黄瓜CsTCTP蛋白表达调控相关信号途径分析及酵母双杂交体系构建

发布时间：2018-02-03 15:31

本文关键词： 黄瓜白粉病 TCTP 生物信息学基因表达酵母双杂交载体构建　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：黄瓜白粉病[Sphaerotheca fuliginea(Schlecht)Poll.]是一种广泛发生的叶部病害,常造成极大的经济损失。目前除应用化学法防治白粉病外,主要依靠选育抗性品种,但长期种植,植株抗性水平逐渐下降。因此,深入研究黄瓜-白粉病互作的分子机制并寻找新的抗性基因资源具有重要意义。本实验室前期研究表明,翻译控制肿瘤蛋白CsTCTP1在黄瓜响应白粉病菌侵染过程中起到重要的调节作用,且CsTCTP1的表达受到外源物质ABA、乙烯、雷帕霉素(TOR抑制剂)等的调控,但CsTCTP1在黄瓜响应白粉病侵染过程中是如何执行其生物学功能的尚不清楚。基于此,本试验利用生物信息学方法,分析预测了 CsTCTP的结构与功能;应用实时荧光定量PCR技术,检测了 TOR、ABA和乙烯信号途径的关键基因在白粉病菌胁迫下的表达变化,初步分析黄瓜CsTCTP1与这些信号通路的相关性;同时,成功构建了CsTCTP1和CsTCTP 的酵母双杂交诱饵表达载体和过表达载体,为深入研究黄瓜CsTCTP蛋白在应答白粉病菌胁迫中的作用与机制奠定基础。本研究主要结果如下:(1)对黄瓜TCTP进行了生物信息学分析。黄瓜有2个TCTP,分别为CsTCTP1和CsTCTP2,CDS区长度均为504bp,且编码168个氨基酸,具有2个TCTP特征结构区(TCTP-1和TCTP-2)。黄瓜TCTP亚细胞定位于细胞质上,为亲水性蛋白,且表现为酸性,α螺旋为TCTP蛋白二级结构中的主要结构。蛋白功能初步预测黄瓜TCTP在翻译、氨基酸的生物合成、能量代谢和胁迫响应等方面发挥主要作用。对不同植物TCTP构建进化树,结果显示黄瓜CsTCTP1与甜瓜的TCTP亲缘关系最近,不同植物TCTP的氨基酸序列一致性高达85.96%,且都具有相同的已知保守结构域。(2)应用实时荧光定量PCR技术,初步研究了黄瓜在白粉病菌胁迫下,黄瓜TOR信号通路相关基因TOR、SnRK1,ABA信号通路关键基因PP2C、SnRK2.1、ABI5和乙烯信号通路基因CTR1、EIN2的表达变化情况。结果表明,这些基因受黄瓜白粉病菌诱导表达,且与CsTCTP1在白粉病菌胁迫下的表达模式相一致。推测黄瓜CsTCTP1响应白粉病菌胁迫可能与ABA、TOR、乙烯等信号通路相关。(3)构建CsTCTP1和CsTCTP2的诱饵表达载体,转化酵母细胞,并验证细胞毒性及自激活活性。通过PCR扩增得到CsTCTP1和CsTCTP2完整的CDS区,并将其重组到诱饵表达载体PGBKT7上,成功构建了诱饵表达载体pGBKT7-CsTCTP1和pGBKT7-CsTCTP2,并将诱饵表达载体转化到酵母细胞Y2HGold中。经验证,诱饵表达载体的融合蛋白对酵母细胞没有细胞毒性,且不具有自激活作用,可用于后续进一步研究,为深入研究黄瓜TCTP的作用机制奠定基础。(4)成功构建了 CsTCTP1和CsTCTP2的过表达载体。通过Gateway技术,将CsTCTPs的编码区构建到过表达载体pB7FWG2中,测序结果显示与已知序列完全一致,说明过表达载体构建成功,可用于下一步研究,且该过表达载体含有GFP标签,可为后续黄瓜TCTP精细定位、瞬时及稳定转化提供实验材料。
[Abstract]:Cucumber powdery mildew. [Sphaerotheca fuliginea SchlechtPoll. is a widespread leaf disease. In addition to chemical control of powdery mildew, it mainly relies on breeding resistant varieties, but long-term planting, plant resistance level gradually decreased. It is of great significance to study the molecular mechanism of cucumber powdery mildew interaction and to search for new resistance gene resources. Translation control tumor protein CsTCTP1 plays an important role in regulating cucumber response to powdery mildew infection, and the expression of CsTCTP1 is affected by exogenous substances ABA and ethylene. The regulation of rapamycin TOR inhibitor, etc., but it is unclear how CsTCTP1 performs its biological function in cucumber response to powdery mildew infection. The structure and function of CsTCTP were analyzed and predicted by bioinformatics. The expression changes of key genes of TORA ABA and ethylene signaling pathway under stress of powdery mildew were detected by real-time fluorescence quantitative PCR. The correlation between CsTCTP1 and these signaling pathways in cucumber was preliminarily analyzed. At the same time, the yeast two-hybrid bait expression vector and over-expression vector of CsTCTP1 and CsTCTP were successfully constructed. In order to further study the role and mechanism of cucumber CsTCTP protein in response to powdery mildew stress, the main results of this study are as follows: 1). The bioinformatics analysis of cucumber TCTP was carried out. There were 2 TCTP in cucumber. The length of CDS region of CsTCTP1 and CsTCTP2 was 504bpand encoding 168 amino acids. Cucumber TCTP subcells were located in cytoplasm and expressed as hydrophilic protein and acidic. 伪 helix is the main structure in the secondary structure of TCTP protein. The function of the protein preliminarily predicts the translation of TCTP and the biosynthesis of amino acids in cucumber. The energy metabolism and stress response played a major role. The results showed that the relationship between cucumber CsTCTP1 and muskmelon TCTP was the closest. The amino acid sequence of TCTP in different plants was consistent as high as 85.96, and all of them had the same known conserved domain. Under the stress of powdery mildew, the key gene PP2CnSnRK2.1 of TOR signaling pathway in cucumber was studied. The expression changes of ABI5 and ethylene signaling pathway genes CTR1 and EIN2. The results showed that these genes were induced by powdery mildew of cucumber. And the expression pattern of CsTCTP1 was consistent with the stress of powdery mildew. It was inferred that the response of cucumber CsTCTP1 to powdery mildew stress might be related to ABA TOR. The decoy expression vectors of CsTCTP1 and CsTCTP2 were constructed and transformed into yeast cells. The complete CDS region of CsTCTP1 and CsTCTP2 was obtained by PCR amplification and was recombined into the decoy expression vector PGBKT7. Bait expression vectors pGBKT7-CsTCTP1 and pGBKT7-CsTCTP2were successfully constructed and transformed into yeast cell Y2HGold. The fusion protein of the decoy expression vector has no cytotoxicity to yeast cells and has no self-activation effect, so it can be used for further study. The overexpression vectors of CsTCTP1 and CsTCTP2 were successfully constructed by means of Gateway technique, which laid a foundation for further study on the action mechanism of cucumber TCTP. The coding region of CsTCTPs was constructed into the overexpression vector pB7FWG2, and the sequencing results were consistent with the known sequence, indicating that the overexpression vector was successfully constructed and could be used in the next research. The overexpression vector contains GFP tag, which can provide experimental materials for subsequent TCTP fine location, transient and stable transformation of cucumber.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.421;Q943.2

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