海岛棉乙烯响应因子GbERF1-like在抗黄萎病反应中的功能解析

发布时间：2018-05-02 13:19

本文选题：棉花 + 黄萎病　；参考：《华中农业大学》2016年博士论文

【摘要】：棉花黄萎病是影响棉花生产的主要病害,乙烯信号路径在棉花与黄萎病菌的互作中扮演着重要的角色。明确乙烯在棉花抗黄萎病反应中的调节机制,并调控乙烯信号路径来改良棉花的抗病性对棉花抗病育种和生产具有重要意义。本研究从海岛棉品种"7124"中克隆了乙烯信号路径的一个ERFs类转录因子基因GbERF1-like,并对其在棉花抗黄萎病反应中的作用机制开展了研究,取得的主要结果有：1、克隆了GbERF1-like的全长序列,其ORF框为537 bp,编码179个氨基酸的多肽,含有一个59个氨基酸的ERFs类转录因子保守AP2/ERF结构域。序列聚类显示GbERF1-like与GhERF1 (AY181251.1), GhERF8 (JN656957.1), TbERF (XM_007035031.1), GbERF5 (JN003808.1)和AtERF1 (AB008103)具有较高的同源性。表达分析显示GbERF1-like在棉花的不同组织都有表达,并能快速响应ET、MeJA处理。相对于感病的陆地棉,GbERF1-like在抗病的海岛棉接种黄萎病菌"V991”后上调表达迅速,并且持续的时间长。2、通过农杆菌介导的遗传转化获得了GbERF1-like在棉花中的超量表达系COV20、COV5与抑制表达系Ci8、Ci6,以及GbERF1-like在拟南芥中的超量表达纯系AOV12、AOV5。接种鉴定分析表明,GbERF1-like的超量表达增强了转基因棉花和拟南芥的抗病性,而抑制GbERF1-like表达的转基因棉花抗病性降低。基因表达分析表明GbERF1-like在棉花中能正调控PR3、PR4的表达,负调控PR1、PR5的表达。但在拟南芥中GbERF1-like能促进AtPR3、AtPR4及AtPDF1.2的表达,但抑制AtPR2的表达。此外,表达分析还显示木质素合成路径相关基因表达受GbERF1-like表达水平的影响,GhPAL5、GhC4H1、GhC3H1、GhHCT1、 GhCCoAOMT1和GhF5H2等基因在GbERF1-like超量表达棉花中表达上升,而在其抑制转基因系中表达量下降。类似的结果在拟南芥中也有发现,这暗示GbERF1-like可能在植物中参与木质素合成的调控。此外,与Mock相比,接种黄萎病菌后GbERF1-like超量表达的转基因棉花、拟南芥中木质素合成路径相关基因的表达上调更为显著。用Wiesner方法对棉花茎的木质素染色结果显示,Mock处理及"V991”处理后的各棉花材料均可被染为红色,所有接种处理的着色明显比Mock深。在Mock处理的各材料间,GbERF1-like超量表达的转基因棉花染色最深,而GbERF1-like抑制表达的转基因棉花染色最浅。木质素总量测定与染色结果相符,接菌处理后,WT、转基因系茎的木质素总量均比Mock处理对应材料显著增加。在Mock及"V991”接种处理后,GbERF1-like超量表达的转基因棉花、拟南芥中木质素总量分别比WT增加6.9%-44.3%,而抑制GbERF1-like表达的转基因棉花中木质素总量比WT降低13.2%-23.4%。木质素单体测定表明,与Mock相比,各材料的G和S单体在接菌后都增加。在Mock及"V991”接种处理后,棉花G单体含量在超表达材料中比WT高但没有达到显著水平,RNAi系的G单体含量比WT显著降低,在5.9%-12.4%之间。转基因拟南芥与WT相比,G单体含量增加幅度在11.2%-31.6%之间。各棉花材料的S单体含量在两种处理中,超表达材料含量较野生型增加10.8%-33.6%,而抑制表达的转基因系下降,降低了7.4%-12.9%。拟南芥的S单体增加不明显。3、分析显示GhHCT1和AtPAL3两个基因的启动子序列均含有TAAT-box、 CAAT-box及GCCGCC-box的ERF类转录因子结合顺式元件。利用酵母单杂交分析表明GbERF1-like与GhHCT1和AtPAL3的启动子之间存在互作。利用双分子荧光检测进一步证实GbERF1-like在棉花原生质体中可以结合GhHCT1和AtPAL3基因的启动子,并提高这两个木质素合成关键基因的转录活性。以上结果证明GbERF1-like直接参与了棉花及拟南芥的木质素合成调控。4、为了进一步证明GbERF1-like介导的棉花抗黄萎病增强与木质素合成激活有关,我们利用病毒介导的基因沉默技术对GbERF1-like超量表达棉花材料COV20中的GhHCT1进行了抑制。分子检测、接种鉴定及木质素含量测定表明,在抑制GhHCT1表达后,GbERF1-like超量表达棉花材料的抗病性明显降低,其茎部木质素含量减少。同时木质素合成相关基因的表达均受到影响,木质素合成上游基因,如GhPAL1 (Gbscaffold40957.1)、GhPAL5、(Gbscaffold11559.9)、GhC4H (Gbscaffold12103.1)普遍表现为表达量降低,而木质素合成下游的基因则普遍表现为表达水平上升,如GhCCR (Gbscaffold2152.13), GhCAD (Gorai.009G192400.1), GhFSH (Gbscaffold12698.2)和GhCOMT (Gorai.004G125600.1)。以上结果证明木质素合成增强在GbERF1-like介导的棉花抗黄萎病反应中具有重要作用。5、通过酵母双杂交筛选鉴定到部分与GbERF1-like互作的蛋白因子。部分因子为前期己报道参与棉花抗病反应的蛋白,如MAPK, MLP, DIRIGENT(Cai et al.,2014;Yang et al.,2015; Hosmani et al.,2013)。此外也有部分互作蛋白的功能还未在棉花抗黄萎病反应中有报道,如P450, HSP, ALLERGEN及GLYOXYLASE等。这些蛋白因子与GbERF1-like互作后对调控木质素合成及棉花抗病反应有何影响还有待进一步研究。
[Abstract]:Cotton Verticillium wilt is the main disease affecting cotton production. Ethylene signaling pathway plays an important role in the interaction of cotton and Verticillium wilt. It is of great significance to clarify the regulation mechanism of ethylene in cotton resistance to Verticillium wilt response and to regulate the resistance of cotton to cotton resistance breeding and production by regulating the ethylene signal path to improve the resistance to cotton. A ERFs class transcription factor gene GbERF1-like of ethylene