辣椒疫霉效应分子编码基因的克隆及功能分析

发布时间：2018-05-01 17:19

本文选题：辣椒疫霉 + 效应分子　；参考：《扬州大学》2015年硕士论文

【摘要】：辣椒疫霉是一种寄主范围广的半活体营养型植物病原卵菌,常危害茄科、葫芦科和豆类等蔬菜的安全生产。由于疫霉菌在进化上与真菌相差甚远,现有的杀菌剂大多对植物疫病防治无效,加之品种抗病性利用尚不理想,一直以来还没有有效的辣椒疫霉防控手段。因此,在传统的防治措施之外,从疫霉菌与植物互作的角度出发深入了解疫霉菌致病的分子机理,对开发新的杀菌剂作用靶标、设计植物疫病控制策略具有重要价值。本论文首先研究了辣椒疫霉在侵染前期差异表达的基因种类。通过对侵染前期三个关键阶段(菌丝、游动孢子和萌发休止孢)的转录组测序及生物信息学分析,筛选出98个在这些阶段差异表达的效应分子编码基因,其中包含25个RXLR、9个CRN和16个Elicitin编码基因。利用半定量RT-PCR技术,分析获得的效应分子编码基因在辣椒疫霉侵染阶段(本氏烟接种后1.5、3、6、12、24、36、72h)和侵染前期阶段(菌丝、游动孢子囊、游动孢子和萌发休止孢)的表达水平。结果发现35个基因在辣椒疫霉侵染本氏烟的过程中差异表达,包括RXLR(12个)、CRN(5个)、Elicitin(13个)、PcF/SCR(2个)和NLP(3个)基因。不同类型效应分子在侵染过程中的转录模式不同,同一类型的不同效应分子的转录模式也存在差异。通过高保真扩增技术将这35个侵染阶段差异表达的效应基因克隆到马铃薯X病毒(Potato virus X, PVX)表达载体中,利用农杆菌介导的瞬时表达法分析这些基因是否能够引起本氏烟植物的细胞死亡(Plant cell death, PCD)。结果表明,4个Elicitin、1个NLP以及1个RXLR效应分子均能诱导本氏烟的PCD。为进一步明确效应分子引起PCD的功能区域,对其中的RXLR效应分子Pc129113进行了缺失和替换突变分析,构建了13个突变体。结果显示,第1和第2个W-Y-L domain对其触发本氏烟PCD的功能是必需的,第81-312位氨基酸残基对其维持触发PCD的功能很重要。除上述6个基因之外,其余的基因不能诱导本氏烟产生PCD。本研究分析了RXLR和CRN效应分子对本氏烟PCD的抑制情况,分析其是否具有毒性因子的作用。所选取的触发本氏烟PCD的效应分子包括INF1、BAX、PsCRN63、PsojNIP、PsAvh241、R3a/AVR3a。结果表明,大多数效应分子(15/17)具有抑制寄主植物PCD的毒性功能。其中,效应分子中64.7%能够抑制INF1所诱导的PCD,82.4%能够抑制BAX诱导的PCD,76.5%能够抑制PsCRN63诱导的PCD,88.2%能够抑制PsAvh241和PsojNIP所诱导的PCD,而只有17.6%能够抑制AVR3a/R3a组合所诱导的PCD。预测含有典型的核定位信号(NLS)的RXLR效应分子Pc503142能够抑制本氏烟产生的PCD。为明确该效应分子的功能区域,对其进行了缺失和替换突变分析,构建了4个突变体。结果显示其NLS的缺失导致其抑制PCD毒力功能的丧失,表明该效应分子可能是在寄主细胞核内发挥作用。为进一步明晰辣椒疫霉效应分子是否具有毒性功能,本研究利用辣椒疫霉菌株接种已经表达效应分子的本氏烟叶片,测量在其表达的情况下,病原菌的侵染是否更加剧烈。结果表明,两个CRN效应分子(Pc559084和Pc563418)在本氏烟上的表达能够显著促进辣椒疫霉侵染本氏烟。为明确效应分子在进化过程中面临的寄主选择压力,分析了来自全国不同地区的36个辣椒疫霉菌株中RXLR和PcF/SCR效应分子的序列多态性。结果发现,三个RXLR效应分子(Pe107349、Pc503142和Pc129113)均有丰富的序列多态性,PcF/SCR效应分子Pc96045家族至少含有4个成员。结果说明,这些效应分子可能在侵染寄主植物的过程中起着重要的作用,在进化的过程中面临着很大的寄主选择压力。为进一步分析上述效应分子的功能,在辣椒疫霉中进行基因沉默分析,测定转化子的致病力和生物学性状是否产生变化。对3个RXLR效应分子(Pe107349、Pc503142和Pc129113)进行基因沉默,结果发现这三个基因的沉默均导致辣椒疫霉对本氏烟的致病力降低。而且,基因Pc503142的沉默转化子对外源活性氧的耐受性降低,表明Pc503142可能在抑制本氏烟抗病相关的活性氧途径中发挥作用。类似地,基因沉默实验发现PcF/SCR效应分子Pc96045对辣椒疫霉的致病性具有重要作用。综上所述,基因沉默结果表明,RXLR和PcF/SCR效应分子在辣椒疫霉侵染寄主植物的过程中起着重要的作用。本论文通过转录组测序及RT-PCR分析,获得了35个在生长发育和侵染阶段差异表达的辣椒疫霉效应分子；利用农杆菌浸润法在本氏烟上瞬时表达这些效应分子,明确了效应分子抑制植物免疫反应的毒性功能,或者触发植物免疫反应的无毒性功能；进一步利用CaCl2-PEG介导的原生质体转化技术和dsRNA介导的基因瞬时沉默技术分析效应分子的功能,明确了4个效应分子(Pc107349、Pc503142、Pc129113和Pc96045)对辣椒疫霉的致病性具有重要作用。本论文通过差异表达基因筛选、克隆和功能分析,较为系统地分析了重要效应分子在辣椒疫霉侵染过程中的作用,为全面解析辣椒疫霉的致病机理、有效防控由辣椒疫霉引起的植物疫病提供了坚实的基础。
[Abstract]:Phytophthora capsici is a kind of semi living vegetative oobacteria with wide host range, which often endangering the safe production of vegetables such as Solanaceae, cucurbit and legumes. Because the Phytophthora has evolved far away from fungi, most of the existing fungicides are not effective for plant disease prevention and control, and the use of the disease resistance is still not ideal, and has not yet been found. Effective means of prevention and control of Phytophthora capsici, therefore, in addition to the traditional prevention and control measures, from the angle of Phytophthora and plant mutual understanding of the molecular mechanism of Phytophthora pathogenicity, the development of a new bactericide target target, the design of plant disease control strategy is of