通过GbF3'H基因单独沉默及其与GbCHI和GbDFR基因共沉默研究其在海岛棉中抗枯萎病功能

doi:10.11963/1002-7807.lyxcqj.20200109

摘要/Abstract

摘要：

【目的】通过沉默海岛棉GbF3'H基因及共沉默GbF3'H、GbCHI和GbDFR基因,研究其在海岛棉抗枯萎病中的作用。【方法】以海岛棉抗病材料06-146为研究对象,GhCLA1基因为阳性对照,空载体为阴性对照,构建海岛棉TRV₂-GbF3'H沉默载体,协同课题组前期构建的TRV₂-CHI和TRV₂-DFR载体,利用病毒诱导的基因沉默技术（Virus-induced gene silencing,VIGS）分别进行GbF3'H基因单独沉默以及GbF3'H、GbCHI和GbDFR这3种基因共沉默试验。通过实时荧光定量聚合酶链式反应（Quantitative real time-polymerase chain reaction,qRT-PCR）分析各处理样品中基因沉默情况;设置室内接种枯萎病菌试验测定病情指数,分析各沉默材料对枯萎病的抗性差异。【结果】qRT-PCR结果显示,海岛棉GbF3'H基因沉默后其在海岛棉根、茎和叶中的表达量比空载体对照低,GbF3'H、GbCHI和GbDFR这3种基因共沉默后其在海岛棉根、茎和叶中的表达量均比空载体对照低。病情指数调查结果显示,野生型<空载体对照<GbF3'H单基因沉默组<3种基因共沉默组,说明共沉默材料对枯萎病的抗性明显低于单基因沉默材料,单基因沉默材料明显低于空载体对照和野生型。【结论】初步确定GbF3'H基因对提高海岛棉枯萎病抗性有一定作用,且可与GbCHI、GbDFR基因协同作用增强抗病性,这可为海岛棉功能基因组学研究提供参考。

关键词: 海岛棉; GbF3'H; 枯萎病抗性; 病毒诱导的基因沉默（VIGS）; 共沉默; 实时荧光定量聚合酶链式反应

Abstract:

[Objective] The aim of this study is to study the effect of silencing GbF3'H gene and co-silencing GbF3'H, GbCHI and GbDFR genes on resistance of Gossypium barbadense to Fusarium wilt. [Method] A disease resistant material 06-146 of G. barbadense was taken as the research objective, with GhCLA1 gene as the positive control, and the empty vector (TRV::00) as the negative control. The G. barbadense TRV₂-GbF3'H silencing vector was constructed, and the TRV₂-CHI and TRV₂-DFR vectors were constructed in cooperation with the research group in the early stage. The GbF3'H gene was silenced separately and co-silenced with the GbCHI and GbDFR by using the virus-induced gene silencing technology. The gene silencing status in each treatment samples were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). The differences of resistance of each treatment materials to Fusarium wilt were analyzed by investigating the disease index after Fusarium oxysporium inoculation. [Result] The results of qRT-PCR showed that the expression level of GbF3'H gene in the roots, stems and leaves of G. barbadense after its silencing was lower than that of the empty vector control, and the expression level of GbF3'H, GbCHI and GbDFR genes in the roots, stems and leaves of G. barbadense after co-silencing was lower than that of the empty vector control. The disease index investigation showed wild type<TRV::00<GbF3'H single gene silencing group<three genes co-silencing group, which indicates the resistance of co-silencing materials to Fusarium wilt was significantly lower than that of single gene silencing materials, and the resistance of single gene silencing materials was significantly lower than those of the empty vector control and wild type. [Conclusion] The results showed that GbF3'H gene had a certain effect on the resistance of G. barbadense to Fusarium wilt, moreover, synergistic effect with GbCHI and GbDFR genes to enhance disease resistance. These results provide a reference for functional genomics of study G. barbadense.

Key words: Gossypium barbadense; GbF3'H; Fusarium wilt resistance; virus-induced gene silencing (VIGS); co-silencing; quantitative real time-polymerase chain reaction (qRT-PCR)

李玉霞,曲延英,艾海提·艾合买提,王慧敏,黄启秀,陈琴,陈全家. 通过GbF3'H基因单独沉默及其与GbCHI和GbDFR基因共沉默研究其在海岛棉中抗枯萎病功能[J]. 棉花学报, 2020, 32(1): 1-10.

Li Yuxia,Qu Yanying,Aihaiti Aihemaiti,Wang Huimin,Huang Qixiu,Chen Qin,Chen Quanjia. Through Single Silencing GbF3'H Gene and Its Co-Silencing with GbCHI and GbDFR Genes to Study Their Function in Resistance to Fusarium Wilt in Gossypium barbadense[J]. Cotton Science, 2020, 32(1): 1-10.

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基因 Gene	引物序列 Primer sequence (5'-3')	用途 Purpose
GbF3'H	F: GCTCTAGATCGGGATTTGGTAGCCGCAGCC	构建GbF3'H基因沉默载体所用引物（下划线为酶切位点）
	R: GGGGTACCGCATGTGGAGGTTACTCAGTTT	Primer for construction of GbF3'H gene silencing vector
GbF3'H	F: GTTATTTCATCCGTCGTCTTC	GbF3'H实时荧光定量聚合酶链式反应所用引物
	R: AGGTTCCCATTTTGAGGTAC	GbF3'H expression analysis by qRT-PCR
GhUBQ7	F: GACCTACACCAAGCCCAAGAAG	内标对照
	R: TGAGCCCACACTTACCACAATAGT	Reference gene as control
TRV₂	F: GACATTGTTACTCAAGGAAGCAC	空载体
	R: AACCTAAAACTTCAGACACGGAT	Control vector
GbCHI	F: GGAAATCCAAGGGCAGTT	GbCHI实时荧光定量聚合酶链式反应所用引物
	R: TCTCAAAATCACCTGTAACGAC	GbCHI expression analysis by qRT-PCR
GbDFR	F: GACAATGGCAGAGCAAGC	GbDFR实时荧光定量聚合酶链式反应所用引物
	R: GCAGTTATGAGGCTTGGC	GbDFR expression analysis by qRT-PCR