《植物生理学报》 2015, 51(4): 546-552

疫霉侵染辣椒基因差异表达的cDNA-AFLP分析

贺俐¹, 徐波^2,3, 黄子君¹, 何水林^2,4,*, 吴杨^1,*

¹井冈山大学生命科学学院, 江西吉安343009; 福建农林大学²作物遗传育种与综合利用教育部重点实验室, ³生命科学学院, ⁴作物科学学院, 福州350002

通信作者：何水林；E-mail: wuyangfenghao@126.com; shlhe201304@aliyun.com；Tel: 0796-8100493

摘　要：

采用cDNA-AFLP (complementary DNA-amplified fragment length polymorphism)技术, 以高抗疫霉病辣椒品种‘L11’接种疫霉菌的叶片为材料, 对6叶期辣椒幼苗灌根接种疫霉菌侵染后6个时间点的叶片基因表达谱进行了差异分析。利用86对引物组合, 共筛选出了363条差异表达的cDNA片段, 对其中100个表达差异显著的片段进行测序, 最终得到73个差异片段的核苷酸序列。经Blastx比对和功能分类分析, 其中45个序列与已报道的功能基因具较高同源性, 其功能涉及蛋白质代谢、信号转导、防御反应、碳水化合物代谢和光合作用等。在差异片段序列基础上设计特异引物, 采用基于PCR技术的96孔文库筛选方法, 从文库中分离得到2个差异基因的全长cDNA, 荧光定量PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。本研究利用cDNA-AFLP技术筛选到的一批与抗疫霉病相关的基因片段, 可进一步用于功能鉴定和分子育种。

关键词：辣椒(Capsicum annuum); 疫霉; cDNA-AFLP

收稿：2015-01-20   修定：2015-02-15

资助：国家自然科学基金(31260482和31060263)、江西省科技厅科技支撑计划项目(20122BBF60135)和江西省教育厅科技计划项目(GJJ13543和GJJ13544)。

cDNA-AFLP Analysis of Differentially Expressed Genes in Pepper Infected by Phytophthora capsici

HE Li¹, XU Bo^2,3, HUANG Zi-Jun¹, HE Shui-Lin^2,4,*, WU Yang^1,*

¹College of Life Sciences, Jinggangshan University, Ji’an, Jiangxi 343009, China; ²Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, ³College of Life Sciences, ⁴College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Corresponding author: HE Shui-Lin; E-mail: wuyangfenghao@126.com; shlhe201304@aliyun.com; Tel: 0796-8100493

Abstract:

Pepper cultivar ‘L11’ with the high resistance to Phytophthora capsici was used as the experimental material, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to analyze gene differential transcription profiling of six-leaf-stage seedlings of pepper infected by root-irrigating P. capsici. Eighty-six primer combinations were used to investigate 363 cDNA fragments. After sequencing of 100 ESTs, the nucleic acid sequences of 73 ESTs were obtained. Blastx analyses and functional annotations were then performed and the results revealed that 45 ESTs showed homologous to the known function genes of other species. The function of homologous genes involved in different pathways such as protein metabolism, signal transduction, defense responding, carbohydrate metabolism and photosynthesis. Using specific primers designed from the sequences of TDFs, two differential expression gene full-length cDNA were isolated by the method of PCR-based 96-hole screening, which were chosen for further real-time PCR expression patterns and confirmed the cDNA-AFLP profiles. Gene expression profiling in response to P. capsici and differentially expressed genes of pepper were identified via cDNA-AFLP analysis. These novel genes could be used in future for functional analysis and strategies of molecular breeding.

Key words: pepper (Capsicum annuum); Phytophthora capsici; cDNA-AFLP