摘 要

以毛竹（Phyllostachys edulis）是实验材料，对影响SSR-PCR扩增效果的Taq酶、引物浓度、dNTPs及MgCl2等因素进行L9（3⁴）正交试验筛选，建立适宜于毛竹SSR-PCR分子标记反应体系，所有反应采用Touchdown 退火。结果表明：在20ul体系中， 10×PCR buffer2ul、Taq酶1.5U、Mg2 1.0mM、dNTPs 0.1mM、引物 0.44Μm是毛竹的最适SSR反应体系。

本研究对来源于NCBI公共数据库中非冗余10608条毛竹cDNA进行简单序列SSR搜索，其中425条序列符合SSR设计条件，从中随机挑选设计了44对毛竹SSR引物，以毛竹为DNA为模板进行初筛选，有20对引物有较清晰且稳定的目标扩增产物；以此对12个毛竹群体代表进行复筛选，结果表明有5对引物的产物呈现多态性，多态位点百分率为25%；随机挑选3对引物进行四个毛竹群体96个样品扩增，平均每对引物可扩增出条带4.3，其中Phe44和Phe51可扩增出条带接近于5，多态丰富。结果表明，这两对引物可用于可为今后毛竹种内遗传多样性研究，也为竹类植物遗传作图、遗传多样性分析、功能基因的发掘与定位等方面的研究奠定良好基础。

关键词：毛竹，SSR-PCR，反应体系，引物筛选

Establishment of Phyllostachys edulis SSR marker reaction system and selection of primers

Abstract

The SSR reaction system of Phyllostachys edulis was established through optimizing some factors including Taq polymerase, the concentrations of Primer, dNTPs and Mg2 by L9(3⁴) test. The results indicated that the optimized SSR-PCR system for this bamboo was 10×PCR buffer2ul、Taq 1.5U、1.0mM Mg2 、0.1Mm dNTPs, 0.44Μm Primer. A modified touchdown PCR protocol was as follow: 94℃ for 5 min; 15 cycles of 94℃ for 30 s, 65℃decreasing to 50℃ at 1℃ per cycle for 30 s, 72℃ for 30 s; 20 cycles of 94℃ for 30 s, 50℃ for 30 s, 72℃ for 30 s; and a final extension at 72℃ for 5 min.

The availability of cDNAs in GenBank became an alternative source of mining simple sequence repeat (SSR). In the paper, uni-cDNAs of Ph. edulis from the database of NCBI were screened using SSRIT software and 490 SSRs(≥20bp) in 10608 cDNAs were mined out. 44 primer pairs were designed by Primer Primer 5 programme, which 20 of them resulted in PCR products of the expected size and 5 SSR markers could have products with polymorphism from 12 Ph. edulis samples of 12 separate populations. Phe 44/51/87 were used to assess the genetic diversity of 96 Ph. edulis samples from 4 different provinces or regions and Phe44/51 presented rich polymorphism with the average of 5 polymorphism bands. These results indicated that it is an available approach to develop cDNA-SSR markers based on cDNA sequence from Ph. edulis, and Phe 44 and 51could be used in Ph. edulis genetic diversity analyzing.

Key words: Phyllostachys edulis; SSR-PCR; reaction; primer screening

目 录

1 文献综述 1

1.1 毛竹及其分子生物学特性 1

1.1.1 毛竹资源概况 1

1.1.2 毛竹分子生物学特性 1

1.2 PCR的反应体系 1

1.2.1 PCR标准反应体系 1

1.2.2各个因子对PCR反应体系的影响 2

1.3 SSR标记及其特点 2

1.3.1分子标记 2

1.3.2 SSR标记的产生 2

1.3.3 SSR标记的特点 3

1.4竹类植物的SSR研究 3

1.5研究目的、内容 4

1.5.1研究目的及意义 4

1.5.2主要研究内容 4

2材料和方法 5

2.1供试材料 5

2.2主要仪器、试剂及溶液配制 5

2.3试验方法 6

2.3.1植物总DNA提取方法 6

2.3.2 SSR-PCR反应体系正交优化试验 6

2.3.3 SSR引物开发 7

2.3.4 PCR扩增 7

2.3.5聚丙烯酰胺电泳 8

2.3.6银染技术 8

3结果与分析 9

3.1体系正交试验结果 9

3.2 SSR-引物设计 9

3.3引物初筛选 12

3.4引物复筛选 13

3.5四个毛竹群体SSR扩增 14

4结论与讨论 15

致 谢 16

参考文献 17

1 文献综述

1.1 毛竹及其分子生物学特性