An ISSR-PCR reaction system for <i>Paris polyphylla</i>

TIAN Fengguang; HE Yingfei; DUAN Chengli

doi:10.11833/j.issn.2095-0756.2014.06.023

Volume 31 Issue 6

Nov. 2014

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TIAN Fengguang, HE Yingfei, DUAN Chengli. An ISSR-PCR reaction system for Paris polyphylla[J]. Journal of Zhejiang A&F University, 2014, 31(6): 983-989. doi: 10.11833/j.issn.2095-0756.2014.06.023

Citation:

TIAN Fengguang, HE Yingfei, DUAN Chengli. An ISSR-PCR reaction system for Paris polyphylla[J]. Journal of Zhejiang A&F University, 2014, 31(6): 983-989. doi: 10.11833/j.issn.2095-0756.2014.06.023

An ISSR-PCR reaction system for Paris polyphylla

doi: 10.11833/j.issn.2095-0756.2014.06.023

The Nurturing Station for the Key Laboratory of Subtopical Silviculture, Zhejiang A & F University, Lin'an 311300, Zhejiang, China

Received Date: 2014-01-19
Rev Recd Date: 2014-03-24
Publish Date: 2014-12-20

Abstract

In order to select a stable inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) system for Paris polyphylla, a single factor experiment and an orthogonal design were combined and applied in this study. Concentrations for parameters were determined in a 25.0 μL system. Results revealed an optimized reaction system with concentrations of 0.2 mmol·L^-1 dNTPs, 0.75×16.67 nkat (0.75 U) Taq DNA polymerase, Mg²⁺ 2.0 mmol·L^-1, 0.6 μmol·L^-1 primer, and 40 ng DNA template. The optimal annealing temperature was 52℃ for primer ISSR2. This study of an ISSR-PCR reaction system with high stability, repeatability, and detective polymorphism ability has laid a technical foundation for germplasm resource identification and genetic diversity analyses of P. polyphylla.
- botany,
- Paris polyphylla,
- ISSR-PCR,
- reaction system

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References


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[4]	ZHOU Zhixiu, CHEN Liang, LOU Luhuang, et al. Medicinal plants from Mount Longwang Nature Reserve in Zhejiang[J]. J Zhejiang For Coll, 2000, 17(4):378-383.
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七叶一枝花Paris polyphylla是百合科Liliaceae重楼属Paris多年生草本植物，为中国特有的名贵药材重楼的基源植物之一，以根茎入药，具有清热解毒、消肿止痛、凉肝定惊等功效，民间多用于治疗无名肿毒和毒蛇咬伤，以及流行性腮腺炎、扁桃体炎、乳腺炎、咽喉肿痛、跌打伤痛、惊风抽搐等症^[1]。现代药理研究表明，重楼还具有抗癌止血、祛痰抑菌、止咳平喘、镇静镇痛、抗早孕杀灭精子和抗细胞毒等功效，是中国云南白药、宫血宁、百宝丹、热毒清、季德盛蛇药、抗病毒颗粒、总皂苷片等许多著名中成药的主要原料，具有广阔的开发应用前景^[2-3]。据《浙江植物志》和《浙江省药用资源名录》记载，七叶一枝花作为浙江的珍贵野生药材资源，在浙江各地均有分布^[4-5]。但长期以来，掠夺式的过度采挖以及生态环境的恶化，严重危及七叶一枝花的自然生长与繁衍，其野生种群的自我更新能力也受到极大破坏。目前浙江多处七叶一枝花资源已非常稀少，难以寻见，濒于灭绝的境地^[6-10]。因此，七叶一枝花的研究工作逐步为人们重视，主要包括基于16S rDNA和28S rDNA基因间隔序列(ITS序列)亲缘关系、药理活性、化学成分分析、快繁及人工栽培技术等研究^[11-14]。ISSR(Inter-simple sequence repeat，简单重复序列间扩增)是由Zietkiewicz等^[15]于1994年创建的一种基于聚合酶链式反应(PCR)的新型分子标记，它以能快速高效灵敏地检测出基因组DNA的多态性，并具有良好的重复性，操作简便、成本低、DNA用量小、安全性较高等特点，而被广泛应用于品种鉴定、遗传关系及遗传多样性、基因标记、植物基因作图、指纹图谱的建立等多方面的研究^[16]。近年来，ISSR技术迅速在药用植物的种源鉴定、种质遗传多样性分析、亲缘关系分析、药用植物辅助育种等研究中得到应用，取得了较好的效果，也越来越受到研究者的重视^[17-18]。ISSR是基于PCR反应的一种分子标记，影响反应的因素较多，不同物种所需的各种技术参数也存在一定差异。为了在七叶一枝花研究中获得稳定可靠、重复性好的分析结果，本研究利用单因素试验初筛和正交设计实验筛选结合的方法，对影响PCR反应的dNTPs，TaqDNA聚合酶，引物，模板DNA和镁离子(Mg²⁺)浓度及引物退火温度等技术参数进行优化筛选，以建立适合七叶一枝花ISSR-PCR的最佳反应体系和反应条件，为后续的种源鉴定及遗传多样性评价奠定基础。

