黄萎病菌侵染下陆地棉Dirigent-like蛋白基因表达差异分析

doi:10.3864/j.issn.0578-1752.2014.22.001

Abstract

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

GUO Bao-sheng, SHI Gong-yao, WANG Kai-hui, LIU Su-en, ZHAO Cun-peng, WANG Zhao-xiao, GENG Jun-yi, HUA Jin-ping. Expression Differences of Dirigent-Like Protein Genes in Upland Cotton Responsed to Infection by Verticillium dahliae[J].Scientia Agricultura Sinica, 2014, 47(22): 4349-4359.

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

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