黄瓜低霜霉威残留性相关基因CsDIR16的鉴定及功能分析

发布时间：2018-09-07 14:06

【摘要】：黄瓜(Cucumis sativus L.)是主要的温室蔬菜之一,对水分、肥力、温度、湿度等生长条件要求较高。由于温室内湿度、温度较高,容易引发病虫等非自然灾害,霜霉病是比较常见且危害严重的病害之一。霜霉威是一种低毒杀菌剂,具有局部内吸作用能够快速被植物吸收,有效防治病害的发生,半衰期短,毒性低,为我国农业部推荐的可以在无公害农产品生产中使用的农药品种之一。但是由于菜农的不正确过量地使用,会导致黄瓜进入市场销售时,霜霉威残留量严重超标,危害消费者健康。研究黄瓜低农药残留问题,对提高我国黄瓜产品的品质和质量,保障人民群众的饮食安全,对提升我国蔬菜产品的在国际上的竞争能力方面有着非常重要的理论意义和现实意义。在无法避免施用农药的情况下,选择农药吸收量小、有效降解农药的品种,可以从根本上解决农残量超标问题。本研究在Solexa测序霜霉威胁迫后低霜霉威残留品种D0351转录组基础上,筛选出符合|log2Ratio|≥1、P-Value≤0.01、FDR≤0.001,在D0351和D9320中表达模式存在明显差异的Cs DIR16基因,以D0351和高残留品系D9320为研究对象,利用生物信息学、分子生物学等手段进行了深入研究。主要结果如下:(1)成功克隆Cs DIR16,含588个碱基共编码195个氨基酸,分子式为C977H1515N259O277S7,分子量是21545.7,理论等电点为9.36,是一个具有弱疏水性的稳定蛋白。Cs DIR16具有Dirigent蛋白的保守区属于Dirigent-like蛋白家族,与甜瓜的Cm DIR7同源性最高达到99%,具有DIR典型的β-桶结构,是DIR-b/d亚家族基因,具有一个跨膜结构,一个信号肽切割位点和N-糖基化位点(分泌蛋白的主要特征)。黄瓜基因组中一共筛选出23个Cs DIR家族基因,归属于a、b、d、e四个亚家族,响应霜霉威胁迫4个Cs DIR家族基因中Cs DIR16响应霜霉威胁迫差异表达最大。(2)采用q RT-PCR分析了霜霉威胁迫下D0351和D9320中Cs DIR16随时间的表达模式。结果发现,霜霉威胁迫下,Cs DIR16的表达存在品种间特异性。Cs DIR16在D0351中的达量随时间逐渐增大6 h时达到最高,9 h后减小,在各个时间点均表现为明显上调表达;在D9320中表达量随时间逐渐增大3 h时达到最高,6 h后减小,但是只有9h时与对照相比呈现明显上调趋势,其余各个时间点与对照相比均差异不显著。(3)采用q RT-PCR分析了霜霉威胁迫下D0351和D9320不同组织中Cs DIR16的表达。组织特异性分析显示,Cs DIR16在D0351和D9320的表达存在组织表达特异性,Cs DIR16在D0351的叶、茎中显著上调表达,在根中差异不显著,在D9320的叶和茎中均表现为与对照相比明显下调,在根中差异不显著。Cs DIR16主要在D0351的叶、果皮和茎中发挥作用。(4)成功构建Cs DIR16-p GII-EGFP绿色荧光蛋白融合表达载体,Cs DIR16主要定位于细胞核中,是核蛋白。(5)成功构建植物正义表达载体p CXSN-Cs DIR16(+)和反义表达载体p CXSN-Cs DIR16(-),并利用农杆菌介导将重组质粒转入D9320,获得抗性植株。通过对抗性植株检测共得到转正义Cs DIR16(+)黄瓜植株13株,转反义Cs DIR16(-)黄瓜植株15株。选择表达量基本一致的各6株,经霜霉威胁迫处理后残留量检测转Cs DIR16(+)的T0植株中,6h以后霜霉威的残留量与未转基因植株相比明显下降;转Cs DIR16(-)的T0植株中霜霉威的残留量与野生型基本保持一致。说明Cs DIR16基因的过表达对降低D9320中霜霉威的残留量起到了积极作用。(6)对转基因T1植系进行生理生化检测,Cs DIR16(+)与可溶性糖和POD之间存在相互作用关系,而与可溶性蛋白、SOD、GST、GR之间不存在明显相互关系。MDA含量在转Cs DIR16(+)植株中的快速降低,证实Cs DIR16(+)基因的转入对膜脂具有修复能力,有效降低了霜霉威胁迫造成的伤害。(7)推测Cs DIR16基因降低霜霉威残留的途径为:霜霉威胁迫会导致ROS在植物体内累积,从而激活苯丙烷代谢通路,POD酶活性与Cs DIR16相互作用生成木质素,增加木质素积累,从而降低霜霉威胁迫造成的活性氧对质膜造成的伤害,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.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S642.2;S481.8

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