基于COⅠ基因的二化螟种群遗传多样性检测方法
Genetic diversity of Chilo suppressalis (Walker) as determined by partial COⅠgene sequencing and DGGE
李 晴1 梁玉勇2 厉建蕾1 程正新2 熊焕保2 刘雨芳3 桂芳艳3 马 伟1
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DOI：10.7679/j.issn.2095-1353.2014.147
作者单位：1. 上海交通大学生命科学技术学院，微生物代谢国家重点实验室，上海 200240；2. 江西省农科院植物保护研究所，南昌 330200； 3. 湖南科技大学 生命科学学院，湘潭 411201
中文关键词：二化螟，转基因水稻，COⅠ，遗传多样性，DGGE
英文关键词：Chilo suppressalis , transgenic rice, COⅠ, genetic diversity, DGGE
中文摘要：
【目的】 建立二化螟Chilo suppressalis（Walker）遗传多样性检测与分析方法，以研究转基因水稻是否会对二化螟（靶标昆虫）种群遗传多样性产生显著影响。【方法】 从江西、湖南两地采集的二化螟样本中，各随机挑选12只，提取基因组DNA，克隆COⅠ基因的35~ 692 bp 区段（658 bp）进行测序；同时，采用PCR-DGGE技术分析江西3个样本的COⅠ基因遗传多样性，以获取样本群体遗传多样性信息。【结果】 对158个COⅠ基因克隆测序结果分析发现，在658 bp的区段中，共有173个位点存在多态，江西二化螟种群的单倍型多样度（h）为0.820，而湖南二化螟种群的单倍型多样度（h）仅为0.542，江西二化螟COⅠ基因多态要比湖南样本丰富。对COⅠ基因1 278~1 493 bp 区段（266 bp）进行DGGE分析，共获得5条清晰条带，将分析的3只二化螟样本分成两类，该结果与基因测序结果一致。【结论】测序方法可以获得丰富、详细的二化螟目标基因多态信息，但工作量、实验周期及成本较高；DGGE方法虽然信息量较小，但有通量大、实验周期短、成本低等优点，因此该方法适用于二化螟等昆虫大样本种群遗传多样性研究。本研究建立的方法可以为判断转基因水稻是否会对靶标、非靶标昆虫的遗传多样性产生影响提供可靠的分子依据。
英文摘要：
[Objectives] To investigate whether or not transgenic rice will affect the genetic diversity of Chilo suppressalis (Walker). [Methods] 12 individuals of C. suppressalis were randomly selected respectively from specimens collected in Jiangxi and Hunan. After extraction of genomic DNA, PCR and cloning of 658 bp fragment from the COⅠgene ( 35 - 692 bp), 158 clones were sequenced. Meanwhile three worms were analyzed by PCR-DGGE. [Results] 173 polymorphic sites were identified in the 658 bp fragment. The haplotype diversity (h) of Jiangxi samples was 0.820 whereas that of Hunan samples was only 0.542, suggesting greater COⅠ gene polymorphism in Jiangxi than Hunan population. DGGE analysis of a 266-bp-fragment in the COⅠ gene (1 278-1 493 bp) resulted in five distinct bands. Three worms could be clustered into two groups, which is consistent with result of gene sequencing. [Conclusion] Sequencing can obtain more detailed information on the genetic diversity of target genes in C. suppressalis but is higher in cost and is relatively time consuming. DGGE provides less information but is a high-throughput method. If the DGGE system is well optimized and used properly, large numbers of samples can be analyzed in a reasonable period of time. So DGGE is suitable for investigating whether or not transgenic rice will affect the population genetic diversity of target insects, i.e. C. suppressalis, and non-target insect, i.e. rice plant hoppers.