【作者】 杨国峰；

【导师】 周鹏；

【作者基本信息】 海南大学 ， 作物遗传育种， 2012， 博士

【摘要】 番木瓜环斑病毒(Papaya Ringspot Virus, PRSV)是属于马铃薯Y病毒(genus Potyvirus, family Potyviridae)的一种植物病毒,为单链正义RNA, PRSV引起的番木瓜环斑病病毒病在全球各生长区给番木瓜种植业造成了严重的损失,病毒编码的复制酶(NIb)、辅助蛋白(HC-Pro)和外壳蛋白(CP)等参与蚜虫介导的传毒、病毒在宿主细胞内复制、积累及细胞间运动等过程,在病毒为害番木瓜的过程中发挥着重要作用。人们在生产实践中积累的控制PRSV的常规方法大多是限制蚜虫介导的传播或销毁病株,仅能在一定程度上控制病毒的蔓延,并不能直接消除病毒,非常被动。番木瓜杂交抗病毒育种因其种内缺乏抗病资源、种间杂交不亲和等原因,一直进展缓慢,尚未得到适合大面积推广应用的高抗品系。基因工程技术克服了番木瓜抗病毒种质资源缺乏的困难,充分利用了来自病毒和其他种属植物的抗病毒基因,为番木瓜抗病毒育种开辟了新的有效途径。但转基因抗病毒番木瓜的抗性谱较窄,能高抗PRSV某一株系的转基因番木瓜品系往往对其它地区的其他PRSV株系无效或只有低度抗病性,且转基因番木瓜植株潜在的生态风险和食品安全问题也饱受争议,限制了基因工程技术的应用。番木瓜生产中迫切需要既适合于大田生产、又行之有效的PRSV防治方法。本研究利用dsRNA介导的RNA沉默抗病毒机制,构建了PRSV编码基因同源dsRNA的原核表达系统,并使用诱导表达的同源dsRNA处理番木瓜植株,研究了利用原核表达的dsRNA防治番木瓜环斑病毒病的可行性。通过对已报道PRSV株系的NIb基因、HC-Pro基因和CP基因的序列同源性比对,发现这3个编码基因的3’端序列的同源性均较高。本研究通过PCR扩增得到了PRSV HN株系NIb基因、HC-Pro基因和CP基因3’端的N312、N501、N809、H315、H489、 H824、C279、C432、C867等9个核苷酸序列,利用OZ-LIC法分别构建了与N312、N501、 N809、H315、H489、H824、C279、C432、C867同源的发夹RNA编码结构(含有PDK内含子),以M-JM1091acY菌株为宿主菌分别构建了高效的PRSV编码基因同源dsRNA原核表达系统M-Jm109LacY/pSP73-RNAi-N312、M-Jm109LacY/pSP73-RNAi-N501、 M-Jm109LacY/pSP73-RNAi-N809、M-Jm109LacY/pSP73-RNAi-H315、 M-Jm109LacY/pSP73-RNAi-H489、M-Jm109LacY/pSP73-RNAi-H824、 M-Jm109LacY/pSP73-RNAi-C279、M-Jm109LacY/pSP73-RNAi-C432、 M-Jm109LacY/pSP73-RNAi-C867。经IPTG诱导成功利用原核系统表达了dsRNA,并证明dsRNA不被DNase I和RNase A降解,稳定性较好。本研究分别用PRSV Nib基因、HC-Pro基因、CP基因构建了GFP瞬时植物融合表达载体pNIb-GFP、pHC-GFP、pCP-GFP。对经Trizol法提取的dsRNA和对应GFP瞬时融合表达载体共转化的番木瓜叶片原生质体的共聚焦显微镜观察及通过半定量RT-PCR对其中mRNA表达量的分析结果证实：融合基因NIb-GFP、HC-GFP、CP-GFP的表达均发生了不同程度的下调,说明dsRNA在原生质体中引发了针对同源基因的沉默。本研究采用高压细胞破碎法处理经IPTG诱导的dsRNA原核表达工程菌细胞制备不同的dsRNA粗制品,利用dsRNA粗制品分别在接种PRSV前对番木瓜植株进行保护性处理、在表现环斑病症状后对番木瓜植株进行治疗性处理,并实施了田间的保护性处理实验。保护性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRNA、 H315-dsRNA、H489-dsRNA、H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组分别表现出了27%、50%、60%、20%、44%、50%、27%、40%、54%的抗病性,ELISA分析和Real-time PCR结果证实实验组番木瓜叶片中的病毒积累受到了不同程度的抑制,Northern杂交实验结果说明其抗性是由RNA沉默引起的抗性。治疗性处理实验结果显示,N312-dsRNA、N501-dsRNA、N809-dsRN、H315-dsRNA、H489-dsRNA、 H824-dsRNA、C279-dsRNA、C432-dsRNA、C867-dsRNA实验组在喷洒后dsRNA后第3天均检测到病毒积累量的小幅下降,第10天左右有部分染病植株叶片出现回绿或新长出的叶片没有症状,但随后病毒积累量又恢复至对照水平,回绿叶片重新花叶,无症状叶片也表现出症状,Northern杂交结果表明病毒积累量的下降是RNA沉默所致。田间保护性实验结果显示,定期喷洒N809-dsRNA粗制品使番木瓜植株获得了稳定抵抗PRSV的能力,在近距离生长的对照植株发病的情况下处理植株仍能保持不发病,长期保持较好长势,效果良好,证明该方法是一种绿色、廉价、便捷、有效的番木瓜环斑病毒防治方法。以上这些研究结果为利用dsRNA引发RNA沉默防治番木瓜环斑病毒的田间应用奠定了基础。更多还原

