【作者】 刘坤；

【导师】 李付广；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 棉花是重要的经济作物,各种病害特别是黄萎病严重影响着棉花生产,基因工程技术的发展为棉花抗病育种提供了新途径。ERF族转录因子通过ERF域和GCC-box等顺式元件相互作用,调控下游基因如病程相关蛋白基因的转录过程,从而改变植物抗病、抗逆性状,在植物抵抗生物和非生物胁迫中扮演重要角色。ERF族转录因子分为不同组类,其中B3亚组成员通过上调启动子中含有GCC-box元件基因的转录活性,增强植物体抗病性,而B1亚组转录因子对启动子中含有GCC-box基因的转录活性起抑制作用,具有防止下游基因过度激活的功能。克隆这两亚组基因、探究其生物学功能,对农作物抗病性状的遗传改良具有重要意义。为了克隆与棉花黄萎病抗性相关的B3/B1亚组转录因子基因,本研究首先利用陆地棉EST库进行电子克隆,获得20个B3亚组和9个B1亚组陆地棉ERF族基因。然后根据海岛棉和陆地棉的同源性及ERF族转录因子的保守性,以黄萎病处理的海岛棉幼苗cDNA为模板,利用PCR技术扩增出29个具有完整阅读框的同源基因,其中27个是第一次从海岛棉分离出的新基因。利用实时荧光定量技术,对大丽轮枝菌孢子液处理的海岛棉幼苗根组织中各基因转录本量分析发现:GbB3002、GbB3003、GbB3009、GbB3016-20、GbB1006、GbB1007等多个基因明显增加,上调幅度在7倍以上;GbB3012和GbB3015两个基因只有处理前的三分之一左右;GbB3010基本没有变化;其它基因虽有上升,但上调幅度较小,都在5倍以下。利用RACE技术扩增基因的5’-和3’-UTR,获得4个基因全长cDNA序列,分别命名为GbEREB3（GbB1007）、GbEREB4 （GbB1006）、GbEREB5 （GbB3009）和GbEREB6 （GbB3015）,并提交到NCBI GenBank数据库,注册号分别是JN003806,JN003807,JN003808,JN003809。在利用生物信息学对4个基因分析的基础上,构建GFP瞬时表达载体转化洋葱表皮细胞进行亚细胞定位分析,结果表明四个转录因子均分布于核内;利用BL21（DE3）原核表达菌株,表达GST和转录因子融合蛋白,纯化后进行凝胶阻滞实验,证明它们和GCC-box能够特异结合;构建超表达载体转化烟草,对T₀代植株进行半定量和荧光定量分析发现, GbEREB3/4和GbEREB5/6在转基因阳性植株中均得到异位表达,而防卫基因NtCHI-B和NtPR2的转录本量相应地减少和增加,该结果表明GbEREB3/4和GbEREB5/6分别具有下调和上调NtCHI-B和NtPR2基因转录活性的功能,暗示这些基因参与防卫反应过程。另外,构建超表达载体将B1亚组（Gb）EREB2（GbB1004）基因转入陆地棉,实时荧光定量分析表明T₀阳性棉株中几丁质酶（CHI）和β-1,3-葡聚糖苷酶（BGL）基因的转录活性下降;测量GbEREB2转基因阳性棉株的离体叶片失水率发现比受体叶片更容易失水,表明GbEREB2基因不仅参与棉花的防卫反应过程,而且与植物的水分代谢生理过程密切相关。研究表明这些基因参与棉花对黄萎病菌防卫反应的应答过程,可能参与棉花对黄萎病抗性的调控。有关转基因阳性株系对黄萎病菌抗性水平的鉴定分析研究,正在进行之中。本研究为抗黄萎病相关基因的筛选奠定了基础。更多还原

【Abstract】 Cotton is an important cash crop, however, a variety of diseases, especially Verticillium wilt have exerted serious impact on cotton production. The development of genetic engineering technique provides a new way for cotton disease resistance breeding. Transcription factors of ERF (Ethylene responsive factor) family play an important role in resistance and/or tolerance to biotic and/or abiotic stresses. All members of ERF family contain a highly conservative DNA binding domain, which endow the transcription factors with the capacity to recognize and combine with the GCC-box and nonGCC-box. Via these interactions, they are able to regulate the expression of downstream genes such as PRs (pathogenesis-related protein genes) which contain GCC-box in their promotors, thereby changing the plant traits of disease resistance and stress tolerance. ERF family transcription factors can be divided furhter into many different groups or subgroups. The transcription factors of B3 subgroup are able to serve as activators and up-regulate the transcription activities of GCC-box-promoted genes, and in turn enhance the plant resistance to diseases. On the contrary, B1 subgroup members can inhibit the expression of GCC-box-promoted genes, for preventing overactivation of defense genes. To clone genes of the two subgroups and research their function have significance in genetic improvement of the disease resistance of crops.For the purpose to isolate the B3 and B1 subgroup genes related to resistance to verticillium wilt, in this study, we firstly cloned in silico 29 genes including 20 B3 and 9 B1 members utilizing the EST (Expression sequence tag) data of Gossypium hirsutum. Then, according to the homology between Gossypium barbadense and Gossypium hirsutum, and the conservative characteristics of ERF family transcription factors, we cloned 29 genes, containing complete coding region homologous to upland cotton, by PCR using cDNA extracted from Sea Island cotton seedlings infected with verticillium dahliae as templates, and 27 of the 29 genes are novel. RT-qPCR(Real-time Fluorescence Quantitative Polymerase Chain Reaction)analysis showed that, compared with the control without inoculation, after treated with Verticillium dahliae, the expression of GbB3002、GbB3003、GbB3009、GbB3016-20、GbB1006 and GbB1007 genes in roots tissue were up-regulated obiviously, more than 7 times; however, the GbB3012 and GbB3015 genes were down-regulated at 2-3 fold, and the GbB3010 didn’t basically change, and the transcripts accumulation of other genes had a slight increase less than 5 fold. Then, we cloned the full length cDNAs of the four genes of GbEREB3(GbB1007)、GbEREB4(GbB1006)、GbEREB5(GbB3009) and GbEREB6(GbB3015) by RACE (Rapid amplification of cDNA end) technique, and submmitted them to NCBI GenBank data and received the accession numbers : JN003806, JN003807, JN003808 and JN003809.On the basis of bioinformatics analysis, constrution of over-expression vectors inserted respectively with the four genes fragaments and transformation into onion epidermis cell were performed for transient expression analysis, and the results showed that they were all nucleus-localized proteins, and meanwhile, the results of EMSA (Electrophoretic mobility shift assay) proved that they were all able to combined with GCC-box. Semi-quantitative PCR and RT-qPCR analyses in transgenic tobacco showed that GbEREB3/4/5/6 had the capacity to activate or inhibit the expression of PRs. Furthermore, the results of RT-qPCR analysis also showed that the expression levels of both chitinase (CHI)andβ-1,3-glucanase (BGL)genes decreased in transgenic cotton plants, in which GbEREB2 (GbB1004) over-expressed. In addition, experiments demonstrated that the leaf water loss rate of transgenic cotton plants with GbEREB2 increased compared with non-transgenic cotton lines. These data implied that (Gb)EREB2, a member of B1subgroup genes, involved in the resistance to diseases and water metabolism.This study shows that these genes are involved in the precesses of cotton defense responses to attacks from Verticillium dahliae, and may regulate the resistance of cotton to verticillum wilt. The identification and analysis of cotton resistance against Verticillium dahliae are in progress. And this study provides the basis for screening of genes relative to cotton resistance to verticillium wilt.更多还原