柑橘衰退病毒基因p23 RNAi载体的构建及转化

doi:10.3864/j.issn.0578-1752.2016.20.007

摘要/Abstract

摘要： 【目的】构建柑橘衰退病毒（Citrus tristeza virus，CTV）含p23的RNAi载体，以获得具有抗性的柑橘转基因植株。【方法】基于转化病毒基因介导抗性，根据NCBI公布的CTV基因组序列，查找p23保守序列，设计并克隆两条不同长度的片段。对两条片段和植物表达载体pBI 121进行双酶切和连接来构建RNAi载体。初步预测所构建的载体发生RNAi抗病毒的可行性。利用农杆菌介导的瞬时表达技术将含RNAi载体的农杆菌注射入CTV指示植物墨西哥莱蒙的叶片，利用GUS组织化学染色法观察叶片中载体发生瞬时表达的情况。发生瞬时表达的叶片接种CTV T36基因型，利用酶联免疫反应（ELISA）检测病毒含量。同时，提取叶片的RNA并反转录为cDNA，利用实时荧光定量PCR（q-PCR）检测CTV p20，通过该基因的表达量反映叶片中的病毒含量。通过农杆菌介导的遗传转化将RNAi载体转入大红甜橙实生苗上胚轴节间茎段，抗生素筛选得到的芽嫁接至枳橙实生试管苗。提取大红甜橙叶片的DNA，通过PCR扩增确定其是否为转基因阳性；目的基因检测为阳性的植株二次嫁接至温室保存的酸橙实生苗；根据插入的p23基因序列设计q-PCR引物，检测转基因植株中p23的表达情况。取 CTV T36基因型寄主的带皮芽，用腹接法接种大红甜橙转基因植株。取接种后新萌发枝梢上的叶片，用检测瞬时表达叶片同样的方法分析植株的抗病性。对于第1次接种后未检测出病毒感染的植株，进行第2次接种并检测分析。【结果】克隆得到CTV p23 513 bp的长片段和291 bp的短片段，与载体pBI121连接后成功构建含发夹结构的来自病原且能靶向目的基因的RNAi载体，命名为p23-RNAi。注射p23-RNAi的墨西哥莱蒙叶片经GUS染色后能够产生蓝色斑点，表明农杆菌p23-RNAi可以在叶片中发生瞬时表达；接种CTV后第15和30天，瞬时表达p23-RNAi的墨西哥莱蒙叶片ELISA检测结果均为阴性，同时q-PCR检测结果显示其CTV p20的积累水平和增加速度明显低于对照植株，表明瞬时表达的p23-RNAi在一定时间内可以对CTV的侵染产生抑制。p23-RNAi经农杆菌介导遗传转化大红甜橙获得抗性芽，通过普通PCR的扩增结果证明得到7个转基因植株；q-PCR检测结果进一步表明7个转基因植株间p23的含量呈现一定差异，植株E的含量最高，其次是C、F、H、A、B和G。接种CTV后，p20的表达量在7个转基因植株间也表现出一定差异，表达量最高的是植株A，其次是G、F、E、B、H、C，且与对照植株相比，呈现不同程度的抗病性。转基因植株对病毒的抗性与外源基因的表达水平没有相关性，外源基因表达水平最高的植株E并没有表现强的CTV抗性。经过两次病毒接种，转基因植株C在接种后具有完全抗性。【结论】p23-RNAi载体能引起植物抗柑橘衰退病毒；瞬时表达技术可快速鉴定RNAi载体的抗病性，有利于筛选高效率的RNAi载体。

