棉花<i>GhMYB113</i>基因克隆及在黄萎病抗性中的功能分析

doi:10.7668/hbnxb.20196180

摘要/Abstract

摘要：

以棉花GhMYB113基因为对象,旨在探究其在棉花黄萎病抗性中的功能。GhMYB113编码249个氨基酸,分子质量29.07 ku,等电点6.46。通过系统进化和亚细胞定位分析,发现其与大豆GmMYB113同源性最高,且定位在细胞核。接着利用实时荧光定量PCR(qPCR)分析其表达模式,发现根组织中表达量最高,且受大丽轮枝菌、水杨酸(SA)和茉莉酸(JA)诱导表达升高,暗示其参与抗病反应。通过VIGS技术沉默GhMYB113后,发现植株叶片花青素累积量显著减少,花青素合成途径基因表达降低,表明GhMYB113正调控棉花花青素合成。进一步通过GUS酶报告系统,验证其可激活GhDFR启动子。最后对沉默植株接菌分析,发现其病害症状严重,病情指数值高,病株率增加,茎组织内部褐化现象严重,真菌生物量显著高于对照植株,证实GhMYB113正调控棉花黄萎病抗性。综上所述,GhMYB113在棉花花青素合成及抗黄萎病方面发挥重要作用,为棉花抗病育种提供了理论依据。

关键词: 棉花, MYB113, 黄萎病, 大丽轮枝菌, 抗性, 花青素

Abstract:

This study investigated the function of the cotton gene GhMYB113 in resistance to Verticillium wilt.GhMYB113 encoded a 249 amino acid protein with a molecular weight of 29.07 ku and an isoelectric point of 6.46.Phylogenetic and subcellular localization analyses revealed that GhMYB113 shared the highest homology with soybean GmMYB113 and was localized to the nucleus.Expression profiling by qPCR showed that GhMYB113 was predominantly expressed in roots and was upregulated upon infection by Verticillium dahliae as well as by treatment with jasmonic acid(JA)and salicylic acid(SA),suggesting a role in disease resistance signaling.Silencing of GhMYB113 via virus-induced gene silencing(VIGS)led to a significant decrease in leaf anthocyanin accumulation and downregulated the expression of key anthocyanin biosynthesis genes,indicating that GhMYB113 positively regulates anthocyanin synthesis in cotton.This was further confirmed using a GUS reporter system,which demonstrated that GhMYB113 activates the promoter of GhDFR.Inoculation assays performed on GhMYB113-silenced plants resulted in more severe disease symptoms,higher disease index(DI)values,increased disease incidence,enhanced stem browning,and significantly greater fungal biomass in stems compared with control plants,demonstrating that GhMYB113 positively regulates cotton resistance to Verticillium wilt.In summary,GhMYB113 plays a critical role in both anthocyanin biosynthesis and Verticillium wilt resistance in cotton,providing a theoretical foundation for breeding disease-resistant cotton varieties.

Key words: Cotton, MYB113, Verticillium dahliae, Verticillium wilt, Defense, Anthocyanin

中图分类号:

钟贵买, 廖婵娟, 李丹, 吴琪, 胡广. 棉花GhMYB113基因克隆及在黄萎病抗性中的功能分析[J]. 华北农学报, 2026, 41(4): 215-221. doi: 10.7668/hbnxb.20196180.

ZHONG Guimai, LIAO Chanjuan, LI Dan, WU Qi, HU Guang. Cloning and Functional Analysis of the Cotton GhMYB113 Gene in Verticillium Wilt Resistance[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 215-221. doi: 10.7668/hbnxb.20196180.

