Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.     

Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.     

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 （Pseudomonas sp.）

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.     

Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced. The nucleotide sequence of this gD gene was 1 251 bp,encoding 417 amino acids. Comparied with the published P8-2 strain ,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%. The results indicated that gD of BHV-1 was highly conservative.     

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz.et Migusch)

A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dyl0.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dyl0.1 from seed.     

新疆稻麦低分子量谷蛋白亚基基因序列分析

根据普通小麦低分子量谷蛋白亚基基因保守区序列,设计了一对引物(P1和P2),采用PCR法对新疆稻麦(Triticum petropavlovskyiUdacz.et Migusch)的基因组DNA进行扩增,获得1条约900 bp的片段,纯化后克隆到载体pMD18-T后,对筛选阳性克隆测序,获得1个基因LMWXJ-1(Genbank登录号:AY695380)。序列分析的结果表明LMWXJ-1具有典型的低分子量谷蛋白亚基基因的基本结构。推导的氨基酸序列比较结果表明,LMWXJ-1与Glu-A3和Glu-D3位点的低分子量谷蛋白基因具有较高的相似性(最高相似性分别为82%和80%),而与Glu-B3位点的差异较大(最高相似性仅有68%)。     

TAPHS1基因序列多态性与小麦生殖发育稳定性的相关分析

TAPHS1基因是MFT-like基因,可调控小麦成熟期的籽粒休眠(影响穗发芽抗性)且与开花的发育控制相关,位于小麦3A染色体短臂上。为探究该基因序列多态性与小麦生殖发育稳定性的关系,设计PCR引物扩增TAPHS1基因的2个高频变异区,获得86个品种相应基因区段的DNA序列信息;以不同播期之间抽穗期相差时间为指标调查评价各品种的生殖发育稳定性。结果表明:在TAPHS1基因的高频变异区存在5种多态性,将其命名为A类、B类、C类、D类、E类;A类、B类、C类属非编码区多态性,D类及E类同时涉及编码区和非编码区多态性;D类及E类序列多态性与生殖发育稳定性相关,并根据D类序列信息开发了分子标记。     

小麦TaSPL17基因的克隆、组织特异性表达及原核表达分析

具有SBP结构域的SPL(SQUAMOSA promoter-binding protein-like)蛋白是一类植物特有的转录因子,在植物的生长发育过程中发挥重要的调控作用。为了进一步研究小麦基因TaSPL17的功能,本研究通过同源克隆的方法,从普通小麦品种‘小偃22’中克隆得到TaSPL17基因全长CDS序列。生物信息学分析表明,该基因开放阅读框长度1 158bp,编码385个氨基酸,理论分子质量40.25ku,理论等电点为9.12。半定量RT-PCR结果显示,TaSPL17在小麦根、茎基部、叶片、幼穗中均有表达,在幼穗和茎基部中表达量最高,根次之,在叶片中表达量最少,表明TaSPL17基因可能与小麦的分蘖和穗部的发育有关。将基因TaSPL17与载体pGEX6P-1连接构建原核重组表达载体pGEX6P-1-TaSPL17,然后将其转入大肠杆菌DH5α并对诱导表达的时间、IPTG浓度、温度进行优化,SDS-PAGE分析表明,融合GST标签的TaSPL17在温度37℃,IPTG浓度为0.4mmol/L,诱导3h后表达量最大,有助于进一步纯化TaSPL17蛋白质。     

野生二粒小麦低分子量谷蛋白基因序列分析

根据低分子量谷蛋白亚基(LMW-GS)基因编码区保守序列设计引物,用PCR方法从蛋白质含量低至13.17%的D81和高达27.20%的D42 2份野生二粒小麦(Triticumdicoccoides)中克隆得到2个LMW-GS基因序列LMW-D81和LMW-D42(GenBank上的序列号分别为FJ461691和FJ461690)。它们具有小麦低分子量谷蛋白基因的典型结构特征,其长度分别为1053bp和1011bp,并分别编码350和336个氨基酸残基的成熟蛋白。LMW-D42和LMW-D81的氨基酸序列估算分子量分别为38kDa和39kDa,说明二者均为C型亚基编码基因。LMW-D42和LMW-D81的N-末端序列都为METSHIP-,表明这2个C型亚基编码基因归属LMW-m型。同源性比对和聚类分析揭示,LMW-D81和LMW-D42均属于Glu-B3位点编码基因。LMW-D42和LMW-D81的核苷酸序列和推导的氨基酸序列一致性分别为93.94%和92.57%。与LMW-D81相比,LMW-D42除发生了22处间断性的碱基替换外,还存在一段42个碱基的缺失。对推导氨基酸序列进行的二级结构预测显示,LMW-D81和LMW-D42的蛋白质二级结构高度一致。其α-螺旋主要位于信号肽和C-末端,少量的α-螺旋和不规则卷曲构成了N-末端。大多数不规则卷曲位于重复区,仅有的一段β-折叠则出现在C-末端。同时,它们的编码区均具有分布一致的8个半胱氨酸残基,且第一和第七个半胱氨酸残基均位于无规则卷曲中。这些结构特点对小麦加工品质改良具有一定意义。     

3份小麦品种（系）Gsp 1基因的克隆及序列分析

根据已报道的谷类作物硬度基因grain softness protein-1(Gsp-1)保守DNA序列设计一对特异性引物,以优质小麦品种(系)中国春、SZ-56及M9876基因组DNA为模板进行基因扩增、克隆和序列测定,获得636 bp的Gsp-1全长特异性片段。序列分析显示其含有495 bp完整的开放阅读框,编码全长为164 aa的蛋白,该蛋白含有Gsp-1典型的19 aa信号肽,10个保守的半胱氨酸以及2个保守的色氨酸。序列比对表明,Gsp-1基因不仅含有Gsp-1 a、Gsp-1 b和Gsp-1 c这3种类型,还含有一种新的类型,将其命名Gsp-1 d。进一步对其结构预测分析发现,Gsp-1蛋白可形成5个链间二硫键,含有4~5个α-螺旋。进化树分析显示Gsp-1基因与硬度主效基因Pina和Pinb有着紧密关系。     

小麦低分子量麦谷蛋白基因XYGluD3-LMWGS1在毕赤酵母中表达的探索

以已构建的含有小麦低分子量麦谷蛋白基因XYGluD3-LMWGS1的重组质粒pGEM-XYGluD3-LMWGS1为模板,利用设计的序列特异引物,扩增基因XYGluD3-LMWGS1编码区,构建成巴斯德毕赤酵母表达载体pPIC3.5K-XYGluD3-LMWGS,将其线性化后用电激法导入甲醇型酵母（Pichia.pastoris）菌株GS115中;利用pPIC3.5K特征引物进行PCR鉴定,筛选得到重组转化子,将重组转化子进行蛋白诱导表达,经SDS-PAGE电泳检测,XYGluD3-LMWGS1基因未能在毕赤酵母中成功表达。