signal pathway was cloned from the island cotton variety "7124", and the mechanism of its action in cotton resistance to Verticillium wilt was studied. The main results were as follows: 1, the full-length sequence of GbERF1-like was cloned, the ORF frame was 537 BP, and the polypeptide of 179 amino acids was encoded. A 59 amino acid ERFs transcriptional factor conserved the AP2/ERF domain. Sequence clustering showed that GbERF1-like had high homology with GhERF1 (AY181251.1), GhERF8 (JN656957.1), TbERF (XM_007035031.1), GbERF5 (JN003808.1) and AtERF1. Fast response to ET, MeJA treatment. Compared with susceptible land cotton, GbERF1-like was up to up expression after inoculation of Verticillium wilt "V991" in disease resistant island cotton, and it continued to be.2 for a long time. Through agrobacterium mediated genetic transformation, GbERF1-like was obtained in the super expression line of GbERF1-like in cotton, COV20, COV5 and inhibitory expression lines Ci8, Ci6, and GbERF1-like. The overexpression of pure line AOV12 in Arabidopsis, AOV5. inoculation analysis showed that the overexpression of GbERF1-like enhanced the resistance of transgenic cotton and Arabidopsis, and the resistance of transgenic cotton with GbERF1-like expression decreased. The gene expression analysis showed that GbERF1-like could regulate PR3, PR4 expression, negative regulation of PR1, PR5 in cotton flower. But in Arabidopsis, GbERF1-like can promote the expression of AtPR3, AtPR4 and AtPDF1.2, but inhibit the expression of AtPR2. In addition, the expression analysis also shows that the gene expression of lignin synthesis pathway is affected by the level of GbERF1-like expression. The genes of GhPAL5, GhC4H1, GhC3H1, GhHCT1, GhCCoAOMT1 and GhF5H2 are in the super expression of cotton. The expression in the transgenic lines decreased. Similar results were found in Arabidopsis, suggesting that GbERF1-like may be involved in the regulation of lignin synthesis in plants. In addition, the GbERF1-like overexpression of GbERF1-like after inoculation of Verticillium Wilt in cotton and the pathway correlation of lignin synthesis in Arabidopsis thaliana after inoculation of Verticillium wilt. The expression of the expression was more significant. The results of lignin staining of cotton stems by Wiesner method showed that all cotton materials treated with Mock and "V991" could be dyed red, and the coloring of all the treatments was much deeper than that of Mock. Among the materials treated with Mock, the GbERF1-like super expression of transgenic cotton was dyed the most, and GbERF1-li Ke inhibited the expression of transgenic cotton with the most light staining. The total lignin content was in accordance with the staining results. After the treatment, the total lignin content of the transgenic lines was significantly higher than that of the Mock treatment. After the Mock and "V991" inoculation, the total amount of lignin in the transgenic cotton was higher than that of the WT. The total lignin in Arabidopsis thaliana was increased respectively than that of WT after the Mock and "V991" inoculation. Adding 6.9%-44.3%, the