论文部分内容阅读
【目的】棉花曲叶病是棉花生产上的一种重要的病毒病害,在巴基斯坦和印度等国家地区大面积流行,造成严重的经济损失。近年在中国广西南宁的棉花田间发现了棉花曲叶病害,在广西的黄秋葵中也发生了曲叶病,二者的病原均为木尔坦棉花曲叶病毒(Cotton Leaf Curl Multan Virus,CLCuMV),为了对这2个病害有更深的了解,本文对该双生病毒伴随的DNA小分子进行测序分析。【方法】分别从广西南宁地区感染CLCuMV的3棵棉花和3棵黄秋葵中提取总DNA,用CLCuMV DNAβ的特异引物进行PCR扩增,将产物分离纯化并克隆测序,进行序列比对分析。【结果】从棉花曲叶病害中分离得到了1384 nt的新型重组DNA分子,以及从黄秋葵曲叶病害中分离得到了754 nt的新型缺失型DNA分子。研究结果表明1384 nt重组分子是由CLCuMV GX1的DNA-A和DNAβ重组而成。重组分子大部分来源于CLCuMV的DNA-A,包含基因间隔区,附近的部分AV2和AC1基因,以及反向互补的部分AC3基因。其余部分来源于伴随的DNAβ,包含A-rich区域。分析拼接片段的附近序列,发现接头部分含有2-3个共同碱基,推测为重组作用发生的位点。与以前报道的在实验室中产生的CLCuMV重组分子进行比较显示,DNA-A的基因间隔区和DNAβ的A-rich区在重组过程中非常保守。另外,754 nt的重组小分子是由CLCuMV Okra1 DNAβ缺失突变产生,缺失了大部分的编码C1蛋白开放阅读框(Open Reading Frame,ORF)以及小部分的A-rich区。【结论】本研究在自然条件下分离到了来源于CLCuMV和卫星DNAβ的重组分子,以及DNAβ缺陷型分子。这2种重组小分子以前未见报道,这也是在中国发现的棉花曲叶病毒中首次发现重组分子。这种基因组变异现象在棉花曲叶病毒的进化和寄主适应过程中可能有重要的意义。
【Objective】 Cotton leaf curl is an important virus disease in cotton production. It is widely spread in Pakistan, India and other countries and regions, causing serious economic losses. In recent years, cotton leaf curl has been found in cotton fields of Nanning, Guangxi, China. Leaf curl disease has also occurred in okra of Guangxi, both of which are Cotton Leaf Curl Multan Virus (CLCuMV) In order to have a deeper understanding of these two diseases, we sequenced the small DNA molecules that accompany the twin virus. 【Method】 Total DNA was extracted from 3 cotton cultivars and 3 okra cultivars infected with CLCuMV in Nanning, Guangxi. The PCR products were amplified by PCR using primers specific to CLCuMV DNAβ. The products were isolated, purified and cloned for sequence analysis. 【Result】 A novel 1384 nt recombinant DNA molecule was isolated from the cotton leaf curl disease and a novel 754 nt deletion DNA molecule was isolated from the leaf curl of Okra. The results showed that the 1384 nt recombinants were recombined by DNA-A and DNAβ of CLCuMV GX1. The majority of recombinant molecules are derived from DNA-A of CLCuMV, including the intergenic region, portions of nearby AV2 and AC1 genes, and partially complementary AC3 genes. The rest comes from the accompanying DNAβ, which contains the A-rich region. Analysis of the sequence near the splicing fragment revealed that the linker portion contained 2-3 common bases, presumably the site of recombination. Comparisons with previously reported CLCuMV recombinant molecules in the laboratory showed that the DNA-A intergenic region and the DNA-rich A-rich region are highly conserved during recombination. In addition, the 754 nt recombinant small molecule was generated by deletion of the CLCuMV Okra1 DNAβ deletion, most of which lacked the Open Reading Frame (ORF) encoding the C1 protein and a small portion of the A-rich region. 【Conclusion】 In this study, recombinant molecules derived from CLCuMV and satellite DNAβ and DNAβ-deficient molecules were isolated under natural conditions. These two kinds of recombinant small molecules have not been reported before, which is the first discovery of the recombinant molecule in the cotton leaf curl virus discovered in China. This genomic variation may have important implications in the evolution and host adaptation of cotton curl.