【作者】 阳佳位；

【导师】 钟广炎；

【作者基本信息】 西南大学 ， 果树学， 2010， 硕士

【摘要】 柑橘产业是我国南方农村的重要支柱产业,而每年由柑橘衰退病病毒(Citrus tristeza virus, CTV)造成的经济损失巨大。柑橘衰退病病毒属于正义单链RNA病毒。由于利用RNAi技术进行抗植物RNA病毒已有大量成功的报道,所以应用RNAi原理进行抗CTV育种可能成为解决CTV防治的有效方法。RNA病毒侵染植物时,一般表达沉默抑制因子使宿主植物细胞中RNAi机制钝化从而达到成功侵染植物的目的。因此阻断病毒的沉默抑制因子的表达将有助于植物抵抗病毒的侵染,从而达到抗病毒的效果。本试验将CTV中三个沉默抑制因子p25、p20和p23作为RNAi靶标序列,目的是要获得对柑橘CTV有广谱抗性的柑橘品种。(1)RNAi载体的构建、转化和转基因植株的验证根据CTV基因组RNA中的沉默抑制因子p25、p20和p23的序列保守区域设计引物,经RT-PCR成功克隆相关基因片段。利用重叠延伸PCR拼接法(Gene Splicing by Overlap Extension, SOE)拼接PCR产物,获得两条多基因大片段P2520、P252023。为了便于克隆操作,在正向片段下游引物末端加上限制性内切酶BglⅡ识别序列；在巢式PCR上游引物末端加上KpnⅠ识别序列,下游引物加上BglⅡ识别序列,经TA克隆得到含两个沉默抑制因子pe5和p20基因片段的反向重复序列载体pTZ57R-HP2520。应用同样的方法得到含三个沉默抑制因子pe5,p20和p23基因片段的反向重复序列载体pTZ57R-HP252023。载体pTZ57R-HP2520与pCAMBIA-2301G经SacⅠ和BamHⅠ双酶切,胶回收目的片段后用T4-DNA连接酶连接两个片段,然后转入大肠杆菌DH5α中,提取质粒后用Sad和BamHⅠ双酶切检测目的片段。然后将此质粒转化入农杆菌LBA4404菌株。含有质粒pCAMBIA2301-HP2520的农杆菌被用来与锦橙上胚轴共培养获得转基因植株。本试验共获得了抗性不定芽20个,嫁接后再生成活7株,利用T-DNA区域内的GUS基因特异引物PCR检测6株为阳性,同时用NPTⅡ基因特异引物PCR检测7株为阳性。应用同样的方法获得RNAi双元质粒表达载体pCAMBIA2301-HP252023,农杆菌介导转化锦橙上胚轴后诱导获得抗性不定芽1个,嫁接再生成活1株,经特异引物检测发现GUS基因和NPTⅡ基因均为阳性。(2) pCAMBIA2301-HP20转基因酸橙植株的评价分两步对本实验室已经构建好的针对沉默抑制因子p20的单基因片段RNAi (pCAMBIA2301-HP20)转基因酸橙植株进行评价。首先,8株转基因植株经过GUS组织化学染色鉴定和针对p20基因的特异引物PCR检测,证实所有的转基因植株都成功的转入了目的片段。其次,将转基因植株嫁接备份后做攻毒试验,即对其中5株转基因植株嫁接接种CTV强毒系TRL514。分别在接毒30d、50d和60d后用RT-PCR检测CTV存在情况,结果发现对照植株和其中两个转基因植株中的检查结果呈阳性,其余三株转基因植株迄今为止没有检测出CTV病毒RNA。不过,这三株未检测到CTV的转基因植株还需要再次进行攻毒试验,因此,它们是否真正对CTV具有持续稳定的抗性还需要进一步的研究。更多还原

【Abstract】 Citrus industry is an important pillar industry in South China areas. However, the industry has been threatened by Citrus tristeza virus (CTV), a positive-sense single-stranded RNA virus. Since many examples of using RNAi to control plant RNA viruses have been reported, it is possible to RNAi to control CTV in citrus.When plant RNA viruses infect plants, in order to survive, they usually inhibit host’s RNAi mechanism by expressing gene-silencing suppressor genes. Our rationale is that we would be able to fight against plant viruses by inactivating the expression of their gene-silencing suppressors. In this study, three suppressor factor genes p25, p20 and p23 of CTV were targeted by RNA interference.(1) Construction of RNAi vectors, transformation and verification of transgenic plantsPCR primers were designed according to the conserved sequences of CTV genomic RNA that encode the three suppressor genes p25, p20 and p23, and RT-PCR was performed to amplify the target sequences. Sequence overlap extension (SOE) method was used to join the amplified products to form inverted repeats. In this way, two multi-gene fragments P2520 and P252023 were obtained.To facilitate the following cloning steps, restriction site BglⅡwas added to the down stream sense primer, and KpnⅠand BglⅡrestriction sites were also added to the two nested primers, the upstream primer and the downstream primer, respectively. After TA cloning, the pTZ57R-HP2520 construct, in which HP2520 represents the inverted repeat fragment sequences of CTV’s p25 and p20 genes, was obtained. In similar way, the multi-gene construct containing the three suppressor genes was obtained.The pTZ57R-HP2520 and pCAMBIA-2301G were both digested by SacⅠand BamHⅠrestriction enzymes. The purified HP2520 and pCAMBIA-2301G were then ligated and transformed into E. coli DH5a. The transformation was verified by double digestion of the plasmid with SacⅠand BamHⅠextracted from the transformed cells. The plasmid was then transferred into Agrobacterium strain LBA4404.Transgenic plants were obtained by co-culturing the epicotyls of a sweet orange cultivar Jingcheng with the pCAMBIA2301-HP2520-containing Agrobacterium. Consequently,20 kanamicin resistant adventitious buds were regenerated and 7 of them survived the following grafting operation. PCR was performed using GUS and NPTⅡgene-specific primers to detect the transgenes in the transgenic plants. The results showed that six plants were GUS gene positive while all 7 were NPTⅡgene positive.The RNAi binary expression plasmid vector pCAMBIA2301-HP252023 was also obtained by same method. Transgenic plants were obtained but only 1 survived after grafting. PCR result demonstrated that the transgenic plant contained both GUS and NPTⅡgenes.(2) Evaluation of pCAMBIA2301-HP20 transgenic sour orange plants:Seven transgenic sour orange plants containg pCAMBIA2301-HP20, which were obtained previously in the lab, were evaluated. First, GUS staining and PCR amplification of p20 fragment were performed to validate the transgenes. The results showed all of them are true transgenic plants. Second, wild type sour orange plants (as control) and five transgenic plants were inoculated with TRL514, a CTV severe strain, to test and compare their anti-CTV activity. CTV virus RNA detection was performed by RT-PCR 30d,50d and 60d after inoculation. The results showed that CTV RNA was found in control and two transgenic plants but not in the rest three transgenic plants. Therefore, the three CTV RNA undetected transgenic plants may exhibit anti-CTV activity. Further inoculation and study are needed to verify their anti-CTV activity.更多还原