小麦Beclin1类似基因的分子克隆与鉴定

以小麦 簇毛麦 (Triticumaestivum Haynal diavillosa) 6VS/6AL易位系 92R1 3 7为材料 ,应用mRNA差异显示和快速扩增cDNA末端 (rapidam plificationofcDNAends,RACE)技术对在白粉菌(Blumeriagraminis)诱导后表达增强的基因进行了克隆。分离到一个与拟南芥Beclin1类似基因同源的全长cDNA克隆 ,暂定名为小麦Beclin1类似基因。它编码 441个氨基酸组成的多肽。二级结构推导显示与人类Beclin相似 ,具有螺旋结构。Northern杂交分析表明 ,小麦Beclin1类似基因在白粉菌诱导后表达增强。Southern分析证明 ,小麦Beclin1类似基因为单拷贝基因     

一粒小麦抗白粉病和条锈病基因的分析

一粒小麦是普通小麦抗性改良的宝贵资源.本研究对24份一粒小麦分别进行了白粉病和条锈病混合菌种苗期接种鉴定,进一步分别用一套白粉病菌菌株(15个)对2份乌拉尔图小麦和条锈病菌小种(21个)对1份栽培一粒小麦进行接种鉴定,其中乌拉尔图小麦UR206能抵抗所有供试白粉菌菌株,UR204除对白粉菌菌株E11感病外,对其余菌株表现抗性;栽培一粒小麦MO205对不同条锈菌小种表现出不同的抗性反应,研究表明乌拉尔图小麦UR206、UR204和栽培一粒小麦MO205分别含有与已知抗白粉病和抗条锈病基因不同的新基因.对乌拉尔图小麦UR204、UR206和栽培一粒小麦MO205分别进行抗白粉和条锈病基因的遗传分析,结果表明乌拉尔图小麦UR204和UR206分别含有一对显性抗白粉病基因,栽培一粒小麦MO205含有两对独立遗传的显性抗条锈病基因.     

小麦黄色素合成途径中Psy 基因的克隆及分子特性小麦黄色素合成途径中Psy 基因的克隆及分子特性

对普通小麦( Ttiticum aestivum) 黄色素(YP) 合成途径中的首要限速酶———八氢番茄红素合成酶(Psy) 基因进行克隆和测序, 并和玉米Psy 基因进行序列比对。结果表明, 在高和低YP 含量小麦品种中均扩增出一条长1 192 bp 的Psy 基因片段, 该片段包含一条可编码78 个氨基酸的小麦Psy 基因的外显子, 与玉米Psy 基因第4 外显子的核苷酸序列同源率为80 . 74% , 同源区域内有47 个SNPs , 但仅11 个SNPs 导致氨基酸编码序列的改变, 二者氨基酸序列的同源率达85 . 89% , 推测Psy 基因在不同物种中的表达具有较高的保守性。BLAST 聚类分析表明, 禾谷类植物Psy 基因的分类与物种的亲缘关系存在明显的相关性,小麦Psy 基因在系统进化中比禾谷类其他植物更为高级。     

小麦硫代硫酸硫转移酶类似基因的克隆与定位

小麦-簇毛麦6VS/6AL易位系92R137含有抗白粉病基因Pm21。为了研究该易位系的抗病机理,应用mRNA差异显示和快速扩增cDNA未端（Rapid Amplification of cDNAEnd,RACE)技术对在白粉菌诱导后表达增强的基因进行了克隆,分离到1个命名为TaTST的全长cDNA序列。Northern杂交分析表明,TaTST基因在白粉菌诱导后表达明显增强,24h达到峰值,氨基酸序列同源性分析表明,TaTST与Datisca glomerata的硫代硫酸硫转移酶基因（rho-danese,EC,2.8.1.1)序列有64%相同,80%相似,用中国春缺体/四体系和端体系Southern杂交和基因特异性引物扩增（gene specific primer-PCR)将TaTST基因定位在小麦6B染色体短臂上,Southern杂交表明,该基因为单拷贝基因,由于在杨麦5号和6VS/6AL易位系间存在明显多态,可以推测在6VS上有TaTST的同源基因,TaTST是从小麦中分离的新基因。白粉菌诱导后的表达变化提示;TaTST与小麦抗白粉病反应有关。     