关键词: 柑橘衰退病毒, p23, RNAi, 大红甜橙, 瞬时表达, 遗传转化

Abstract: 【Objective】 The objective of this study is to construct RNAi vector containing p23 gene of Citrus tristeza virus (CTV), and obtain transgenic orange plants with virus resistance. 【Method】 Based on the pathogen-derived resistance and the CTV genome sequences published by NCBI, two specific conserved fragments of p23 with different lengths were cloned. Two segments and vector pBI 121 were double-digested and connected for RNAi construction. Subsequently, to initially estimate the antiviral feasibility, the Mexican lime (CTV indicator plant) leaves were injected with Agrobacterium contained the RNAi vector for transient expression, and observed using GUS histochemical staining method. The leaves were inoculated with CTV T36 isolate, and detected by enzyme-linked immunosorbent assay (ELISA). The RNA of leaves was also extracted and reverse transcript to cDNA. Quantitative real-time PCR (q-PCR) was performed to observe the CTV p20 gene expression which could reflect the virus in hosts. The RNAi vector was also transferred into the epicotyl stem of ‘DA HONG’ sweet orange seedlings via Agrobacterium-mediated transformation. Resistant buds were engrafted onto Carrizo Citrange in vitro seedlings. DNA extracted from the ‘DA HONG’ sweet orange leaves was used as the PCR template to identify the transgenic plants. Plants containing the target gene were re-engrafted onto sour orange seedlings and stored in the greenhouse. The expression of the p23 within the transgenic plants was evaluated by q-PCR. The skin buds of CTV T36 isolate hosts were collected and inoculated onto the transgenic sweet orange. The leaves from the sprouted branch tips were collected and analyzed the pathogen resistance capability with the same method that the transient expression leaves detected. Plants without detectable virus infection after the first inoculation were also inspected and analyzed by the same manner in the second round. 【Result】 Long (513 bp) and short (291 bp) fragments of the p23 were cloned. These p23 fragments are then cloned into the pBI121 vector, named p23-RNAi. This p23-RNAi vector was then delivered in the Mexican lime leaves using the Agrobacterium-mediated transient expression assay. The leaves were identified based on the presence of blue stains after GUS staining, indicating that the Agrobacterium contained vector p23-RNAi may induce transient expression in Mexican lime leaves. On the 15th and 30th day after the CTV inoculation, the ELISA detection results for the transgenic Mexican lime leaves in p23-RNAi plants were all negative, whereas the q-PCR detection results showed that the accumulation level of p20 expression was significantly lower than that of the control plants. It indicated that the transient expression of p23-RNAi may, in a defined period, inhibit the CTV infection. Introduction of p23-RNAi via Agrobacterium-mediated genetic transformation led to the production of resistant buds for the ‘DA HONG’ sweet orange, and PCR amplification confirmed a total of seven transgenic plants. The expression of the p23 in all seven transgenic plants was further confirmed by q-PCR amplification, and the gene expression level exhibited a certain degree of difference, with expression level in plant E being the highest, followed by C, F, H, A, B, and G. After inoculation with CTV, the level of expression of p20 varied among these seven transgenic plants, with plant A having the highest level of p20 expression, followed by G, F, E, B, H and C. The transgenic plants showed higher pathogen resistance, albeit to different degrees, when compared to the control plants. However, the virus resistance degrees in the transgenic plants were not closely related to the expression levels of the exogenous gene. For example, plant E, which had the highest expression level of the exogenous gene, did not exhibit powerful CTV resistance. By contrast, the transgenic plant C displayed complete resistance after the two rounds of virus inoculation. 【Conclusion】The p23-RNAi construct that generated confers plant disease resistance to CTV. Transient expression assay can be applied for the high efficiency identification of resistance and screening for high efficiency RNAi vectors.

Key words: Citrus tristeza virus (CTV), p23, RNAi, ‘DA HONG&rsquo, sweet orange, transient expression, genetic transformation

李芳，邓子牛，赵亚，李大志，戴素明. 柑橘衰退病毒基因p23 RNAi载体的构建及转化[J]. 中国农业科学, 2016, 49(20): 3927-3933.

LI Fang, DENG Zi-niu, ZHAO Ya, LI Da-zhi, DAI Su-ming. Construction and Transformation of RNAi Vector for Citrus tristeza virus Gene p23[J]. Scientia Agricultura Sinica, 2016, 49(20): 3927-3933.

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