total lignin content in transgenic cotton with inhibition of GbERF1-like expression was lower than WT 13.2%-23.4%. lignin monomers. Compared with Mock, the G and S monomers of each material increased after inoculation. After Mock and "V991" inoculation, the content of G monomer in cotton was higher than that of WT but did not reach significant level. The content of G monomer in I system was significantly lower than that of WT. Compared with WT, the content of G monomer in transgenic Arabidopsis was increased by 11.2%-31.6%. The content of S monomer in each cotton material was in two treatments, the content of super expression material increased 10.8%-33.6% compared with the wild type, while the transgenic line decreased and the 7.4%-12.9%. South was reduced. The increase of S monomer in mustard was not significant.3. Analysis showed that the promoter sequences of two genes of GhHCT1 and AtPAL3 all contained TAAT-box, CAAT-box and GCCGCC-box ERF transcription factors combined with cis elements. It was confirmed that GbERF1-like could combine the promoter of GhHCT1 and AtPAL3 gene in the cotton protoplast and improve the transcriptional activity of the two key lignin synthesis genes. The above results show that GbERF1-like directly participates in the synthesis and regulation of.4 in cotton and Arabidopsis lignin synthesis, in order to further prove that GbERF1-like mediated cotton resistance to Verticillium wilt is increased. Strongly related to the activation of lignin synthesis, we used virus mediated gene silencing technology to inhibit GhHCT1 in the GbERF1-like overexpressed cotton material COV20. Molecular detection, inoculation identification and lignin content determination showed that after inhibition of GhHCT1 expression, the resistance of GbERF1-like overexpressed cotton material was significantly reduced, its stem The content of lignin in the xylem decreased and the expression of lignin related genes were affected. The upstream genes of lignin synthesis, such as GhPAL1 (Gbscaffold40957.1), GhPAL5, (Gbscaffold11559.9), and GhC4H (Gbscaffold12103.1), generally showed a decrease in expression, while the genes downstream of lignin synthesis were generally expressed as the level of expression, such as Gh. CCR (Gbscaffold2152.13), GhCAD (Gorai.009G192400.1), GhFSH (Gbscaffold12698.2) and GhCOMT (Gorai.004G125600.1). The above results show that lignin synthesis has an important role in GbERF1-like mediated cotton resistance to Verticillium wilt reaction,.5, and the protein factor interacting with GbERF1-like is identified by yeast two hybrid screening. Some factors are the proteins that have been reported to participate in the resistance of cotton in the earlier period, such as MAPK, MLP, DIRIGENT (Cai et al., 2014; Yang et al., 2015; Hosmani et al., 2013). Besides, some of the functions of the mutual protein are not reported in the cotton resistance to Verticillium wilt. The effect of interaction on lignin biosynthesis and cotton disease resistance remains to be further studied.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.62

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