小麦黄色素合成途径中Psy基因的克隆及分子特性

对普通小麦(Ttiticum aestivum)黄色素(YP)合成途径中的首要限速酶——八氢番茄红素合成酶(Psy)基因进行克隆和测序,并和玉米Psy基因进行序列比对。结果表明,在高和低YP含量小麦品种中均扩增出一条长1192bp的Psy基因片段,该片段包含一条可编码78个氨基酸的小麦Psy基因的外显子,与玉米Psy基因第4外显子的核苷酸序列同源率为80.74,同源区域内有47个SNPs,但仅11个SNPs导致氨基酸编码序列的改变,二者氨基酸序列的同源率达85.89,推测Psy基因在不同物种中的表达具有较高的保守性。BLAST聚类分析表明,禾谷类植物Psy基因的分类与物种的亲缘关系存在明显的相关性,小麦Psy基因在系统进化中比禾谷类其他植物更为高级。     

小麦TOM7蛋白类似基因的克隆与分析

通过RT—PCR技术分离了编码小麦线粒体蛋白转运酶TOM7亚基(Translocase of Outer Mitochondrial Membrane subunit 7)的cDNA,并克隆了该基因编码区的基因组DNA,将该基因命名为TaTOM7。TaTOM7是一个没有内含子的基因,其编码的多肽具有一个位于中部的疏水跨膜区和两个分别位于N-端和C-端的亲水区域。来源于真菌、动物和植物的TOM7类似蛋白在疏水跨膜区高度保守,而在亲水区变异很大。在系统发育树中,TOM7类似蛋白可以按动物、植物和真菌分为3个类群。小麦TaTOM7在基因组中以寡拷贝形式存在,它的表达在Ms2近等基因系之间和不同组织之间存在差异。中国春缺体一四体及端二体系分析表明,TaTOM7位于小麦第3染色体同源群。     

Genetic and Molecular Characterization of the VRN2 Loci in Tetraploid Wheat

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.     

小麦矮秆基因Rht3的RAPD和RFLP标记分析

利用PCR和RFLP技术分析了小麦赤霉酸反应不敏感的矮秆基因Rht3的近等基因系及其分离群体,RAPD分析从310条随机引物中,筛选出3个引物可以在高矮亲本中稳定地扩增出多态带,连锁分析表明仅S10601900和S10602000扩增片段与Rht3;连锁,遗传距离分别为7．1cM和9．2cM.RFLP分析选用了主要位于第4部分同源群短臂上的53个探针,其中Xper584,XksuF8和Xcdo38 3个探针在高矮亲本中揭示出多态性,;连锁分析表明仅Xper584与Rht3基因连锁,遗传距离为8．0cM。     

A SNP-Based Molecular Barcode for Characterization of Common Wheat

Wheat is grown as a staple crop worldwide. It is important to develop an effective genotyping tool for this cereal grain both to identify germplasm diversity and to protect the rights of breeders. Single-nucleotide polymorphism (SNP) genotyping provides a means for developing a practical, rapid, inexpensive and high-throughput assay. Here, we investigated SNPs as robust markers of genetic variation for typing wheat cultivars. We identified SNPs from an array of 9000 across a collection of 429 well-known wheat cultivars grown in China, of which 43 SNP markers with high minor allele frequency and variations discriminated the selected wheat varieties and their wild ancestors. This SNP-based barcode will allow for the rapid and precise identification of wheat germplasm resources and newly released varieties and will further assist in the wheat breeding program.     

小麦抗白粉病基因定位与分子标记

对小麦抗白粉病基因的遗传定位与分子标记进行了综述,介绍了小麦抗白粉病的遗传,并对今后的研究方向进行了讨论。     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.     

