中国农业科学 ›› 2014, Vol. 47 ›› Issue (22): 4349-4359.doi: 10.3864/j.issn.0578-1752.2014.22.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

黄萎病菌侵染下陆地棉Dirigent-like蛋白基因表达差异分析

郭宝生1, 2,师恭曜1,王凯辉2,刘素恩2,赵存鹏2,王兆晓2,耿军义2,华金平1   

  1. 1 中国农业大学农学与生物技术学院植物遗传育种系/杂种优势研究与利用教育部重点实验室/作物遗传改良北京市重点实验室,北京 100193
    2 河北省农林科学院棉花研究所/农业部黄淮海半干旱区棉花生物学与遗传育种重点实验室,石家庄 050051
  • 收稿日期:2014-03-11 修回日期:2014-07-09 出版日期:2014-11-16 发布日期:2014-11-16
  • 通讯作者: 华金平,Tel:010-62734748;E-mail:jinping_hua@cau.edu.cn
  • 作者简介:郭宝生,E-mail:guobaosheng111@126.com
  • 基金资助:
    国家重大专项“转基因优质纤维棉花新品种培育”(2011ZX08005-003)

Expression Differences of Dirigent-Like Protein Genes in Upland Cotton Responsed to Infection by Verticillium dahliae

GUO Bao-sheng1,2, SHI Gong-yao1, WANG Kai-hui2, LIU Su-en2, ZHAO Cun-peng2WANG Zhao-xiao2, GENG Jun-yi2, HUA Jin-ping1   

  1. 1Department of Crop Genetics & Breeding,College of Agronomy and Biotechnology, China Agricultural University/Key Laboratory of Crop Heterosis and Utilization of Ministry of Education/Beijing Key Laboratory of Crop Genetic Improvement, Beijing 100193
    2Cotton Research Institute, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Cotton in Huanghuaihai Semiarid Area, Ministry of Agriculture, Shijiazhuang 050051
  • Received:2014-03-11 Revised:2014-07-09 Online:2014-11-16 Published:2014-11-16

摘要:

【目的】发掘陆地棉Dirigent-like蛋白基因家族,明确不同黄萎病抗性品种间该基因家族成员的多态性及表达差异。【方法】利用Illumina Hiseq 2000测序平台,通过RNA-Seq测序技术,获得黄萎病菌侵染陆地棉抗病品种和感病品种的Dirigent-like蛋白基因家族成员Unigene序列;采用生物信息学软件对其进行核苷酸序列及编码蛋白的多重比对和系统进化分析;针对该家族成员设计特异的quantitative real-time PCR引物,Applied Biosystems 7500 Real Time PCR System上使用SYBR green PCR试剂盒标记反应产物,棉花GhACTIN为内标,采用2-ΔΔCt法分析抗病品种冀79、感病品种TM-1中该基因家族成员在0 h及受侵染1、8、24、48、72和96 h后的表达差异。【结果】获得12个陆地棉Dirigent-like蛋白基因家族成员;该家族成员分属3个进化群,基因间存在4个保守结构域。其同源基因在D5基因组染色体上,主要集中于第2、3、6、7、9、10、13染色体陆地棉受黄萎病侵染后,Dirigent-like蛋白基因抗病、感病品种间差异表达,抗病品种冀79受黄萎病菌侵染1 h时上调2倍以上的有GhDIR4GhDIR6GhDIR7GhDIR9GhDIR10GhDIR11,其中,GhDIR9上调6.5倍;侵染8 h时GhDIR1GhDIR4GhDIR7GhDIR11上调,有2个上调2倍以上;侵染24 h时仅GhDIR4上调2倍以上;侵染48 h时GhDIR1GhDIR4GhDIR7GhDIR11上调达到2倍以上;侵染72 h时仅GhDIR9上调2倍以上,达到6.4倍;侵染96 h时仅GhDIR4上调2倍以上。感病品种TM-1受侵染1 h时仅GhDIR7上调2倍以上;受侵染8 h时GhDIR7上调325倍,GhDIR10上调2倍以上;侵染24和48 h时GhDIR4GhDIR5GhDIR6GhDIR7GhDIR4GhDIR7GhDIR9GhDIR7GhDIR9GhDIR10在感病品种TM-1中上调显著。此外,抗病、感病品种间GhDIR1GhDIR3GhDIR5GhDIR8GhDIR10GhDIR12存在核苷酸多态性。【结论】Dirigent-like蛋白与黄萎病菌侵入后棉花植株的抗性反应GhDIR11值得关注;GhDIR6GhDIR9GhDIR10急剧上调,GhDIR7GhDIR9GhDIR10上调10倍以上;受侵染72 h时GhDIR4GhDIR7还保持较高的表达量;侵染96 h时GhDIR5GhDIR6GhDIR7GhDIR9GhDIR10GhDIR11保持较高的表达量。由此看出,受黄萎病侵染时抗病种质冀79 Dirigent-like蛋白基因家族成员上调表达要早于感病品种,但是,该基因家族成员在感病品种受侵染8 h后上调倍数远远高于抗病品种,而且到96 h时其表达量还较高;抗病品种上调的Dirigent-like蛋白家族成员相关

