外源水杨酸诱导RNAi与MAPK3级联信号抗番前黄化曲叶病毒研究

发布时间：2018-03-07 05:30

本文选题：番茄黄化曲叶病毒　切入点：水杨酸　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：番茄黄化曲叶病毒病(Tomato yellow leaf curl disease,TYLCD)由番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)引起,严重威胁番茄安全生产。目前番茄抗TYLCV研究主要通过将野生材料中抗性基因、标记渗透到栽培品种中,或者通过RNAi(RNA interference)技术干扰病毒基因序列以达到抑制病毒基因组复制的目的。前者耗时长,抗性不稳定,后者涉及到转基因技术,目前转基因技术还没有被消费者广泛接受。诱导植物抗性为我们提供了一条新思路。诱导抗性是指植物在一些非生物或生物激发子刺激下,对随后病原的侵染产生更快更强的防御反应。植物诱导抗性主要包括两种:一种是诱导系统获得性抗性(ISR),另外一种是系统获得性抗性(SAR)。ISR抗性途径依赖于茉莉酸(JA)和乙烯(ET)信号。SAR可以通过前期病原侵染,也可以通过化学物质,如水杨酸(SA)或水杨酸衍生物苯丙噻二唑(BTH)诱导植物SAR的发生。因此,我们提出通过SA提高番茄对TYLCV诱导抗性的思路。诱导抗性需要MAPK级联信号系统参与,而植物抗病毒需要RNAi信号参与;MAPK级联信号、RNAi在SA介导抗性中到底发挥什么作用。本试验通过外源叶面喷施SA,检测番茄植株TYLCV抗性指标,确定SA诱导番茄植株的最适浓度;然后通过最适SA浓度研究RNAi通路关键基因SlDCL2和SlDCL4以及诱导抗性关键基因MAPK3在番茄抗TYLCV防御中的作用,进一步研究它们与SA信号的关系,对SA诱导抗性在抗TYLCV的作用机理做了一定研究,为植物诱导抗性抗病毒提供理论依据。主要结果如下:(1)外源叶面喷施SA提高番茄植株对TYLCV耐性的最适浓度为0.5mM。用不同浓度的SA前处理感病材料‘TTI112B-2’,2天后人工接种TYLCV侵染性克隆,一个月后统计发病率(%)和发病指数。结果显示0.5mM SA前处理植株的发病率(51.1%)和发病指数(29.3)显著低于对照(68.8%,35.7),进一步研究发现RNAi通路相关基因被诱导表达,TYLCV对植物细胞膜的破坏降低,植物抗氧化能力提高、光合能力增强,植物叶片中病毒的相对含量降低,植株病症减缓,经检测系统性抗性被诱导。(2)DCL2和DCL4在番茄抗TYLCV防御方面至关重要。利用qRT-PCR技术检测25个RNAi通路基因在抗感材料‘Y19’和‘TTI112B-2’中的差异,结果显示:在抗感病材料中,大多数RNAi相关基因都可以被TYLCV诱导上调表达,但是抗性材料中表达相对较高。通过VIGS技术沉默SlDCL2和SlDCL4,发现抗性材料TYLCV含量增加,病情指数增加,抗性被破坏,而感病材料提前发病,TYLCV含量同样增加。(3)SA诱导植物对TYLCV抗性需要DCL2和DCL4参与。用0.1mMSA前处理拟南芥dcl2,dcl4和dcl2/4突变体,24h后人工接种TYLCV,14天后qPCR、ELISA和半定量检测TYLCV含量。结果显示SA前处理可以抑制野生型拟南芥Col-中TYLCV的复制,但是不能够降低dcl2、dcl4和dcl2/4突变中的病毒含量。(4)MAPK3参与调控番茄植株对TYLCV的耐性。TYLCV接种抗性番茄材料‘Y19’,qPCR、ELISA以及Westernblot检测结果显示SlMAPK3参与番茄植株的抗TYLCV响应。通过VIGS和植物过表达技术证明MAPK3参与调控SA、JA防御信号通路基因的SlPR1/SlPR2,SlLapA/SlPI-I/SlPI-II表达,提高了番茄植株的抗氧化能力,最终提高了植株对TYLCV的耐性。(5)SA信号参与TYLCV病原诱导的MAPK3响应。用0.1 mM SA前处理SA信号中断突变体npr1,24 h后,人工接种接种TYLCV,12 h后,Western blot检测MAPK3活性,结果显示野生型Col-中TYLCV诱导的MAPK3磷酸化增幅大于npr1中的。0.1mMSA前处理不能抑制mapk3中TYLCV的复制,但是可以降低野生型Col-中的TYLCV含量。
[Abstract]:TYLCD (Tomato yellow leaf curl disease, TYLCD) by tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) caused a serious threat of tomato production safety. Current research of TYLCV resistance is mainly through resistance of wild materials in gene markers penetrated into cultivars, or through the RNAi (RNA interference) interference virus gene sequence to inhibit the replication of the virus genome. The purpose of the former is time-consuming, the resistance is not stable, which relates to transgenic technology, transgenic technology has not been widely accepted by consumer. Plant resistance provides a new idea for us. The induced resistance refers to the plants in some non biological or biological elicitor stimulation, defense faster and stronger on the infection of the pathogen. The plant induced resistance mainly includes two types: one is the induction of acquired resistance system (ISR), another is the systemic acquired resistance (SAR) resistant.ISR pathway dependent on jasmonic acid (JA) and ethylene (ET) signal.SAR through early pathogen infection can also, by chemical substances, such as salicylic acid or salicylic acid derivatives (SA) phenyl thiophene (BTH) two were induced by plant SAR. Therefore, we propose to improve the induced resistance of tomato TYLCV way through SA. MAPK signal cascade induced resistance system in plant antiviral RNAi signals in MAPK; cascade signal, RNAi in the end what is the role of SA mediated resistance. In this experiment, exogenous