黄瓜LRR型抗病相关蛋白基因的表达分析与载体构建

发布时间：2019-03-06 15:43

【摘要】：黄瓜在世界范围内广泛种植,其产量与品质深受重视。致病菌尖孢镰刀菌黄瓜转化型(Fusarisum oxysporum f.sp. cucumerinum)引起的黄瓜枯萎病很难防控,严重限制了黄瓜的产量与质量,因此对其抗病机制的研究显得尤为重要。前期实验在研究枯萎病菌胁迫下的黄瓜抗、感品系根系差异蛋白质组时,一个与黄瓜枯萎病抗性显著相关的蛋白被鉴定出来(CsaUNM029170.1),该蛋白注释为LRR型蛋白家族成员,暂命名为CsLRR-pf1,其CDS区长1317bp (GenBank登录号：gi|449450244)。本试验克隆与分析了CsLRR-pfl基因的启动子序列,利用技术分析了CsLRR-pfl的基因表达特性,同时构建了黄瓜CsLRR-pfl的原核表达载体和GFP表达载体,以期为深入研究该LRR型抗病相关蛋白在黄瓜防御反应中的作用及机制奠定基础。克隆了CsLRR-pfl基因的编码区与启动子序列,并应用PlantCARE在线软件对CsLRR-pfl启动子区域序列分析。结果表明,在CsLRR-pfl基因起始密码子ATG上游的启动子正反义链中预测到以下调控元件：1个高水平表达相关元件5UTR Py-rich stretch;1个增强木质部中及花瓣中表达而阻抑韧皮部表达相关元件AC-Ⅱ；4个厌氧诱导的顺式作用元件ARE；1个ATBP-1结合相关AT-rich元件；1个TGAGTCA motif相关顺式作用元件ATGCAAAT motif;1个生长素响应相关顺式作用元件AuxRR-core;1个真菌激发子相关顺式作用元件Box-W1;光响应元件G-box 2个、GA-motif2个、GATA-motif1个、GT1-motif 1个、MNF11个、Spl2个；2个热胁迫相应相关顺式作用元件HSE；2个光响应相关MYB结合位点MRE；37个CAAT-box；88个TATA-box；4个防御与胁迫响应元件TC-rich repeats;1个损伤与病原菌响应元件W box;1个胚乳表达所需元件Skn-1-motif;1个生物节律控制相关元件circadian;1个赤霉素响应元件P-box; MeJA响应相关顺式作用元件CGTCA-motif与TGACG-motif各2个。这些调控元件在启动子序列上基本呈均匀分布。通过定量分析了CsLRR-pfl基因在GA3、MeJA、 SA、ETH、 ABA等胁迫下的表达模式。根据预测结果,选择了5种外源激素处理以及枯萎病菌侵染黄瓜幼苗,分析了CsLRR-pf1基因的表达情况。枯萎病菌接种后F9(高抗枯萎病)、995(高感枯萎病)的CsLRR-pf1基因的表达量都被诱导表达,表达高峰分别为第11天、13天,CsLRR-pf1基因在F9中的大量积累早于995,推论这可能是导致其抗感性状差异的主要原因。同时也说明CsLRR-pf1可能参与黄瓜对枯萎病菌的响应；GA3、 MeA与SA处理后,F9中CsLRR-pfl基因均被诱导表达且变化趋势相同,可能是因为就是SA、 GA3、 MeJA的信号通路交叉而在诱导植物防御响应过程中互相协同。然而,ABA与ETH处理后,F9与995中CsLRR-pfl基因响应不同处理的表达模式有相似也有差异,这说明了生物响应外界不同环境时的机制极为复杂。构建了黄瓜CsLRR-pfl原核表达载体,菌落PCR鉴定后用Nde I/Hind Ⅲ双酶切鉴定确定其原核表达载体构建成功。同时,构建了此基因与GFP融合表达载体,通过菌落PCR与Xba Ⅰ/ BamH Ⅰ双酶切鉴定确定融合表达载体构建成功,为后续深入研究该LRR型抗病相关蛋白在胁迫响应中的作用等奠定基础。
[Abstract]:The cucumber is widely cultivated in the world, and its yield and quality are of great importance. Fusarium oxysporum f. sp. The cucumber fusarium wilt is difficult to control, and the yield and quality of the cucumber are severely restricted, so the research on the disease resistance mechanism of the cucumber is particularly important. In the early-stage experiment, when the root system of cucumber was resistant to Fusarium oxysporum under the stress of Fusarium oxysporum, a protein related to the resistance of cucumber fusarium wilt was identified (CsA UNM029170.1), and the protein was expressed as a member of the LRR-type protein family, and it was designated as CsLRR-pf1, and its CDS was 1317bp (GenBank accession number: Gi | 449450244). The promoter sequence of CsLRR-pfl was cloned and analyzed, and the expression of CsLRR-pfl was analyzed by using the technique. The prokaryotic expression vector and the GFP expression vector of CsLRR-pfl were constructed. In order to study the role and mechanism of the LRR-type disease-related protein in the defense of cucumber. The coding region and the promoter sequence of the CsLRR-pfl gene were cloned and the region sequence analysis of the CsLRR-pfl promoter was carried out using the PlantCARE on-line software. The results showed that the following regulatory