great value. This paper first studied the poor early infection of Phytophthora capsici in the early infection By sequencing and bioinformatics analysis of the three critical stages (mycelia, spores and germinating spore), 98 effector genes, including 25 RXLR, 9 CRN and 16 Elicitin encoding genes, were screened by the sequencing of the transcriptional group of the early infecting stage (mycelia, spore and germinating spore), and the semi quantitative RT-PCR technique was used. The expression level of the effector molecular coding gene was analyzed in the infecting stage of Phytophthora capsici (1.5,3,6,12,24,36,72h after inoculation of benthia paprika) and the stage of early infection (mycelium, sporo sporsa, spores and germinating spore). The results showed that the 35 genes were expressed differently in the process of infection by Phytophthora capsici, including RXLR (12), C RN (5), Elicitin (13), PcF/SCR (2) and NLP (3) genes. The transcriptional patterns of different types of effector molecules are different in the infection process, and the transcription patterns of the same type of different effector molecules are also different. By high fidelity amplification, the effect genes of the 35 infection stages are cloned to the potato X virus (Potato viru). S X, PVX) expression vector, using Agrobacterium mediated transient expression to analyze whether these genes can cause cell death of Plant cell death (PCD). The results show that the PCD. of 4 Elicitin, 1 NLP and 1 RXLR effector molecules can induce the functional region of PCD. 13 mutants were constructed by the deletion and replacement mutation analysis of the RXLR effect molecule Pc129113. The results showed that first and second W-Y-L domain were necessary to trigger the function of the PCD, and the 81-312 amino acid residue was important for its maintenance of the function of triggering PCD. In addition to the above 6 genes, the remaining genes could not be induced. The effect of RXLR and CRN effect molecules on the PCD of benfic smoke was analyzed and the effects of the toxic factors were analyzed. The selected effector molecules that trigger the PCD of PCD. include INF1, BAX, PsCRN63, PsojNIP, PsAvh241, and R3a/AVR3a. results show that most of the effector molecules (15/17) have inhibition of host plants. Among them, 64.7% of the effector molecules can inhibit the PCD induced by INF1, 82.4% can inhibit BAX induced PCD, 76.5% can inhibit PCD induced by PsCRN63, 88.2% can inhibit PCD induced by PsAvh241 and PsojNIP, and only 17.6% can inhibit PCD. prediction induced by AVR3a/R3a combination containing typical nuclear positioning signal (NLS) The effector molecule Pc503142 can inhibit the PCD. produced by Benedict smoke to clarify the functional region of the effector molecule, and the deletion and substitution mutation analysis of the effector molecule is used to construct 4 mutants. The result shows that the deletion of NLS leads to the loss of its inhibition of PCD toxicity, indicating that the effector molecule may play a role in the host nucleus. One step is to clarify whether the Phytophthora capsicum