木尔坦棉花曲叶病毒基因重组和缺失产生DNAβ相关的新型小分子

Novel DNA-β associated molecules produced by sequence recombination and deletion of cotton leaf curl Multan virus complex

【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 disease （CLCuD） is a major constraint to cotton production, causing great economic losses in Pakistan and India. In China, CLCuD has been discovered in the field of Nanning, GuangXi. To better understand this disease, we sequenced the virus-associated small DNA molecules. [ Methods] We purified total DNA from cotton and okra plants exhibiting leaf curl symptoms; PCR amplified and sequenced CLCuMV satellite DNA （DNAβ）-related small DNA molecules. [ Results] We identified 2 novel recombinant DNA molecules with 1384 nucleotides in cotton and 754 nucleotides in okra. The 1384 nt molecule contains partial DNA-A and DNAβ of CLCuMV GX1. It includes the intergenic region, adjacent AV2 and AC1 coding sequences, and reverse complementary AC3 of DNA-A and A-rich region of DNAβ. Common nucleotides were found around the junction points of DNA-A and DNAβ sequences, suggesting they were the sites of recombination. Comparison with previous reported CLCuMY recombinants produced in lab showed that the intergenic region of DNA-A and A-rich region of DNAB were conserved on the recombination process. The 754 nt molecule was produced by deletion of CLCuMV DNAβ in the C1 open reading frame and A-rich region. [ Conclusion] We identified a novel recombinant molecule originated from CLCuMV DNA-A and DNAβ and a small defective molecule of DNAβ. This is the first report of sequence recombination and deletion of CLCuMV in China, which may be helpful to understand the CLCuMV evolution and host adaptation.