發(fā)布時間：2018-09-07 14:06

【摘要】：黃瓜(Cucumis sativus L.)是主要的溫室蔬菜之一,對水分、肥力、溫度、濕度等生長條件要求較高。由于溫室內(nèi)濕度、溫度較高,容易引發(fā)病蟲等非自然災(zāi)害,霜霉病是比較常見且危害嚴(yán)重的病害之一。霜霉威是一種低毒殺菌劑,具有局部內(nèi)吸作用能夠快速被植物吸收,有效防治病害的發(fā)生,半衰期短,毒性低,為我國農(nóng)業(yè)部推薦的可以在無公害農(nóng)產(chǎn)品生產(chǎn)中使用的農(nóng)藥品種之一。但是由于菜農(nóng)的不正確過量地使用,會導(dǎo)致黃瓜進(jìn)入市場銷售時,霜霉威殘留量嚴(yán)重超標(biāo),危害消費者健康。研究黃瓜低農(nóng)藥殘留問題,對提高我國黃瓜產(chǎn)品的品質(zhì)和質(zhì)量,保障人民群眾的飲食安全,對提升我國蔬菜產(chǎn)品的在國際上的競爭能力方面有著非常重要的理論意義和現(xiàn)實意義。在無法避免施用農(nóng)藥的情況下,選擇農(nóng)藥吸收量小、有效降解農(nóng)藥的品種,可以從根本上解決農(nóng)殘量超標(biāo)問題。本研究在Solexa測序霜霉威脅迫后低霜霉威殘留品種D0351轉(zhuǎn)錄組基礎(chǔ)上,篩選出符合|log2Ratio|≥1、P-Value≤0.01、FDR≤0.001,在D0351和D9320中表達(dá)模式存在明顯差異的Cs DIR16基因,以D0351和高殘留品系D9320為研究對象,利用生物信息學(xué)、分子生物學(xué)等手段進(jìn)行了深入研究。主要結(jié)果如下:(1)成功克隆Cs DIR16,含588個堿基共編碼195個氨基酸,分子式為C977H1515N259O277S7,分子量是21545.7,理論等電點為9.36,是一個具有弱疏水性的穩(wěn)定蛋白。Cs DIR16具有Dirigent蛋白的保守區(qū)屬于Dirigent-like蛋白家族,與甜瓜的Cm DIR7同源性最高達(dá)到99%,具有DIR典型的β-桶結(jié)構(gòu),是DIR-b/d亞家族基因,具有一個跨膜結(jié)構(gòu),一個信號肽切割位點和N-糖基化位點(分泌蛋白的主要特征)。黃瓜基因組中一共篩選出23個Cs DIR家族基因,歸屬于a、b、d、e四個亞家族,響應(yīng)霜霉威脅迫4個Cs DIR家族基因中Cs DIR16響應(yīng)霜霉威脅迫差異表達(dá)最大。(2)采用q RT-PCR分析了霜霉威脅迫下D0351和D9320中Cs DIR16隨時間的表達(dá)模式。結(jié)果發(fā)現(xiàn),霜霉威脅迫下,Cs DIR16的表達(dá)存在品種間特異性。Cs DIR16在D0351中的達(dá)量隨時間逐漸增大6 h時達(dá)到最高,9 h后減小,在各個時間點均表現(xiàn)為明顯上調(diào)表達(dá);在D9320中表達(dá)量隨時間逐漸增大3 h時達(dá)到最高,6 h后減小,但是只有9h時與對照相比呈現(xiàn)明顯上調(diào)趨勢,其余各個時間點與對照相比均差異不顯著。(3)采用q RT-PCR分析了霜霉威脅迫下D0351和D9320不同組織中Cs DIR16的表達(dá)。組織特異性分析顯示,Cs DIR16在D0351和D9320的表達(dá)存在組織表達(dá)特異性,Cs DIR16在D0351的葉、莖中顯著上調(diào)表達(dá),在根中差異不顯著,在D9320的葉和莖中均表現(xiàn)為與對照相比明顯下調(diào),在根中差異不顯著。Cs DIR16主要在D0351的葉、果皮和莖中發(fā)揮作用。(4)成功構(gòu)建Cs DIR16-p GII-EGFP綠色熒光蛋白融合表達(dá)載體,Cs DIR16主要定位于細(xì)胞核中,是核蛋白。(5)成功構(gòu)建植物正義表達(dá)載體p CXSN-Cs DIR16(+)和反義表達(dá)載體p CXSN-Cs DIR16(-),并利用農(nóng)桿菌介導(dǎo)將重組質(zhì)粒轉(zhuǎn)入D9320,獲得抗性植株。通過對抗性植株檢測共得到轉(zhuǎn)正義Cs DIR16(+)黃瓜植株13株,轉(zhuǎn)反義Cs DIR16(-)黃瓜植株15株。選擇表達(dá)量基本一致的各6株,經(jīng)霜霉威脅迫處理后殘留量檢測轉(zhuǎn)Cs DIR16(+)的T0植株中,6h以后霜霉威的殘留量與未轉(zhuǎn)基因植株相比明顯下降;轉(zhuǎn)Cs DIR16(-)的T0植株中霜霉威的殘留量與野生型基本保持一致。說明Cs DIR16基因的過表達(dá)對降低D9320中霜霉威的殘留量起到了積極作用。(6)對轉(zhuǎn)基因T1植系進(jìn)行生理生化檢測,Cs DIR16(+)與可溶性糖和POD之間存在相互作用關(guān)系,而與可溶性蛋白、SOD、GST、GR之間不存在明顯相互關(guān)系。MDA含量在轉(zhuǎn)Cs DIR16(+)植株中的快速降低,證實Cs DIR16(+)基因的轉(zhuǎn)入對膜脂具有修復(fù)能力,有效降低了霜霉威脅迫造成的傷害。(7)推測Cs DIR16基因降低霜霉威殘留的途徑為:霜霉威脅迫會導(dǎo)致ROS在植物體內(nèi)累積,從而激活苯丙烷代謝通路,POD酶活性與Cs DIR16相互作用生成木質(zhì)素,增加木質(zhì)素積累,從而降低霜霉威脅迫造成的活性氧對質(zhì)膜造成的傷害,加速霜霉威的降解。
[Abstract]:Cucumis sativus L. (Cucumis sativus L.) is one of the main greenhouse vegetables, which requires higher growth conditions such as water, fertility, temperature and humidity. Because of the high humidity and temperature in the greenhouse, it is easy to cause diseases and insects and other non-natural disasters, downy mildew is one of the most common and serious diseases. It is one of the pesticides recommended by the Ministry of Agriculture that can be used in the production of Non-polluted Agricultural products. However, due to improper and excessive use by vegetable farmers, the residue of Methomyl in Cucumber will seriously exceed the standard and endanger consumption when it enters the market. It is of great theoretical and practical significance to study the problem of low pesticide residues in cucumber for improving the quality and quality of cucumber products in China and ensuring the safety of people's diet. It is also of great theoretical and practical significance to enhance the international competitiveness of vegetable products in China. In this study, we screened out CSDIR16 gene which accorded with | log2Ratio | 1, P-Value | 0.01, FDR < 0.001, and had obvious difference in expression patterns between D0351 and D9320 by sequencing the transcript of D0351, a low residue variety threatened by downy mildew. The main results are as follows: (1) CSDIR16 was cloned successfully, which contains 588 bases encoding 195 amino acids. The molecular formula is C977H1515N259O2 77S7, the molecular weight is 21545.7, and the theoretical isoelectric point is 9.36. It is a weak hydrophobicity. CSDIR16 is a stable protein. It has a conserved domain of Dirigent protein belonging to the Dirigent-like protein family. It has a 99% homology with CMDIR7 of melon. It has a typical beta-barrel structure of DIR. It is a DIR-b/d subfamily gene. It has a transmembrane structure, a signal peptide cleavage site and a N-glycosylation site (the main feature of secretory proteins). Twenty-three genes belonging to four subfamilies of a, b, D and E were screened out from the genome. The difference of expression of CDIR16 in response to downy mildew threat was the largest among the four genes of the CDIR family. (2) The expression patterns of CDIR16 in D0351 and D9320 under downy mildew threat were analyzed by Q RT-PCR. The expression of CSDIR16 in D0351 was up-regulated at all time points. The expression of CSDIR16 in D9320 was up-regulated at 3 h and down-regulated at 6 h, but it was up-regulated at 9 h. The expression of CSDIR16 in different tissues of D0351 and D9320 under downy mildew threat was analyzed by Q RT-PCR. The tissue-specific analysis showed that the expression of CSDIR16 was tissue-specific in D0351 and D9320. The expression of CSDIR16 was significantly up-regulated in D0351 leaves, stems and poor in roots. There was no significant difference between D9320 and D9320, but there was no significant difference between D9320 and D9320. CDIR16 mainly played a role in D0351 leaves, pericarp and stem. (4) The fusion expression vector of CDIR16-p GII-EGFP green fluorescent protein was successfully constructed. CDIR16 was mainly located in the nucleus and was a nuclear protein. (5) The plant was successfully constructed. Sensitive expression vector p CXSN-Cs DIR16 (+) and antisense expression vector p CXSN-Cs DIR16 (-) were used to transfer the recombinant plasmid into D9320 to obtain resistant plants. Residues of clotrimazole in transgenic plants T0 treated with downy mildew threat were significantly lower than those in non-transgenic plants after 6 h. Residues of clotrimazole in transgenic plants T0 and wild-type plants were basically the same. The overexpression of CDIR16 gene played an important role in reducing the residues of clotrimazole in D9320. Polar effect. (6) The physiological and biochemical tests of transgenic T1 plants showed that there was an interaction between the soluble sugar and POD, but no significant correlation between the soluble protein, SOD, GST and GR. The rapid decrease of MDA content in transgenic plants confirmed that the transfection of the gene had the ability to repair membrane lipids. (7) It was speculated that CSDIR16 gene could reduce the residual of downy mildew by activating the phenylpropane metabolic pathway and the interaction of POD enzyme activity with CSDIR16 to produce lignin, thus increasing the lignin accumulation and decreasing the activity of downy mildew. The damage caused by sex oxygen to the plasma membrane accelerated the degradation of the cream.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S642.2;S481.8

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