关键词: 棉花, Dirigent-like蛋白基因, 黄萎病菌, 表达差异

Abstract: 【Objective】The objective of this study is to explore the Dirigent-like protein gene family and to figure out polymorphism of genes and expression differences between resistant and susceptible varieties of Upland cotton (Gossypium hirsutum) in responding to infection by Verticillium dahliae.【Method】Dirigent-like protein genes were acquired using Illumina Hiseq 2000 RNA-Seq transcriptional analysis. Multiple sequence alignment of encoding proteins was carried using online sequence analysis softwares and bioinformatics analysis tools for phylogenetic analysis. Quantitative real-time PCR with gene-specific primers was employed to identify the expression differences between resistant variety ‘Ji 79’ and susceptible variety ‘TM-1’, at 0, 1, 8, 24, 48, 72 h, and 96 h after inoculation. The quantitative reactions were performed on the ABI 7500 Real-Time PCR System with 7500 System Software (Applied Biosystems, USA) using the SYBR Green Master Mix. The comparative 2-ΔΔCt method of quantification was used with cotton GhACTIN as the reference.【Result】Totally 12 members of this gene family were found in Gossypium hirsutum, and phylogenetic analysis demonstrated 3 distinct subfamilies and 4 relatively conservative domains existed. The members of Dirigent-like protein genes were located on chromosomes 2, 3, 6, 7, 9, 10 and 13 in Gossypium raimondii, and some located in clusters on the same chromosome of D5 genome. Differential expression analysis indicated that dynamic response existed in different varieties. There were 6 members (GhDIR4, GhDIR6, GhDIR7, GhDIR9, GhDIR10 and GhDIR11) up-regulated (RQ≥2) in resistant variety Ji 79 after 1h inoculation, and RQ of GhDIR9 reached about 6.5; and 4 members (GhDIR1, GhDIR4, GhDIR7 and GhDIR11) up-regulated (RQ≥1) after 8h inoculation, only GhDIR4 up-regulated (RQ≥2) after 24h inoculation, GhDIR1, GhDIR4, GhDIR7 and GhDIR11 up-regulated (RQ≥2) after 48h inoculation; only GhDIR9 up-regulated (RQ=6.4) after 72h inoculation, up to 96 h inoculation, only GhDIR4 up-regulated (RQ≥2). GhDIR7 showed up-regulated (RQ≥2) in susceptible variety TM-1 after 1h inoculation. GhDIR7 and GhDIR10 up-regulated, and RQ of GhDIR7 increased to 325 after 8h inoculation. GhDIR4, GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10 up-regulated significantly after 24h or 48h inoculation,and the RQ of GhDIR7, GhDIR9, GhDIR10 were more than 10. GhDIR4 and GhDIR7 still increased after 72h inoculation. Up to 96 h inoculation, GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10 and GhDIR11 were highly expressed. So the results showed that the expression of GhDIR in response to infection by V. dahlia in resistant variety Ji 79 was earlier than that in susceptible variety TM-1. The expression of GhDIR members in susceptible variety became stronger after 8h vaccination by V. dahlia and continuously went up after 96h inoculation. Significant up-regulation of GhDIR members GhDIR4, as well as GhDIR7, GhDIR9 and GhDIR11, were observed in resistant variety, GhDIR7 and 3 other members (GhDIR5, GhDIR6 and GhDIR10) expressed in susceptible variety. And nucleotide polymorphism of 6 GhDIR genes (GhDIR1, GhDIR3, GhDIR5, GhDIR8, GhDIR10 and GhDIR12) was detected between resistant variety and susceptible variety. 【Conclusion】Dirigent-like protein plays an important role in Upland cotton in response to V. dahlia.

Key words: cotton, Dirigent-like protein gene, Verticillium dahliae, expression differences