棉花GhWRKY3基因的分离及其表达特性分析

Isolation and Expression Analysis of A Novel Gene, Ghwrky3, in Cotton (gossypium Hirsutum L.)

作者: 专业:生物化学与分子生物学 导师:曹学成 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Cotton (Gossypium hirsutum L.), GhWRKY3, Molecular cloning, Expression analysis, Transgenic tobacco

        WRKY是一类主要存在于植物中的转录因子超家族。自Ishiguro和Nakamura从甘薯中分离到SPF1后,WRKY便开始得到广泛的研究和关注,目前已经从多种植物中分离出了WRKY基因并鉴定了其功能。许多研究表明,WRKY参与了植物体的多种生物学过程,尤其是在植物的抗病反应中发挥了重要的作用。本研究以陆地棉为材料,从中克隆得到一个新WRKY基因即GhWRKY3,并对其进行了表达特性分析及初步的功能鉴定。具体结果如下:1、根据同源蛋白设计简并引物,用RACE和RT-PCR的方法从陆地棉中克隆得到一个新的WRKY基因,即GhWRKY3,GenBank注册号为FJ966887。GhWRKY3全长1,705bp,包含72bp的5′非编码区和109bp的3′非编码区,其开放阅读框共1,524bp,编码了一个含有507个氨基酸的多肽。序列分析发现该基因编码的蛋白含有两个典型的WRKY结构域和两个锌指结构(C-X4-C-X22–23-H-X1-H),并且含有一个核定位信号序列PKRR,属于WRKY转录因子家族第I类成员。亚细胞定位发现,该蛋白位于细胞核内。GhWRKY3的基因组含有3个内含子和4个外显子,并且它的第3个内含子位于羧基端的WRKY结构域内。2、用反向PCR和巢式PCR的方法扩增出了该基因的部分启动子序列,共720bp,用PlantCARE在线软件分析发现,该启动子序列内除含有典型的TATA-box,CAAT-box外,还含有ABA应答元件,胁迫应答元件,光应答元件和胚乳发育相关元件等。根据这些结果推测,GhWRKY3可能在植物的胁迫应答反应中和生长发育过程中发挥了一定的作用。3、半定量RT-PCR分析显示,GhWRKY3在根茎叶中均有表达,不具有组织特异性。GhWRKY3可以被多种信号分子包括水杨酸(SA)、脱落酸(ABA)、茉莉酸甲酯(MeJA)、乙烯(ET)、赤霉素(GA3)和过氧化氢(H2O2)所诱导,而在外源细胞分裂素6-BA,生长素类似物NAA的胁迫下,其表达量没有明显的变化。此外,GhWRKY3在真菌棉花枯萎病菌F. oxysporum f. sp. Vasinfectum、立枯丝核菌R. solani、炭疽菌C. gossypii和伤胁迫下表达量上升,而干旱(15%PEG6000)、高盐(NaCl)和低温(4℃), GhWRKY3的表达量没有发生明显的变化。根据这些结果,可以推测GhWRKY3可能在植物的抗病反应和生长发育中发挥了一定的作用。4、构建了GhWRKY3正义植物表达载体pBI121-GhWRKY3,采用农杆菌介导的方法,转化本生烟草, PCR和RT-PCR鉴定分析表明GhWRKY3在烟草中成功表达。5、选取部分T0代转基因植株的自交种子扩增繁殖,得到T1代转基因植株,在分子鉴定的基础上,初步证明所选择的株系的T1代转基因植株种外源基因基本符合3:1的遗传分离定律。选取两株具有代表性的转基因植物的T1代种子,对种子萌发和萌发早期幼苗的生长情况进行了初步分析,结果显示转基因植株比野生型植株萌发晚。进一步分析了转基因植株在添加不同浓度ABA或GA3的培养基上的萌发和萌发早期幼苗生长情况,结果表明GhWRKY3可能参与调节ABA和GA介导的植物的生长发育过程。
    WRKY transcription superfamily mainly exists in the plant. It had been extensive studied since the first WRKY transcription factor named SPF1 identified from sweet potato by Ishiguro and Nakamura. Now, many WRKY genes had been identified from various plants and their functions had been elucidated using genetic and molecular approaches. The increasing data proved that WRKY transcription factors are involved in various biological processes, especially in disease defense responses. In the present study, we isolated a novel WRKY gene, GhWRKY3, from cotton, and then we analyzed its expression patterns and functions. The main results as follows:1. A novel WRKY gene, named GhWRKY3, was isolated from cotton (Gossypium hirsutum L). The deduced full-length cDNA sequence of WRKY gene consisted of 1,705 nucleotides, containing a 72 bp 5′untranslated region (UTR) and a 109 bp 3′UTR. The GhWRKY3 cDNA contains a 1,524 bp ORF encoding a protein of 507 amino acid residues. The deduced GhWRKY3 was found to possess two WRKY domains and two putative zinc finger motifs(C-X4-C-X22–23-H-X1-H). In addition, the deduced GhWRKY3 protein sequence contains a putative nuclear localization signal (NLS), PKRR. Comparison of the genomic sequence and cDNA sequence of GhWRKY3 gene showed that there were three introns and four extrons.2. Using I-PCR and nested PCR, a 720bp fragment of 5′-flanking region of GhWRKY3 gene was isolated from cotton genomic DNA. Then, several putative cis-acting elements involved in ABA-responsiveness, light responsiveness, defense and stress responsiveness and endosperm expression were predicted in 5′-flanking region using the PlantCARE databases. The basic cis-acting elements TATA-box and CAAT-box also existed in this region. The results suggested that GhWRKY3 may be involved in plant defense responses and development processes.3. Semi-quantitative RT-PCR showed that the transcripts of GhWRKY3 could be up-regulated by SA, ABA, MeJA, ET, GA3 and H2O2. Furthermore, the expression of GhWRKY3 was also induced by wounding and infection with three fungal pathogens including Rhizoctonia solani, Colletotrichum gossypii, and Fusarium oxysporum f. sp. vasinfectum, but not induced by 6-BA, NAA, drought, NaCl, and cold.4. A sense expression vector pBI121-GhWRKY3 was constructed, and was transformed into tobacco. Using PCR and semi-quantitative RT-PCR methods, the identification of the transgenic plants were verified, and the results showed that GhWRKY3 had been expressed successfully in transgenic plant.5. Several T1 transgenic plants were obtained by self-reproduction of T0 transgenic plant seeds. Based on the results of molecular identification, the separation ratio of T1 transgenic plants is 3:1. And the result above is consistent with the law of segregation. Seveal seeds of T1 transgenic plants were selected for analyzing the germination and the growth situations of the seedlings, the results showed that the transgentic plant germinated earlier than wild plant. Analysis of germination and the growth situations of plant on MS with different concentration of ABA or GA3, the results suggested that GhWRKY3 may be involved in regulating plant growth and development processes mediated by ABA and GA.
        