【Abstract】 Papaya ringspot virus (PRSV) is a plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV causes severe economic losses in papaya throughout the tropics and subtropics. PRSV disease management practices include quarantine, eradication, avoidance by planting papaya in areas isolated from the virus, continual rogueing of infected plants, use of tolerant lines to lower the economic losses caused by PRSV, crossprotection and transgenic resistance. Development of PRSV-resistant cultivars through conventional breeding met with limited success because of difficulties in overcoming intergeneric reproductive barriers of wild, related species of papaya. In addition, partial loss of tolerance in back-crosses with the commercial papaya parent also limits the usefulness of this approach. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. We provide a strategy to develop effective and stable PRSV-resistant plants via dsRNAs expressed in Escherichia coli.In this study,9different dsRNA prokaryotic expression vectors carrying different sizes of PRSV-NIb(312,501,809bp), PRSV-HC-Pro(315,489,824bp) and PRSV-CP(279,432,867bp)cDNA were constructed, which were named as, pSP73-RNAi-N312, pSP73-RNAi-N501, pSP73-RNAi-N809, pSP73-RNAi-H315, pSP73-RNAi-H489, pSP73-RNAi-H824, pSP73-RNAi-C279, pSP73-RNAi-C432, pSP73-RNAi-C867respectively. These prokaryotic expression vectors were transformed into the RNaseⅢ-deficient strain M-Jm109LacY, and then were induced with IPTG, respectively. All these E.coli strains deficient for RNaseⅢ could express the predicted sizes of PRSV-NIb-dsRNA, PRSV-HC-Pro-dsRNA and PRSV-CP-dsRNA, respectively.To monitor the silencing efficiency of these9different bacterially expressed dsRNAs derived from PRSV-Nib, PRSV-HC-Pro and PRSV-CP, we developed an effective transient gene silencing system for using the bacterially expressed dsRNA in papaya leaf protoplasts. Three plant transient expression vectors encoding the PRSV-NIb, PRSV-HC-Pro and PRSV-CP with an N-terminal GFP fusion were constructed respectively, which were named as pNIb-GFP, pHC-GFP and pCP-GFP respectively. Protoplasts were co-transfected using the transient GFP-fusion expression vector and the corresponding bacterially expressed dsRNA. The results of GFP fluorescence intensity using a confocal microscope and semi-quantitative RT-PCR analysis of the PRSV-Nib, PRSV-HC-Pro and PRSV-CP mRNA expression showed that the effective bacterially expressed dsRNA-triggered gene silencing was detected. To prove whether the bacterial-produced dsRNA could interfere with PRSV infection, we carry out a protective resistance assay and a therapeutic resistance assay. In the protective resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surface before inoculation of papaya leaves with PRSV. Protective treatment experimental results showed dsRNAs derived from the different functional genes of PRSV could all protect papaya plants from virus infection, and the resistance was obviously different due to different vectors. ELISA analysis and Real-time PCR results confirmed that the virus accumulation could be inhibited in different degree by dsRNA. Northern blot showed that the small RNA fragments specifically complementary to the PRSV-Nib, PRSV-HC-Pro and PRSV-CP antisense probe were detected, which suggested that the resistance is an RNA-mediated virus resistance. In the therapeutic resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surfaces after inoculation of papaya leaves with PRSV. ELISA analysis and Real-time PCR results showed that the virus accumulation declined slightly after spraying dsRNA for three days. Field protective experimental results showed that regular spraying the crude product of the bacterially expressed N809-dsRNA could obtain stable resistance to PRSV.Our work indicated that bacterially expressed double-stranded RNA targeting three viral genes of PRSV could interfere with PRSV infection, which is a potential green and effective approach protecting plants from virus infections compared with the requirements for regenerating PRSV-resistant transgenic plants.更多还原