1. 材料

试验材料为采于浙江省临安市东天目山海拔867 m野生七叶一枝花新鲜叶片，洗净，保存于-80 ℃超低温冰箱中。试验所用ISSR引物由生工生物工程(上海)有限公司合成，PCR反应试剂三磷酸碱基脱氧核苷酸(dNTPs)和Taq DNA聚合酶均购自宝生物工程(大连)有限公司，利用Eppendorf AG PCR仪进行反应扩增。

2. 方法

2.1. 基因组DNA提取与测定

采用改良的4×十六烷基三甲基溴化铵(CTAB)法^[19]提取七叶一枝花的总DNA，然后用10.0 g·kg^-1的琼脂糖凝胶电泳检测其质量，用ND^-1000分光光度计对DNA的浓度进行测定。根据测定结果，用灭菌去离子水将DNA浓度稀释至100 mg·L^-1，并保存至-20 ℃以备用。

2.2. ISSR-PCR反应体系的优化

采用上海生工合成的ISSR引物，通过筛选，选择引物ISSR2(5′-GACAGACAGACAGACA-3′)用于七叶一枝花反应系统的优化。

2.2.1. 单因素试验设计

采用单因素设计方法，参考北重楼Paris vertcillata，黑籽重楼Paris thibetica等^[20-22] ISSR-PCR反应条件，对七叶一枝花ISSR反应体系中的TaqDNA聚合酶，模板DNA，dNTPs，引物，Mg²⁺，退火温度等6种主要因子设置不同浓度水平进行分析(25.0 μL反应体系中各成分优化设计方案见表 1)。

Taq酶/(×16.67nkat)	DNA模板/ng	dNTPs/ (mmol • L^-1)	引物/(μmol.L^-1)	Mg²⁺/(mmol.L^-1)	退火温度/℃
0.25	10	0.05	0.2	1.0	46
0.50	20	0.10	0.3	1.5	48
0.75	30	0.15	0.4	2.0	50
1.00	40	0.20	0.5	2.5	52
1.25	50	0.25	0.6	3.0	54
1.50	60	0.30	0.7	3.5	56

Table 1. Single factors design of ISSR-PCR for Paria polyphylla

2.2.2. 正交设计试验优化

在单因素试验的基础上，采用正交设计方法，选择各因素之间的优化组合。

2.3. PCR扩增与扩增产物的检测

PCR扩增程序为：95 ℃预变性5 min；94 ℃变性40 s；52 ℃退火45 s；72 ℃延伸90 s；40次循环；72 ℃延伸7 min；4 ℃保存。PCR反应产物检测采用15 g·kg^-1的Tris-乙酸(TAE)琼脂糖凝胶，使用杭州纽龙生物科技有限公司生产DNA Marker Ⅱ Plus，用0.5 μg·L^-1溴化乙锭(EB)染色，以1×Tris-乙酸(TAE)为电泳缓冲液在150 V电压下电泳20~25 min，于Alphalmager HP凝胶成像系统进行拍照分析。

4. 讨论

ISSR分子标记技术是基于稳定而可靠的PCR反应体系的一种技术，ISSR-PCR反应易受TaqDNA聚合酶，模板DNA，dNTPs，引物，镁离子(Mg²⁺)浓度及退火温度等多种因素的影响。七叶一枝花是重楼属植物，虽然同属其他植物也进行过ISSR分析研究，但所建立的反应体系却各不相同^[20-22]，说明物种不同，最适的反应条件也存在差异，有必要针对特定物种建立最适的ISSR-PCR反应体系。

本研究在单因素多水平筛选的基础上，进行正交设计试验，既考虑了各因素的最佳条件，又兼顾了因素之间的交互作用，从而筛选出重复性好、多态性高的七叶一枝花最佳ISSR-PCR反应体系，即25.0 μL体系中dNTPs 0.2 mmol·L^-1，TaqDNA聚合酶0.75×16.67 nkat(0.75 U)，引物0.6 μmol·L^-1，Mg²⁺2.0 mmol·L^-1，模板DNA 40 ng，引物ISSR2最适退火温度为52 ℃。该体系的建立为后续的七叶一枝花种质资源鉴定和遗传多样性研究奠定了基础。

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An ISSR-PCR reaction system for Paris polyphylla

doi: 10.11833/j.issn.2095-0756.2014.06.023