-1位点控制的高分子量谷蛋白亚基复杂和丰富。     

烟草高分子量核DNA提取方法比较

高分子量(HMW)核DNA的制备是构建BAC文库关键环节之一。为构建高质量的烟草BAC文库,作者研究了一次选择法和二次选择法提取烟草高分子量核DNA的效果。研究结果表明:虽然二次选择法得到的高分子量核DNA浓度较低,但此法省略了对细胞核连续3￣5次的漂洗,而且得到含DNA的凝胶块(plugs)数量是用一次选择法的3倍,因此得到高分子量DNA的总量更高;同时二次选择法得到的plugs纯度更高,排除了酶切时细胞杂质对HMWDNA的干扰,此外在节约材料等方面也优于一次选择法。     

小麦高分子量麦谷蛋白亚基遗传变异及其与品质和其它农艺性状关系的研究

本研究应用SDS-PAGE技术分析了国内外757份供试材料的高分子量（HMW）麦谷蛋白亚基组成。共发现65种HMW亚基组成，其中53种为常见型，12种为罕见型。国外品种的亚基组合数多于国内品种，国内育成品种又多于国内地方品种。作者还发现了部分罕见亚基，即由Glu-D1编码的亚基2.2+12，2+10，2，12以及前人未曾发现的3个亚基。研究     

小麦远缘杂交后代的高分子量麦谷蛋白亚基组成分析

以P7和郑州9023远缘杂交F2代株系为试材,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium do-decyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE)法鉴定分析了杂交后代的高分子量麦谷蛋白亚基(high-molecular-weight subunits of glutenin)组成。结果表明在Glu-A1,Glu-B1和Glu-D1三个位点上分别检测到2,5和2种亚基类型,3位点结合共出现10种组合类型。其中,在Glu-A1位点上出现了1和N亚基,1优质亚基出现的频率为37.5%;在Glu-B1位点上,7 8优质亚基和7 9亚基出现频率最高(均为31.3%);Glu-D1位点上出现了2 12亚基和被世界公认的优质亚基5 10,其中,5 10亚基出现频率高达68.8%。杂交后代中品质评分高达8分及以上的株系占3/8。     

大麦高分子量基因组DNA提取的优化

高分子量(HMW)DNA的制备是进行生物基因组物理作图等相关基因组学研究至关重要的基础工作。为建立一种高效的提取大麦高分子量DNA的方法,对琼脂糖包埋原生质体和琼脂糖包埋细胞核分离基因组DNA的方法进行了改良和优化。限制酶消化,脉冲电场凝胶电泳(PFGE)和Southern杂交等检测表明,应用优化的实验方法所分离的大麦基因组HMW DNA质量好,数量多。与原方法相比,新改进的方法实验流程缩短,效率提高,所分离HMWDNA能更好地满足后续相关研究的需要。这两种优化的分离大麦HMW DNA技术亦可应用于其它禾谷类植物基因组DNA的制备。     

小麦高分子量谷蛋白亚基的遗传研究 Ⅱ.控制基因的染色体定位及其与SDS沉淀值的关系

通过分析一套中国春(钱尼)染色体代换系的胚乳高分子量谷蛋白亚基和微量 SDS沉淀值表明:高分子量谷蛋白亚基受染色体1B 和1D 控制,可能与4B 染色体有关;本试验不能得知高分子量谷蛋白亚基受染色体1A 控制的情况;亚基3和9在一定条件下对沉淀值具有正效应。     

小麦品种面包烘烤品质与其它品质性状关系之研究

通过对黄淮冬麦区３６个主要小麦品种面包烘烤品质，面筋品质和ＨＭＷ－谷蛋白亚基组成的分析，初步明确了参试小麦品种烘烤品质的现状和遗传基础；揭示了面包体积受吹泡示功仪参数、和面仪参数和小麦籽粒沉降值影响的实质；确定了面包体积与ＨＭＷ－谷蛋白亚基组成的定量关系。     

Assessment of genetic variability in hexaploid wheat landraces of Pakistan based on polymorphism for HMW glutenin subunits

Summary Sixty hexaploid wheat landraces collected from five regions of Pakistan were assessed for genetic variability in terms of high molecular weight (HMW) glutenin subunits as revealed by SDS-PAGE. The germplasm appeared to be diverse and unique on the basis of HMW glutenin subunit compositions. Out of 24 alleles detected at all the Glu-1 loci, four belonged to Glu-A1, 12 to Glu-B1 and eight to Glu-D1 locus. The number of novel HMW glutenin subunits detected were 1, 4 and 6 at the three loci (Glu-A1, Glu-B1, Glu-D1), respectively. The frequency distribution patterns of 24 allelic variants detected at the three Glu-1 loci in 1080 samples analysed for 60 accessions were determined both on the basis of individual accessions and on the basis of regions (accessions pooled across the regions). One allele (null) at the Glu-A1 locus, three alleles (17+18, 7+8, 14) at the Glu-B1 locus and, two alleles (2+12 and 2^**+12) at the Glu-D1 locus were found most frequently distributed in the 60 populations. Maximum variation was observed in the Baluchistan and Gilgit regions of Pakistan in terms of distribution of novel Glu-1 alleles. A higher gene diversity was observed between the populations as compared to the gene diversity within the populations while, a reverse pattern of gene diversity was observed when populations were pooled across the regions (higher within the regions than between the regions). A data base has been generated in this study which could be expanded and usefully exploited for cultivar development or management of gene bank accessions.     

小麦贮藏蛋白中高分子量谷蛋白基因的5＇上游调控序列克隆

以小麦品种东农7742的基因组DNA为模板，用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因（HMW-GS12）的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为917bp，该片段的498～922bp序列与Thomspon报道的同一亚基对应区段序列比较，同源性为97．9％，有9个核苷酸发生了改变。推测的TATA box位于836～841bp，有3个CAAT-like box，分别是693bp处的CCAA；730bp处的CAAAT和808bp处的CCAT。另外，在684bp存在E-box（TGCAAAG），还有2个E-like box，分别是608bp处的TGCAAAC和510bp处的TGCAAAA。认为该元件可能与转录速率和胚乳特异性的表达调控有关。