獐ISSR-PCR反应体系的建立与优化及引物筛选

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摘要

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.

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	张龙龙
	鲍毅新
	于华会
	许婧
	孙波

关键词 ：獐, 正交试验, ISSR, 引物筛选

Abstract：

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.

Key words： Chinese water deer(H.inermis) orthogonal tests ISSR primer screening

收稿日期: 2009-08-25

:	Q953
	Q959.842

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

引用本文:

张龙龙1, 鲍毅新1*, 于华会2, 许婧1, 孙波1. 獐ISSR-PCR反应体系的建立与优化及引物筛选[J]. , 2009, 28(4): 335-341.
ZHANG Long-long1, BAO Yi-xin1, YU Hua-hui2, XU Jing1, SUN Bo1. Establishment and optimization of ISSR-PCR reaction system and primer screening for Chinese water deer(Hydropotes inermis). , 2009, 28(4): 335-341.

链接本文:

http://www.ecolsci.com/CN/Y2009/V28/I4/335

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