生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 127-134.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1519
李秀青1(), 胡子曜2, 雷建峰1, 代培红2, 刘超2, 邓嘉辉2, 刘敏2, 孙玲1, 刘晓东2, 李月2()
收稿日期:
2021-12-07
出版日期:
2022-08-26
发布日期:
2022-09-14
作者简介:
李秀青,男,硕士,研究方向:作物逆境分子生物学;E-mail: 基金资助:
LI Xiu-qing1(), HU Zi-yao2, LEI Jian-feng1, DAI Pei-hong2, LIU Chao2, DENG Jia-hui2, LIU Min2, SUN Ling1, LIU Xiao-dong2, LI Yue2()
Received:
2021-12-07
Published:
2022-08-26
Online:
2022-09-14
摘要:
初步探究陆地棉TIFY家族基因GhTIFY9在棉花黄萎病反应中的功能,为挖掘棉花抗病相关基因及棉花抗病育种奠定基础。通过转录组筛选、克隆获得一个TIFY家族基因GhTIFY9,利用生物信息学的方法分析该基因的理化性质,通过荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)方法分析GhTIFY9在黄萎病诱导下的表达模式。同时构建该基因的VIGS载体,利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术对其在棉花抗黄萎病中的功能进行了初步探究。结果表明,以陆地棉TM-1的cDNA为模板克隆获得GhTIFY9,其开放阅读框(ORF)为594 bp,编码一个含197个氨基酸的碱性亲水性蛋白,相对分子质量为21.65 kD,定位于细胞核,属于TIFY亚家族。RT-qPCR分析结果表明GhTIFY9响应黄萎病菌诱导,抑制该基因表达后沉默植株对黄萎病菌的敏感性显著增强。GhTIFY9是棉花抗黄萎病反应的一个正向调控因子。
李秀青, 胡子曜, 雷建峰, 代培红, 刘超, 邓嘉辉, 刘敏, 孙玲, 刘晓东, 李月. 棉花黄萎病抗性相关基因GhTIFY9的克隆与功能分析[J]. 生物技术通报, 2022, 38(8): 127-134.
LI Xiu-qing, HU Zi-yao, LEI Jian-feng, DAI Pei-hong, LIU Chao, DENG Jia-hui, LIU Min, SUN Ling, LIU Xiao-dong, LI Yue. Cloning and Functional Analysis of Gene GhTIFY9 Related to Cotton Verticillium Wilt Resistance[J]. Biotechnology Bulletin, 2022, 38(8): 127-134.
基因 Gene | 开放阅读框ORF/bp | 氨基酸 Amino acid | 相对分子质量Relative molecular weight/kD | 理论等电点Theoretical isoelectric point | 平均疏水性Average hydrophobicity | 信号肽 Signal peptide | 亚细胞定位 Subcellular localization | 脂肪系数Fat coefficient | 跨膜结构域 Transmembrane domain |
---|---|---|---|---|---|---|---|---|---|
GhTIFY9 | 594 | 197 | 21.65 | 9.08 | -0.477 | 无 None | 细胞核 Nucleus | 76.63 | 无 None |
表1 GhTIFY9的生物信息学分析
Table 1 Bioinformatics analysis of GhTIFY9
基因 Gene | 开放阅读框ORF/bp | 氨基酸 Amino acid | 相对分子质量Relative molecular weight/kD | 理论等电点Theoretical isoelectric point | 平均疏水性Average hydrophobicity | 信号肽 Signal peptide | 亚细胞定位 Subcellular localization | 脂肪系数Fat coefficient | 跨膜结构域 Transmembrane domain |
---|---|---|---|---|---|---|---|---|---|
GhTIFY9 | 594 | 197 | 21.65 | 9.08 | -0.477 | 无 None | 细胞核 Nucleus | 76.63 | 无 None |
图3 黄萎病诱导下GhTIFY9的表达模式分析 *:在0.05水平上存在显著差异,**:在0.01水平上存在显著差异。下同
Fig.3 Analysis of the expression patterns of GhTIFY9 under the treatment of Verticillium dahliae *:Significant difference at the 0.05 level. **:Significant difference at the 0.01 level. The same as below
图4 GhTIFY9的VIGS载体构建 A:GhTIFY9目标片段的PCR扩增;B:TRV﹕GhTIFY9 VIGS载体的酶切鉴定。M:Trans 2K Plus II DNA marker
Fig. 4 VIGS vector construction of GhTIFY9 A:PCR amplification of target fragment of GhTIFY9. B:Identification of TRV﹕GhTIFY9 vectors by restriction enzyme digestion. M:Trans 2K Plus II DNA marker
图5 GhTIFY9的沉默效率检测 A:在棉花中沉默GhCLA1的表型;B:GhCLA1的沉默效率检测;C:GhTIFY9的沉默效率检测
Fig. 5 Silencing efficiency detection of GhTIFY9 gene A:The phenotype of silenced GhCLA1 in cotton. B:Silencing efficiency detection of GhCLA1. C:Silencing efficiency detection of GhTIFY9
图6 GhTIFY9沉默植株的抗病性鉴定 A:GhTIFY9沉默后植株的表型分析;B:病情指数统计;C:剖杆鉴定;D:病原菌恢复培养
Fig. 6 Disease resistance identification of GhTIFY9 gene-silenced plants A:Phenotype analysis of plants after GhTIFY9 gene silencing. B:Disease index statistics. C:Identification by stem anatomy. D:Restoration culture of pathogenic bacteria
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