獐ISSR-PCR反应体系的建立与优化及引物筛选  

Establishment and optimization of ISSR-PCR reaction system and primer screening for Chinese water deer(Hydropotes inermis)

作　　者：张龙龙[1] 鲍毅新[1] 于华会[2] 许婧[1] 孙波[1] 

机构地区：[1]浙江师范大学生态研究所,金华321004 [2]中国林业科学研究院亚热带林业研究所,浙江富阳311400

出　　处：《生态科学》2009年第4期335-341,共7页Ecological Science

基　　金：浙江省重点学科(生态学)基金;浙江省新苗人才计划基金(2007R40G2030152)

摘　　要：利用正交试验L_(16)(4~5)对影响獐(Hydropotes inermis)ISSR-PCR反应的Taq DNA聚合酶浓度、dNTP浓度、引物浓度、Mg^(2+)浓度及模板DNA浓度5个因素在4个水平上进行优化,同时对退火温度进行梯度PCR反应,以建立适合于獐ISSR-PCR反应的最佳体系。最终确定獐25μL ISSR-PCR反应体系为:Taq酶1.25 U·25μL^(-1)、Mg^(2+)浓度2.5 mmol·L^(-1)、引物浓度0.3μmol·L^(-1)、DNA模板量350 ng·25μL^(-1)、dNTP浓度0.15 mmol·L^(-1)。在此基础上,利用优化的反应体系成功筛选出10条用于獐相关研究的ISSR引物并确定了各自的最佳退火温度,为今后利用ISSR技术进行獐的物种鉴定与分类、亲缘关系、系统发育和生理病理学研究奠定了技术基础,也为开展其它大型资源动物如黑麂的保护遗传学研究提供理论基础。Five factors of amplification system, such as concentration of Mg^2＋, dNTPs, primers, Taq DNA polymerase and DNA template were investigated to optimize the ISSR-PCR reaction system of H. inermis through orthogonal tests. The data were analyzed by software DPS. As a result, a satisfactory ISSR reaction system for 1t. inermis with desirable repeatability and polymorphie bands was established. The optimized ISSR-PCR system ofH. inermis included 2.5 mmol·L^-1Mg^2＋, 1.25 U·25 μL^-1 Taq DNA polymerase, 0.3 μmol·L^-1 primer, 350 ng·25 μL^-1 DNA template and 0.15 mmol·L^-1 dNTPs in 25 μL PCR reaction system. Ten primers with stable amplification and rich polymorphism for ISSR were screened using this reaction system and its optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. This optimized ISSR-PCR reaction system would provide the basis for the analysis of genetic diversity, phylogenetic analysis, physiological or pathological events and genetic variation of important traits of H, inermis.

关 键 词：獐 正交试验 ISSR 引物筛选 

分 类 号：Q953[生物学—动物学] Q959.842