elements were predicted in the antisense strand of the promoter upstream of the initiation codon ATG of the CsLRR-pfl gene:1 high-level expression-related element 5UTR Py-rich strecch;1 enhancement xylem and expression in the petal to inhibit the expression of the phloem-related elements AC-II; 4 anaerobic-induced cis-acting elements ARE;1 ATBP-1 binding related AT-rich element;1 TGAGTCA motif-related cis-acting element ATGCAAAT motif;1 auxin response-related cis-acting element AuxRR-core;1 fungal elicitor-related cis-acting element Box-W1; light response element G-box 2, GA-motif2, GATA-motif1, GT1-motif 1, MNF11, Spl2;2 thermal stress-related cis-acting element HSE;2 photoresponse-related MYB binding site MRE;37 CAAT-box;88 TATA-box;4 defense and stress response elements TC-rich repeats;1 injury and pathogen response element W box;1 endosperm expression required element Skn-1-motif; One biorhythm control related element, circle,1 gibberellin response element, P-box, and MeJA response-related cis-acting element CGTCA-motiv and two TGACG-motif. These regulatory elements are substantially uniformly distributed over the promoter sequence. The expression pattern of CsLRR-pfl gene under the stress of GA3, MeJA, SA, ETH and ABA was quantitatively analyzed. The expression of CsLRR-pf1 gene was analyzed based on the results of the prediction and the selection of five exogenous hormones and the infection of cucumber seedlings by Fusarium oxysporum. The expression of CsLRR-pf1 gene of F9 (high resistance to fusarium wilt) and 995 (high-sense fusarium wilt) was induced by Fusarium oxysporum. The expression of CsLRR-pf1 gene in 995 (high-sense fusarium wilt) was induced and the expression of CsLRR-pf1 gene in F9 was earlier than 995. At the same time, CsLRR-pf1 may be involved in the response of cucumber to Fusarium oxysporum. After treatment with GA3, MeA and SA, the CsLRR-pfl gene in F9 is induced to be expressed and the variation trend is the same, probably because the signal paths of SA, GA3 and MeJA cross with each other in the course of inducing plant defense response. However, after the ABA and ETH treatment, the expression patterns of the CsLRR-pfl genes in F9 and 995 were similar to those of the different treatments. The prokaryotic expression vector of the cucumber CsLRR-pfl was constructed and the expression vector of the prokaryotic expression vector was determined by Nde I/ HindIII double-enzyme digestion. At the same time, this gene and the GFP fusion expression vector were constructed, and the construction of the fusion expression vector was determined by the colony PCR and XbaI/ BamH I double-enzyme digestion and identification, and the basis for further study of the role of the LRR-type disease-related protein in the stress response was established.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.421

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