棉花学报 ›› 2020, Vol. 32 ›› Issue (1): 1-10.doi: 10.11963/1002-7807.lyxcqj.20200109
• 研究与进展 • 下一篇
李玉霞(),曲延英,艾海提·艾合买提,王慧敏,黄启秀,陈琴,陈全家(
)
收稿日期:
2019-08-10
出版日期:
2020-01-15
发布日期:
2020-03-25
通讯作者:
陈全家
E-mail:751232545@qq.com;chqjia@126.com
作者简介:
李玉霞(1993―),女,硕士, 基金资助:
Li Yuxia(),Qu Yanying,Aihaiti Aihemaiti,Wang Huimin,Huang Qixiu,Chen Qin,Chen Quanjia(
)
Received:
2019-08-10
Online:
2020-01-15
Published:
2020-03-25
Contact:
Chen Quanjia
E-mail:751232545@qq.com;chqjia@126.com
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。
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.
表1
本研究所用引物"
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 |
图1
GbF3'H基因沉默序列的克隆及VIGS载体构建 A. PCR扩增395 bp的GbF3'H片段。M:5 kbp DNA marker,1~2:395 bp GbF3'H片段。B. pTRV2-GbF3'H重组质粒双酶切验证。M:10 kbp DNA marker,1~2:pTRV2-GbF3'H重组质粒。C. 质粒和菌液PCR验证。M:2 kbp DNA marker,1:pTRV2空载体质粒用TRV2通用引物扩增片段,2:pTRV2空载体GV3101菌液用TRV2通用引物扩增片段,3~4:pTRV2-GbF3'H质粒阳性对照用TRV2通用引物扩增片段,5~6:pTRV2-GbF3'H GV3101菌液用TRV2通用引物扩增片段。D. GbF3'H基因沉默载体示意图。"
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