has a toxic function. This study uses the Phytophthora capsici strain to inoculate the tobacco leaf slices that have expressed the effector molecules. In the case of its expression, the infection of the pathogen is more intense. The results show that the expression of the two CRN effect molecules (Pc559084 and Pc563418) on the tobacco can be shown to be significant. In order to promote the infection of Phytophthora capsici, the sequence polymorphism of RXLR and PcF/SCR effect molecules from 36 Phytophthora capsici strains from different regions of the country were analyzed. The results showed that three RXLR effector molecules (Pe107349, Pc503142 and Pc129113) had a rich sequence. Polymorphisms, the PcF/SCR effect molecule Pc96045 family contains at least 4 members. The results indicate that these effector molecules may play an important role in the process of infecting host plants and face great host selection pressure during the process of evolution. In order to further analyze the function of the above effect, gene silencing in Phytophthora capsici Analysis of the changes in the pathogenicity and biological characters of the transformants. Gene silencing of 3 RXLR effect molecules (Pe107349, Pc503142 and Pc129113) was carried out. The results showed that the silence of the three genes resulted in the decrease of the pathogenicity of Phytophthora capsici and the tolerance to exogenous reactive oxygen species by the silenced transformant of Pc503142. The decrease in sex indicates that Pc503142 may play a role in inhibiting the active oxygen pathway related to the disease resistance of benchic tobacco. Similar, gene silencing experiments have found that the PcF/SCR effect molecule Pc96045 plays an important role in the pathogenicity of Phytophthora capsici. To sum up, the gene silencing results indicate that the RXLR and PcF/SCR effectors infected host plants in Phytophthora capsici. In this paper, 35 Phytophthora capsicum effector molecules were obtained by transcriptional sequencing and RT-PCR analysis, and the effector molecules were instantaneously expressed on the tobacco by Agrobacterium tumefacience method, and the toxic function of the effector molecules to inhibit the plant immune response was clearly defined. Or triggering the nontoxic function of the plant immune response; further using CaCl2-PEG mediated protoplast transformation and dsRNA mediated gene transient silence technique to analyze the function of the effector molecules, it is clear that 4 effector molecules (Pc107349, Pc503142, Pc129113 and Pc96045) have an important role in the pathogenicity of Phytophthora capsici. Through the screening of differentially expressed genes, cloning and functional analysis, the role of important effector molecules in the infection process of Phytophthora capsici was systematically analyzed, which provided a solid foundation for the comprehensive analysis of the pathogenic mechanism of Phytophthora capsici and the effective prevention and control of Phytophthora plague caused by Phytophthora capsici.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

【相似文献】