摘要
玉米是重要的粮食、饲料与工业原料作物。目前我国玉米的种植面积呈逐年增加的趋势,但玉米病害尤其是玉米的矮花叶病和粗缩病成了限制我国玉米生产发展的巨大障碍,造成玉米的大量减产和品质下降。利用基因工程技术培育推广抗病品种和寻找新方法已成为病毒病防治的迫切需求。RNA干涉作为一种特异、高效的反向遗传学手段,在植物的抗病毒育种中表现出了巨大的优势,成为植物抗病毒基因工程的热点。为进一步筛选和建立病毒cp基因dsRNA的原核表达体系和遗传转化体系,探讨RNAi技术在玉米抗病毒基因工程中的应用效果,建立玉米RNAi的分子育种新方法。本文利用大肠杆菌HT115高效表达甘蔗花叶病毒cp基因dsRNA,较系统地研究了外源dsRNA抗甘蔗花叶病毒RNAi调控技术；构建了兼抗甘蔗花叶病毒及粗缩病毒cp基因的RNAi复合表达载体,开展农杆菌介导的玉米原位转化,并对转化株进行了分子确证,在此基础上对转化株RNAi效果和抗病性进行了分析,获得了如下主要结果：
1、根据甘蔗花叶病毒cp基因序列设计特异性引物,RT-PCR扩增甘蔗花叶病毒cp基因特异性干涉片段,构建cp基因反向重复原核表达载体LMCP。利用IPTG进行诱导表达并对诱导表达条件进行优化。结果表明,经过IPTG诱导,LMCP在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的片段,经DNase I和RNase A消化处理,证实为dsRNA。同时IPTG浓度为0.4～0.6mmol/L,诱导表达4h,dsRNA的表达量最高。
2、利用大肠杆菌表达的dsRNA提取液处理玉米8112植株,并对处理参数进行了优化,结果表明当dsRNA稀释1/5浓度以下时处理效果明显；dsRNA喷洒后3d内接种病毒,抗病毒侵染效果最好；另外,加入适宜浓度的渗透剂能提高dsRNA的抗病效果。
3、RT-PCR分别扩增甘蔗花叶病毒和粗缩病毒外壳蛋白基因特异性RNA干扰片段,分别构建成反向重复序列并串联在一起,再与抗除草剂bar基因分别插入到pDTBU的两个T-DNA区段,构建了兼抗甘蔗花叶病毒和粗缩病毒的RNA干涉复合表达载体pDTBU SM。并通过冻融法将该载体直接导入农杆菌,获得了经PCR鉴定的转化子,用于农杆菌介导的玉米原位转化。
4、利用农杆菌介导的原位转化技术将cp基因RNA干涉表达载体pDTBU SM转入玉米8112自交系,对T0代植株进行除草剂Basta筛选,结合PCR扩增筛选得到19株共转化植株,对T1代共转化株系进行PCR检测及Southern杂交分析,证明有3个株系发生了分离,且获得了整合有cp基因RNAi片段的转化株。
5、提取T1代不同转化株的叶片总RNA以及开花期不同器官的RNA,经反转录反应后,进行荧光定量检测cp基因在不同转基因植株以及在同一转基因植株不同器官中的表达量,结果表明转化的cp基因干涉片段在不同转基因植株中的表达量不同。目的基因在雄花和叶中的表达量最高,而在根中的表达量最低。
6、T2代转基因玉米植株4叶期时接种甘蔗花叶病毒,并于接毒后不同时间提取叶片总RNA,荧光定量检测外源病毒诱导对内源cp基因表达的影响,结果表明转化的cp基因干涉片段对甘蔗花叶病毒产生了干涉效应,转入的cp基因RNAi片段在转基因植株中能正确转录,并导致cp基因:mRNA含量的交替变化。
7、对T2代4叶期转基因玉米接种甘蔗花叶病毒,于接毒后14d、21d、30d取叶片,ELISA检测植株的发病情况,结果表明,不同转化株株系的感病情况有差异,但抗病性均不同程度地高于对照。
综上所述,本文利用细菌高效表达的dsRNA处理玉米8112自交系,通过对处理植株的发病情况进行研究,筛选并建立了外源dsRNA的干扰调控技术体系；成功构建了兼抗两种病毒的无选择标记RNAi复合表达载体,利用农杆菌介导的玉米原位转化技术,获得了整合cp基因的RNAi转化株,其RNAi效果显著,转化植株的抗病性得到了提高。其研究结果对建立RNA干扰调控技术体系在玉米抗病毒基因工程中的应用,开辟玉米抗病毒育种的新方法具有重要意义。
Maize is an important agricultural crop with diverse applications, including its primary uses in livestock feed, human consumption, and ethanol production (as well as other biofuels). Maize production in China is second in the world, and has now reached nearly one half that of the United States. However viral diseases, particularly maize dwarf mosaic disease caused by Sugarcane mosaic virus and maize rough dwarf disease are among the most damaging diseases affecting maize production and quality in China. It is urgency to cultivate and popularize disease-resistant cultivar and search for new methods. RNA interference (RNAi), as an important RNA-based reverse-genetic system, has been used for gene function and genetic melioration studies in plants. RNAi has displayed great advantage in antiviral of breeding which has been proved to be a technology distinctiveness, high efficiency, economy, and inhibiting gene expression, and has become the focus of scientist researches. In order to select and establish the prokaryotic expression system and the in planta transformation system of the cp gene from virus, and to investigate effects of RNAi in maize antivirus genetic engineering and to find a new molecular breeding method in maize, we explored a basic principle of inverted-repeat expression vector design, and report an efficient method to produce dsRNA using a bacterial expression system. Two marker-free siRNA complex expression vectors of pDTBU SM from SCMV cp and MRDV cp were constructed, and transformed into Agrobactirium tumefaciens. Our work illustrates some basic principles for protecting plants against viral diseases in vitro and in vivo, as well as the study of gene silencing mechanisms in plant viral infections. The integration of target gene was analyzed, and we investigated the effect of RNAi and amylase content. The main results are summarized as follow:
1. Two fragments of the SCMV cp gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi, and subsequently joined to generate LMCP. The two recombinant plasmids were transformed individually into E.coli HT115, and dsRNA was induced by IPTG. The results showed that the expression products were the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of0.4～0.6mmol/L and induction time of4h was the most optimal expression condition.