http://www.hbnxb.net/CN/Y2026/V41/I4/215

图/表 6

表1 本研究所用引物序列

Tab.1 The primer sequences used in this study

引物 Primer	序列(5'—3') Sequence (5'—3')	用途 Employ
MYB113-GFP-F	CGAGATCTGATGGAGGGCTCATCTTTAAGAG	PCR
MYB113-GFP-R	CGCACTAGTTGGGTTAAACACATTCCACAG	PCR
qMYB113-F	TTGAGAAATATGGTGAAGGGAA	qPCR
qMYB113-R	GGAGGTTATGGAGGCGAATA	qPCR
GhUBI3-F	GAAGGCATTCCACCTGACCAAC	qPCR
GhUBI3-R	CTTGACCTTCTTCTTCTTGTGCTTG	qPCR
GhCHI-F	TGATGCAGAAGCAAAAGCCAT	qPCR
GhCHI-R	AGCTCAGTACCAGAAGGTGGGA	qPCR
GhF3H-F	GGGCCTAGCTTGCAAGCTTCTT	qPCR
GhF3H-R	AAGCAAGAGTGTGATGGTGCCTG	qPCR
GhDFR-F	GACCCTGGCAACTCGAAGAA	qPCR
GhDFR-R	GGACTCGAAGTCCATAGGCG	qPCR
GhANS-F	CCGCAGCTGTGTCTATCAACAC	qPCR
GhANS-R	AGCAAATTTCCAAGCTGCCTT	qPCR
GhANR-F	GAGGCCTAGCGAGCAAAATAC	qPCR
GhANR-R	GTGAGCTTCGACACCGAGAG	qPCR
GhUFGT-F	TCTTCTCCGCATCCCAACTA	qPCR
GhUFGT-R	TCCATTGCCTTCCTCAGATT	qPCR
vMYB113-F	CGGAATTCTGGAGGGCTCATCTTTAAG	VIGS
vMYB113-R	CGGGATCCTGAACTGCCACTTGCACTGATAC	VIGS
VdTUB1-F	AACAACAGTCCGATGGATAATTC	qPCR
VdTUB1-R	GTACCGGGCTCGAGATCG	qPCR
GhACT7-F	TTGCAGACCGTATGAGCAAG	qPCR
GhACT7-R	ATCCTCCGATCCAGACACTG	qPCR
DFRpro-F	CGAAGCTTGATTGTTCATACAGGGACATGACCGAC	PCR
DFRpro-R	CCGGGATCCTGGGAAATAACTTGAACACTCTGCTC	PCR

图1 GhMYB113的系统进化和亚细胞定位 A.棉花MYB转录因子的系统进化分析;B.GhMYB113与其他植物的MYB113的系统进化分析;C.亚细胞定位载体构建;D.在H2B-RFP转基因烟草叶片中观察GhMYB113-GFP融合蛋白瞬时表达荧光。细胞核定位标记使用H2B-RFP转基因本氏烟。比例尺.30 μm。

Fig.1 Phylogenetic analysis and subcellular localization of GhMYB113 A.Phylogenetic analysis of cotton MYB transcription factors;B.Phylogenetic analysis of GhMYB113 alongside MYB113 from various other plant species;C.Construction of the vector for subcellular localization;D.Fluorescence observation of GhMYB113-GFP fusion.H2B-RFP transgenic N.benthamiana plants were used as a nucleus marker.Scale bars.30 μm.

图2 GhMYB113的表达谱不同小写字母表示显著差异(P<0.05)基于Tukey测试。图3—5同。

Fig.2 GhMYB113 expression profiles Different lowercase letters denoted significant differences(P<0.05),based on the Tukey HSD test.The same as Fig.3—5.

图3 GhMYB113调控花青素合成 A.GhMYB113-TRV沉默载体构建;B.GhPDS基因沉默植株代表图;C.沉默植株中GhMYB113的相对表达量;D、E.GhMYB113沉默和对照植株中的花青素积累量;F.花青素合成途径基因表达水平。

Fig.3 GhMYB113 regulates anthocyanin synthesis A.Construction of the GhMYB113-TRV silencing vector;B.Representative diagram of TRV:PDS plants;C.Relative expression of GhMYB113 in silenced plants;D,E.Anthocyanin accumulation in GhMYB113-silenced and control plants;F.The expression levels of anthocyanin synthesis pathway genes.

图4 GhMYB113促进GhDFR转录表达 A.效应子和报告子载体构建示意图;B.本氏烟叶GUS染色;C.本氏烟叶酶活性测定。

Fig.4 GhMYB113 promotes GhDFR transcriptional expression A.Diagram illustrating the construction of effector and reporter vectors;B.GUS staining in tobacco leaves;C.Enzyme activity assays in tobacco leaves.

图5 GhMYB113沉默植株对黄萎病的抗性分析 A.GhMYB113沉默和对照植株的病症代表图;B、C.GhMYB113沉默和对照植株的病害指数(DI)及病株率;D.棉花茎纵向剖面代表图;E.真菌恢复培养;F.通过大丽轮枝菌的TUB1基因与棉花ACT7基因的DNA比值定量真菌生物量。

Fig.5 Resistance analysis of GhMYB113-silenced plants to Verticillium wilt A.Representative images of disease symptoms on GhMYB113-silenced and control plants inoculated with V.dahliae;B,C.Disease index(DI)and the rate of diseased plants of GhMYB113-silencing and control plants;D.Representative images of the longitudinal sections of cotton stems infested with V.dahliae;E.Fungal recovery assay;F.Fungal biomass was quantified as the ratio of V.dahlia TUB1 gene to cotton ACT7 gene DNA levels.

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棉花GhMYB113基因克隆及在黄萎病抗性中的功能分析

Cloning and Functional Analysis of the Cotton GhMYB113 Gene in Verticillium Wilt Resistance

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Cloning and Functional Analysis of the Cotton GhMYB113 Gene in Verticillium Wilt Resistance

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