獐ISSR-PCR反应体系的建立与优化及引物筛选

利用正交试验L₁₆(4⁵)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、Mg²⁺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、Mg²⁺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.

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

Five factors of amplification system,such as concentration of Mg²⁺,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 H.inermis with desirable repeatability and polymorphic bands was established.The optimized ISSR-PCR system of H.inermis included 2.5 mmol·L^-1Mg²⁺,1.25 U·25μL^-1 Taq DNA polymerase,0.3μmol·L^-1 primer,350 ng·25μL^-1 DNA template and 0.15 mmol·L^-1 dNTPs in 25μL 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.