2. The crude extracts of E.coli HT115containing large amounts of dsRNA were sprayed to plants and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (cp1) was more effective than was dsRNA derived from a downstream region(cp2) of the SCMV cp gene. The results showed that plants inoculated with the virus and LMCP preparation, diluted1/5or less, and treated at or before5days prior to viral inoculation, displayed decreased disease symptoms. Besides, dsRNA-containing extracts with penetrating agent of suitable selection concentration could Inhibit virus infection effectively.
3. Based on the coat protein gene sequence of SCMV and MRDV in GenBank and MaizeSeq database, six pairs of specific primers were designed and six specific fragments were amplified by RT-PCR. To prepare siRNA that corresponded to part of the SCMV cp or MRDV cp gene. In the first cloning step, an inverted repeat sequence of pUCCRNAi+2F was constructed. Next, two inverted repeat sequences were inserted into pBluscript SK in series to generate pBluscript+SM. Meanwhile, Ubiquitin promoter and Nos termination were cloned into pDTB to generate pDTBU. In the third step, pDTBU and pBluscript+SM plasmids were digested and joined to generate pDTBU SM. The study presented here provides a valuable tool for plant viral control using RNAi and the PTGS approach.
4. RNA interference expression vector of SCMV cp and MRDV cp gene were transformed into maize inbred lines8112by Agrobacterium tumefaciens and the results of PCR-Southern and Southern blot proved integration of the targeted segment into the8112genome successfully, and four transgenic plants were obtained finally.
5. Total RNA was extracted from different maize leaves4LP (4-leaf-period) or different organs of florescenceand, and cDNA segment of cp was obtained by real-time quantitative PCR, which resulted in a reduction of endogenous cp mRNA. The results showed that the expression of the cp gene fragment in transgenic plants was different. And the highest expression was in male flowers and leaves, while the lowest was in the roots.
6. Transgenic plants of4-leaf-stage in T2generation were inoculated with maize dwarf mosaic virus, and total RNA was extracted from the leaves at different times after inoculation, and the expression of endogenous cp gene was detected by fluorescence quantitative PCR when induced by exogenous virus. The results showed that the cp gene fragment in transgenic plants could be transcribed correctly which leaded to mRNA content in alternate, and the hpRNA structure transcripted by the cp gene could interference SCMV infection subsequently.
7. Transgenic plants of4-leaf-period were inoculated with maize dwarf mosaic virus, and ELISA results were detected at14dpi、21dpi and30dpi (days post inoculation). The results showed that almost transgenic plants could inhibit SCMV infection, while more than87%non-transgenic plants displayed typical disease symptoms.
In summary, maize inbred line8112was inoculated with crude extracts of bacterially expressed dsRNAs to protect maize plants against SCMV infection and viral infection was detected. RNAi technique system of dsRNA in vitro was filtered and constructed. Marker-free siRNA complex expression vector against SCMV and MRDV was constructed and in planta transformation to maize inbred line8112by mediated Agrobacterium tumefaciens. Transgenic plants were obtained and RNAi effect was in evidence. And the resistance of the transgenic plants was improved. Based on those above results, our study provides a reference on utilizing of the RNAi technique to protect maize against SCMV infection and constructing a new breeding method of high resistance maize.
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