利用双链RNA分解酶基因pac1培育广谱抗病毒甘蔗新种质

There are various plant virus diseases on sugarcane leading to its yield and sugar decreased. Mixed infection of multiple plant viruses resulted in increasingly serious harm to sugarcane. Breeding new varieties with broad-spectrum virus-resistant is one of the main goals for sugarcane disease resistance breeding. Sugarcane virus diseases are mainly caused by a variety of RNA viruses. There is a double-stranded RNA replicative form (RF) in the replication cycle of RNA virus. Double-stranded RNA-specific ribonuclease encoded by pac1 gene can specifically recognize and degrade double-stranded RNA. Expression of double-stranded RNA-specific ribonuclease in sugarcane leads to degradation double-stranded RNA RF produced by RNA virus in its replication cycle, thus blocking the virus replication process so that it can’t finish the systemic infection in sugarcane and getting broad-spectrum virus-resistant sugarcane finally. In this project, we intend to express the double-stranded RNA-specific ribonuclease using pac1 gene in prokaryotic cells, test the degradation activity of the ribonuclease for dsRNA of four sugarcane viruses, such as sugarcane mosaic virus, sorghum mosaic virus, sugarcane streak mosaic virus and sugarcane yellow leaf virus in vitro, then introduce the pac1 gene into sugarcane by using Agrobacterium tumefaciens-mediated method and make it express in sugarcane effectively. Then inoculate the transgenic plants with different strains of these four viruses separately to identify their disease resistance, screen and obtain the broad-spectrum virus-resistant germplasms of sugarcane. This project will provide a new pathway for breeding new varieties of sugarcane with broad-spectrum virus-resistant.

甘蔗上存在多种植物病毒病害导致其产量和糖分下降，多种植物病毒的混合浸染，使其危害日益严重。培育广谱抗病毒新品种是甘蔗抗病育种的一个主要目标。甘蔗病毒病主要由多种RNA病毒引起，RNA病毒在复制周期内都存在一个双链RNA复制中间体的形态，pac1基因编码的双链RNA分解酶具有特异识别并分解双链RNA的特性，在甘蔗体内表达pac1基因，浸染甘蔗的RNA病毒在其复制周期产生的双链RNA复制中间体就会被降解，而阻断病毒复制进程使其不能对植物形成系统浸染，达到广谱抗病毒的目的。本项目拟通过原核表达pac1基因并在体外检测其对甘蔗花叶病毒、高粱花叶病毒、甘蔗线条花叶病毒和甘蔗黄叶病毒等4种甘蔗病毒dsRNA降解活性，再利用农杆菌介导法把该基因导入甘蔗使其得到有效表达，然后利用这4种病毒不同株系对转基因植株进行挑战接种鉴定其抗病性并筛选获得广谱抗病毒甘蔗新种质。为甘蔗广谱抗病毒新品种的培育提供新途径。

甘蔗上存在多种植物病毒病害导致其产量和糖分下降，多种植物病毒的混合浸染，使其危害日益严重。培育广谱抗病毒新品种是甘蔗抗病育种的一个主要目标。双链和单链RNA病毒在其复制过程中都存在一个双链RNA复制中间体。由pac1基因表达的双链RNA分解酶具有能特异识别并分解双链RNA的特性，通过在转基因甘蔗植株内表达双链RNA分解酶蛋白，降解病毒的双链RNA复制中间体，进而阻止病毒的复制，有望实现广谱抗病毒育种的目的。本研究通过原核表达获得了纯化的双链RNA分解酶蛋白，并通过试剂盒人工合成方式，获得以高粱花叶病毒（SrMV）、甘蔗条纹花叶病毒（SCSMV）和甘蔗黄叶病毒（SCMV）的RNA序列为模板合成的双链RNA。通过体外降解实验证明，PAC1双链RNA分解酶蛋白对不同序列的双链RNA均有很好的特异降解活性。而后通过植物表达载体构建，将pac1基因连接到植物表达载体pCAMBIA3300上，并通过农杆菌介导的遗传转化，将pac1基因导入到甘蔗主栽品种ROC22中。经筛选及分子鉴定后，将转基因植株移栽入温室，通过接种致病力最强的导致甘蔗产生花叶病的甘蔗条纹花叶病毒（SCSMV），进行转基因植株的抗病性鉴定。结果证明pac1基因提高了转基因植株的抗病性，所有转基因植株花叶病症状均明显较非转基因植株轻，病毒载量显著低于非转基因植株，田间农艺性状也均优于非转基因植株。其中转基因株系T3和T9，抗病性最强，田间农艺性状与未感病的健康植株无显著差异，有进一步利用的价值。本研究为培育广谱抗病毒甘蔗新种质提供了新思路。

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