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点蜂缘蝽效应子新型筛选体系的建立及应用
Establishment and application of a new screening system for Riptortus pedestris effectors
鲁王善,李继芬,董玉妹,沈丹宇,窦道龙,夏 爱
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DOI:
作者单位:南京农业大学植物保护学院
中文关键词:点蜂缘蝽;荧光素酶;大豆基因启动子;昆虫效应子;信号通路;烟草瞬时表达
英文关键词:Riptortus pedestris; luciferase; promotor of soybean gene; insect effector; signal pathway; transient gene expression in Nicotiana benthamiana
中文摘要:

   【目的】 建立以荧光素酶为报告基因且调控不同植物抗性相关通路的点蜂缘蝽效应子筛选新体系,并应用于点蜂缘蝽效应子的研究。【方法】 通过对点蜂缘蝽Riptortus pedestris取食前后的大豆叶片转录组比较分析,挑选出了4个点蜂缘蝽取食后高度上调表达且影响不同信号通路的大豆抗性相关基因。其中Gm2参与异黄酮代谢、Gm3参与生长素和硫苷代谢、Gm6参与免疫信号识别和Gm8参与茉莉酸生物合成。利用PCR扩增4个基因的启动子序列并构建到pGreen-LUC载体上。构建成功的载体转化到大肠杆菌中,再利用根癌农杆菌注射在本氏烟中瞬时过表达建立报告系统。其次,在烟草叶片上表达点蜂缘蝽候选效应子12 h后再分别表达报告系统。通过观察效应子对荧光素酶表达活性的影响来研究效应子的功能。【结果】 琼脂糖凝胶电泳和测序证明了PCR成功扩增了4个抗性相关基因的启动子。荧光素酶报告基因检测、酶标仪酶活检测以及Western Blot验证了4个基因启动子在烟草叶片上成功表达。昆虫取食、机械处理和MeJA处理验证了该报告系统的可行性。最后利用该筛选体系对9个点蜂缘蝽效应子进行了筛选,结果显示9个点蜂缘蝽效应子对4个报告基因的表达有不同的调控作用。【结论】 成功建立了以荧光素酶为报告基因的点蜂缘蝽效应子筛选新体系,该体系可应用于点蜂缘蝽效应子的大量筛选,且具有高效、快速和操作简易等特点,为其他昆虫效应子的研究提供了新思路。

英文摘要:

Abstract  [Aim]  To develop a new screening system for Riptortus pedestris effectors that target different plant resistance related pathways using luciferase as a reporting gene, and apply these it to the study of R. pedestris effectors. [Methods]  Four resistance-related soybean genes that are highly upregulated after R. pedestris damage to soybean plants, and that are involved in different soybean signaling pathways, were selected after comparative analysis of two soybean transcriptomes from R. pedestris infested, and non-infested, leaves. The four genes were, Gm2, which participates in the isoflavone metabolism, Gm3, which is involved in the auxin and glucosinolate metabolism, Gm6, which is related to immune signal recognition and Gm8, which is involved in the biosynthesis of jasmonic acids. All promoter sequences of these four genes were amplified with PCR, then inserted into the pGreen 35s-LUC vector. Successfully constructed vectors were transferred to Escherichia coli, and then transiently expressed in the leaves of Nicotiana benthamiana using the A. tumefaciens infiltration method to establish reporting systems. After each R. pedestris effector had been transiently expressed in the leaves of N. benthamiana for 12 h, the reporting systems were expressed in the same area of each leaf. The function of each effector was determined by observing the impact of each gene on the expression of luciferase. [Results]  Agarose gel electrophoresis and gene sequencing confirmed that four promotors of soybean resistance-related genes were successfully amplified using PCR. The luminous intensity of the luciferase reporter gene and an enzyme activity detection assay using Microplate Reader and Western Blot, demonstrated that all four promoters were successfully expressed in the leaves of N. benthamiana. The results of a feeding assay, mechanical plant damage, and treatment with methyl jasmonate (MeJA), confirm that this new screening system is reliable. The system was subsequently used to screen R. pedestris effectors and indicates that nine R. pedestris effectors have different regulatory functions on the expression of the four reporter genes. [Conclusion]  A new screening system for R. pedestris effectors, using luciferase as reporter, was successfully developed and used to select R. pedestris effectors. This new screening system is easy to operate, efficient and fast. It provides a novel approach for research on other insect effector genes.

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