棉花GhWRKY3基因的分离及其表达特性分析

摘要9-11
Abstract11-12
1 引言13-27
    1.1 病原相关分子模式激发的免疫反应(PTI)13-14
    1.2 效应蛋白激发的免疫反应(ETI)14-15
    1.3 植物激素与植物抗病反应15-18
        1.3.1 水杨酸、茉莉酸与乙烯15-16
        1.3.2 脱落酸16-17
        1.3.3 其它一些与抗病相关的激素17-18
    1.4 转录因子18-25
        1.4.1 WRKY 转录因子的发现及其起源18-19
        1.4.2 WRKY 转录因子的结构特点19-21
        1.4.3 WRKY 转录因子的结合序列21-22
        1.4.4 WRKY 转录因子的分类22
        1.4.5 WRKY 转录因子的生物学功能22-25
    1.5 本研究的目的和意义25-27
2 材料与方法27-51
    2.1 实验材料27-29
        2.1.1 植物材料27
        2.1.2 植物材料培养与处理27-28
        2.1.3 菌株与质粒28
        2.1.4 酶与各种生化试剂28
        2.1.5 PCR 引物28-29
    2.2 实验方法29-51
        2.2.1 RNA 提取29-31
        2.2.2 cDNA 第一链的合成31-32
        2.2.3 cDNA 回收纯化与加尾32
        2.2.4 cDNA 全长序列的获得32-35
        2.2.5 植物总DNA 的提取与纯化35-37
        2.2.6 基因组DNA 的获得和启动子的分离37-38
        2.2.7 目的片段的回收38-39
        2.2.8 目的片段与克隆载体的连接39
        2.2.9 大肠杆菌感受态细胞制备39
        2.2.10 大肠杆菌感受态细胞的转化39-40
        2.2.11 质粒DNA 的提取40-42
        2.2.12 对重组质粒的鉴定42-43
        2.2.13 半定量RT-PCR 检测目的基因的表达43-44
        2.2.14 植物表达载体的构建与转化44-48
        2.2.15 农杆菌介导转化烟草48-49
        2.2.16 转基因植物的鉴定49
        2.2.17 主要的序列分析软件49-51
3 结果与分析51-72
    3.1 棉花GhWRKY3 基因的克隆51-53
        3.1.1 棉花RNA 的提取及反转录51
        3.1.2 GhWRKY3 中间片段的分离51-52
        3.1.3 GhWRKY3 5′片段的分离52
        3.1.4 GhWRKY3 3′片段的分离52
        3.1.5 GhWRKY3 全长cDNA 的分离52-53
    3.2 GhWRKY3 的序列分析53-60
        3.2.1 GhWRKY3 的全长cDNA 序列分析53-55
        3.2.2 GhWRKY3 编码蛋白序列分析55-58
        3.2.3 GhWRKY3 基因组序列分析58-60
    3.3 GhWRKY3 的亚细胞定位分析60-62
    3.4 GhWRKY3 的表达特性分析62-65
        3.4.1 GhWRKY3 在棉花不同的组织器官中的表达特性62
        3.4.2 GhWRKY3 在信号分子的诱导下表达特性分析62-64
        3.4.3 GhWRKY3 在非生物胁迫下的表达特性分析64
        3.4.4 GhWRKY3 在生物胁迫下的表达特性分析64-65
    3.5 GhWRKY3 在烟草中的超表达及功能初探65-72
        3.5.1 转基因正义表达载体的构建65
        3.5.2 转基因植株的获得65-66
        3.5.3 转基因植株的PCR 鉴定66-67
        3.5.4 转基因植株的半定量RT-PCR 分析67
        3.5.5 T_1 代转基因植株的PCR 鉴定67-68
        3.5.6 T_1 代转基因植株的萌发和萌发后早期的生长情况68-72
4 讨论72-76
    4.1 GhWRKY3 蛋白结构特征.72
    4.2 GhWRKY3 基因启动子序列分析72
    4.3 GhWRKY3 在外界环境胁迫下的表达特性分析72-74
    4.4 GhWRKY3 在转基因烟草生长发育过程中的作用初探74-76
5 结论76-77
参考文献77-87
附录87-89
致谢89-90
攻读学位期间发表的学术论文90
        下载全文需10


本文地址:

上一篇:amiRNA介导抗性转基因烟草植株的抗病性分析
下一篇:盐胁迫下高粱新生叶片结构和光合特性的系统调控研究

分享到: 分享棉花GhWRKY3基因的分离及其表达特性分析到腾讯微博           收藏
评论排行
公告