foliar SA, detection of tomato plants TYLCV resistance index sure, the optimum concentration of SA in tomato plants; then the optimum SA concentration of RNAi pathway key genes SlDCL2 and SlDCL4 and induced resistance genes in the MAPK3 anti TYLCV defense role in tomato, further study The relationship with SA signal, do some research on SA induced resistance in the mechanism of anti TYLCV, and provide a theoretical basis for plant induced resistance to antiviral. The main results are as follows: (1) exogenous spraying SA increased tomato plant tolerance to TYLCV the optimal concentration of 0.5mM. with different concentrations of SA before treatment of susceptible materials "TTI112B-2 ', 2 days after the inoculation of TYLCV infectious clone, incidence rate after one month (%) and disease index. The results showed that 0.5mM SA before the onset of treatment plant rate (51.1%) and disease index (29.3) was significantly lower than the control (68.8%, 35.7), further study found that RNAi signaling pathway related genes were induced to express TYLCV, reduce the damage to the plant cell membrane, improve the antioxidant capacity of plant photosynthetic capacity, reduce the relative virus content in leaves of plants, plant disease resistance by slow detection system is induced. (2) DCL2 and DCL4 in tomato TYL It is very important for CV defense. Using qRT-PCR technology to detect 25 genes of the RNAi pathway in resistant and susceptible materials' Y19 'and' TTI112B-2 'differences, results showed that: in the resistant and susceptible materials, most of the RNAi genes can be induced by upregulation of the expression of TYLCV, but the expression of resistance material is relatively high. Through the VIGS technology to silence SlDCL2 and SlDCL4, TYLCV content was found to increase the resistance of materials, the disease index increased, the resistance was destroyed, while the susceptible materials early onset, the content of TYLCV also increased. (3) SA induced plant resistance to TYLCV DCL2 and participation in DCL4. With the Arabidopsis dcl2 treatment before 0.1mMSA, DCL4 and dcl2/4 mutants, 24h after inoculation TYLCV 14 after qPCR, ELISA and semi quantitative detection of TYLCV content. The results showed that TYLCV can inhibit Col- replication in wild type Arabidopsis SA before treatment, but can not reduce the content of dcl2, DCL4 and dcl2/4 mutations in the virus (4. MAPK3) is involved in the regulation of tomato plants to TYLCV tolerance.TYLCV inoculated resistant tomato materials' Y19 ', qPCR, ELISA and Westernblot showed that the anti TYLCV response of SlMAPK3 in tomato plants. Plant expression by VIGS and that MAPK3 is involved in the regulation of SA signal pathway, JA defense gene SlPR1/SlPR2, SlLapA/SlPI-I/SlPI-II expression increased in tomato plant antioxidant ability, and ultimately improve the tolerance of plants to TYLCV. (5) SA signal in TYLCV pathogen induced MAPK3 response. By 0.1 mM SA pretreatment SA signal interruption mutant npr1,24 h after artificial inoculation with TYLCV, 12 h, Western blot MAPK3 activity assay, the results showed that TYLCV induced phosphorylation of MAPK3 growth of wild type Col- in more than NPR1 in.0.1mMSA pretreatment could not inhibit mapk3 TYLCV replication, but can reduce the content of TYLCV of wild type Col-.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.412

【相似文献】