本文關(guān)鍵詞： 黃瓜白粉病 TCTP 生物信息學(xué) 基因表達(dá) 酵母雙雜交 載體構(gòu)建　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：黃瓜白粉病[Sphaerotheca fuliginea(Schlecht)Poll.]是一種廣泛發(fā)生的葉部病害,常造成極大的經(jīng)濟(jì)損失。目前除應(yīng)用化學(xué)法防治白粉病外,主要依靠選育抗性品種,但長期種植,植株抗性水平逐漸下降。因此,深入研究黃瓜-白粉病互作的分子機(jī)制并尋找新的抗性基因資源具有重要意義。本實驗室前期研究表明,翻譯控制腫瘤蛋白CsTCTP1在黃瓜響應(yīng)白粉病菌侵染過程中起到重要的調(diào)節(jié)作用,且CsTCTP1的表達(dá)受到外源物質(zhì)ABA、乙烯、雷帕霉素(TOR抑制劑)等的調(diào)控,但CsTCTP1在黃瓜響應(yīng)白粉病侵染過程中是如何執(zhí)行其生物學(xué)功能的尚不清楚�；诖�,本試驗利用生物信息學(xué)方法,分析預(yù)測了 CsTCTP的結(jié)構(gòu)與功能;應(yīng)用實時熒光定量PCR技術(shù),檢測了 TOR、ABA和乙烯信號途徑的關(guān)鍵基因在白粉病菌脅迫下的表達(dá)變化,初步分析黃瓜CsTCTP1與這些信號通路的相關(guān)性;同時,成功構(gòu)建了CsTCTP1和CsTCTP 的酵母雙雜交誘餌表達(dá)載體和過表達(dá)載體,為深入研究黃瓜CsTCTP蛋白在應(yīng)答白粉病菌脅迫中的作用與機(jī)制奠定基礎(chǔ)。本研究主要結(jié)果如下:(1)對黃瓜TCTP進(jìn)行了生物信息學(xué)分析。黃瓜有2個TCTP,分別為CsTCTP1和CsTCTP2,CDS區(qū)長度均為504bp,且編碼168個氨基酸,具有2個TCTP特征結(jié)構(gòu)區(qū)(TCTP-1和TCTP-2)。黃瓜TCTP亞細(xì)胞定位于細(xì)胞質(zhì)上,為親水性蛋白,且表現(xiàn)為酸性,α螺旋為TCTP蛋白二級結(jié)構(gòu)中的主要結(jié)構(gòu)。蛋白功能初步預(yù)測黃瓜TCTP在翻譯、氨基酸的生物合成、能量代謝和脅迫響應(yīng)等方面發(fā)揮主要作用。對不同植物TCTP構(gòu)建進(jìn)化樹,結(jié)果顯示黃瓜CsTCTP1與甜瓜的TCTP親緣關(guān)系最近,不同植物TCTP的氨基酸序列一致性高達(dá)85.96%,且都具有相同的已知保守結(jié)構(gòu)域。(2)應(yīng)用實時熒光定量PCR技術(shù),初步研究了黃瓜在白粉病菌脅迫下,黃瓜TOR信號通路相關(guān)基因TOR、SnRK1,ABA信號通路關(guān)鍵基因PP2C、SnRK2.1、ABI5和乙烯信號通路基因CTR1、EIN2的表達(dá)變化情況。結(jié)果表明,這些基因受黃瓜白粉病菌誘導(dǎo)表達(dá),且與CsTCTP1在白粉病菌脅迫下的表達(dá)模式相一致。推測黃瓜CsTCTP1響應(yīng)白粉病菌脅迫可能與ABA、TOR、乙烯等信號通路相關(guān)。(3)構(gòu)建CsTCTP1和CsTCTP2的誘餌表達(dá)載體,轉(zhuǎn)化酵母細(xì)胞,并驗證細(xì)胞毒性及自激活活性。通過PCR擴(kuò)增得到CsTCTP1和CsTCTP2完整的CDS區(qū),并將其重組到誘餌表達(dá)載體PGBKT7上,成功構(gòu)建了誘餌表達(dá)載體pGBKT7-CsTCTP1和pGBKT7-CsTCTP2,并將誘餌表達(dá)載體轉(zhuǎn)化到酵母細(xì)胞Y2HGold中。經(jīng)驗證,誘餌表達(dá)載體的融合蛋白對酵母細(xì)胞沒有細(xì)胞毒性,且不具有自激活作用,可用于后續(xù)進(jìn)一步研究,為深入研究黃瓜TCTP的作用機(jī)制奠定基礎(chǔ)。(4)成功構(gòu)建了 CsTCTP1和CsTCTP2的過表達(dá)載體。通過Gateway技術(shù),將CsTCTPs的編碼區(qū)構(gòu)建到過表達(dá)載體pB7FWG2中,測序結(jié)果顯示與已知序列完全一致,說明過表達(dá)載體構(gòu)建成功,可用于下一步研究,且該過表達(dá)載體含有GFP標(biāo)簽,可為后續(xù)黃瓜TCTP精細(xì)定位、瞬時及穩(wěn)定轉(zhuǎn)化提供實驗材料。
[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. 偽 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.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S436.421;Q943.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;黃瓜貯藏中防止衰老的初步研究[J];Journal of Integrative Plant Biology;1976年02期