小麦Mlo及NBS—LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因MlocDNA序列及一个来源于栽培一粒小麦（Triticum monococcumL.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选。结果获得两个表达基因的cDNA克隆。其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%。另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类。白粉菌诱导前后,该片段RT-PCR扩增产物存在差异。表明该片段可能与小麦抗病性相关。利用“中国春”缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上。     

小麦MBD基因家族的鉴定和特征分析

MBD蛋白是一类与甲基化DNA结合的反式作用因子,在植物生长发育调控过程中发挥重要功能。该研究以‘中国春’小麦为材料,利用生物信息学方法分析了小麦基因组中MBD基因家族成员的组成、序列特征、染色体定位和表达模式,利用qRT-PCR技术分析TaMBD6和TaMBD9基因的时空表达模式。结果显示:(1)小麦MBD基因家族包含16个成员(44个基因位点)分布于第1、2、5、6和7号染色体群;聚类分析表明,小麦MBD蛋白分别属于第Ⅰ、Ⅱ、Ⅲ、Ⅴ、Ⅶ和Ⅷ亚类,其中第Ⅱ、Ⅲ和Ⅷ亚类的MBD蛋白含有5个识别甲基化DNA的保守位点;基因结构分析显示,小麦MBD基因家族成员的内含子数目在1～10之间,启动子区域普遍存在光响应和激素应答元件,且基因组结构特征在同一亚类内高度相似。(2)RNA-Seq数据的基因表达谱分析显示,小麦MBD基因家族多数成员在穗和籽粒发育早期均有较高的表达水平,而且部分成员对干旱和热胁迫有明显响应。(3)qRT-PCR分析显示,TaMBD6和TaMBD9的3个部分同源基因在不同组织间差异表达,但均在幼穗中表达量最高。结果表明,小麦MBD基因可能在小麦发育及非生物胁迫响应过程中发挥调控功能,为进一步探讨小麦MBD基因的功能奠定了基础。     

鸭肠炎病毒gB基因的分子特征

鸭肠炎病毒是鸭病毒性肠炎的病原,被划分为疱疹病毒科成员.gB是疱疹病毒感染和复制所必需的糖蛋白,能刺激机体产生中和抗体和细胞免疫应答,在疱疹病毒家族中高度保守.通过PCR技术获得了DEV C-KCE株gB基因的核苷酸序列,首次对DEV gB基因及编码氨基酸进行了较为详尽的生物信息学分析.结果表明,DEV gB基因与α-疱疹病毒的马立克氏病毒属亲缘关系最近;DEV gB蛋白具有与其他疱疹病毒gB相同或相似的结构、N糖基化位点、半胱氨酸残基、硫酸乙酰肝素蛋白聚糖(HSPGs)结合位点、弗林蛋白酶水解位点和胞浆区PACS-1识别位点.     

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.     

小麦淀粉粒束缚淀粉合成酶基因多态性的分子鉴定

运用6％的SDS-PAGE对14个小麦品种成熟籽粒Wx蛋白的多态性进行了鉴定,结果表明,14个小麦品种根据其Wx蛋白的缺失情况可分为6种组合类型。另外,根据Wx-A1、Wx-D1和Wx-D1这3个位点基因序列和变异情况分别设计了PCR引物,扩增结果表明：Wx-A1位点突变材料扩增产物为327bp,正常材料中扩增不到该特异带;在Wx-B1位点扩增出187bp目标带,突变材料没有该扩增产物;在Wx-D1位点扩增出约700bp目标带,突变材料没有该特异带。与前人的研究结果相比,Wx-B1引物在3个位点的扩增产物长度更短,差异更大,在2％琼脂糖胶上即可清楚分开,缩短了鉴定时间,提高了效率,为大规模筛选优质面条小麦品种提供了可能。     

小麦耐盐基因的标记和标记的克隆

普通小麦（Triticum aesticum L）耐盐农家品种茶淀红与盐敏感品种农大85021杂交,对杂交后代141株F2分离个体进行耐盐性鉴定。通过遗传分析和卡方检验证实小麦耐盐农家品种茶淀红含有一个主效耐盐基因,杂交后代符合1:2:1的分离规律。依据混合群体分组分析法（Bulked Segregate Analysis,BSA）建立耐盐基因池和盐敏感基因池,通过RAPD实验,从520个引物中筛选出了一个在两池间具有多态性的引物OPZ09,用双亲、F1、F2单株DNA进行RAPD实验证明了该引物扩增出的特异性片段OPZ09-590是一个与茶淀红耐盐基因连锁的RAPD标记,用JOINMAP Version1.4计算基因与标记间的重组值为5.674%,连锁距离为6.557cM。从琼脂糖凝胶回收OPZ09-590与载体pUCm-T连接,并转入受体菌JM109,对克隆片段测序表明其实际大小为591bp,故此小麦茶淀红耐盐基因的RAPD标记为OPZ09-591。