2 ;保護(hù)地專用黃瓜品種[J];河南科技;2002年22期

3 張圣平;顧興芳;許文連;王燁;;生物技術(shù)用于黃瓜遺傳育種[J];生物技術(shù)通報;2006年02期

4 孫振久;王亞娟;張顯;;黃瓜分子標(biāo)記輔助育種研究進(jìn)展[J];西北植物學(xué)報;2006年06期

5 鄒金美;張國廣;陳亮;;黃瓜轉(zhuǎn)基因研究進(jìn)展[J];廈門大學(xué)學(xué)報(自然科學(xué)版);2008年S2期

6 韓建明;王穎;;生物技術(shù)在黃瓜研究中的應(yīng)用[J];生物技術(shù)通報;2010年08期

7 李鈺;王文宏;段靜霞;毛春云;;不同氧分壓對黃瓜在貯藏中形態(tài)與結(jié)構(gòu)變化的影響的初步觀察[J];Journal of Integrative Plant Biology;1973年01期

8 藍(lán)強(qiáng);周玉意;;黃瓜四個品種的核型研究[J];廣西農(nóng)學(xué)院學(xué)報;1989年01期

9 任志雨,張福墁;不同黃瓜品種對缺鐵的敏感性研究[J];張家口農(nóng)專學(xué)報;1999年01期

10 駱鐵寶,趙洪梅;為什么有的黃瓜吃起來很苦[J];生物學(xué)教學(xué);1999年08期

相關(guān)會議論文 前10條

1 樊榮;周清;;植物聲頻控制技術(shù)對黃瓜生長特性影響的研究[A];紀(jì)念中國農(nóng)業(yè)工程學(xué)會成立30周年暨中國農(nóng)業(yè)工程學(xué)會2009年學(xué)術(shù)年會（CSAE 2009）論文集[C];2009年

2 程奕;孟兆芳;李淑菊;張璽;霍振榮;龐金安;馬德華;;不同黃瓜品種養(yǎng)分吸收特性的研究[A];紀(jì)念天津科潤黃瓜研究所建所二十五周年論文摘要集（2001—2010）[C];2010年

3 周秀艷;秦智偉;;黃瓜品種資源商品性的評價[A];中國園藝學(xué)會第十屆會員代表大會暨學(xué)術(shù)討論會論文集[C];2005年

4 沈鏑;李錫香;王海平;宋江萍;;黃瓜種質(zhì)資源研究進(jìn)展與展望[A];全國蔬菜和薯類種質(zhì)資源研究與利用研討會論文集[C];2006年

5 張亞平;胥愛玲;;黃瓜品種間抗病性與葉綠素含量的關(guān)系及其遺傳性[A];中國園藝學(xué)會首屆青年學(xué)術(shù)討論會論文集[C];1994年

6 繆e，

本文編號：1487728