棉花抗枯萎病基因<i>GhERF14</i>的功能分析

doi:10.7668/hbnxb.20195360

摘要/Abstract

摘要：

探究棉花GhERF14基因与尖孢镰刀菌致病力的关系,解析尖孢镰刀菌致病分子机制,初步探究棉花GhERF14基因对枯萎病的响应及对相关抗病基因的调控作用,为培育抗枯萎病的棉花新品种提供一定的理论依据。利用基因克隆、病毒诱导基因沉默(VIGS)构建了GhERF14 基因非保守结构域干扰载体pTRV2-GhERF14,通过实时荧光定量(qRT-PCR)技术和VIGS技术,研究枯萎病菌胁迫和激素处理后,GhERF14和下游与木质素(Lignin)、乙烯(ET)、茉莉酸(JA)、水杨酸(SA)相关基因,抗氧化酶基因及病程相关蛋白(PR)基因的表达特征,分析其在棉花抗病过程中的作用,表明抑制GhERF14基因的表达可显著降低茉莉酸、水杨酸以及乙烯合成及信号通路相关基因的表达。利用VIGS 技术将 GhERF14 基因沉默后,棉株对枯萎病菌更敏感,表明GhERF14在尖孢镰刀菌致病及宿主-病原体互作过程中可能发挥着重要的作用。

关键词: 棉花, GhERF14, 枯萎病, VIGS, 尖孢镰刀菌

Abstract:

This study explored the relationship between cotton GhERF14 gene and Fusarium oxysporum pathogenicity,analyzed the molecular mechanism of F.oxysporum pathogenicity,and tentatively explored the response of cotton GhERF14 gene to Fusarium wilt disease and its regulatory effect on related resistance genes,to provides some theoretical basis for breeding new cotton cultivars resistant to wilt.Gene cloning and virus-induced gene silencing(VIGS)were used to construct the non-conserved domain interference vector pTRV2-GhERF14.Using Real-time fluorescence quantification(qRT-PCR)technology and VIGS technology,the expression characteristics of GhERF14 and downstream genes related to lignin,ethylene(ET),jasmonic acid(JA),salicylic acid(SA),antioxidant enzymes and disease progression-related protein(PR)were analyzed after F.oxysporum stress and hormone treatment,and the role of GhERF14 in the process of cotton disease resistance was analyzed.The results indicated that inhibition of GhERF14 gene expression could significantly reduce the synthesis of jasmonic acid(JA),salicylic acid(SA)and ethylene(ET)and the expression of genes related to the signaling pathway.After GhERF14 gene silencing by VIGS technology,cotton plants were more susceptible to Fusarium wilt.These results suggested that GhERF14 may play an important role in the pathogenesis and host-pathogen interaction of F.oxysporum.

Key words: Cotton, GhERF14, Blight, VIGS, Fusarium oxysporum

中图分类号:

赵龙飞, 张建华, 赵曾强, 蔡树东, 张薇. 棉花抗枯萎病基因GhERF14的功能分析[J]. 华北农学报, 2025, 40(3): 133-140. doi: 10.7668/hbnxb.20195360.

ZHAO Longfei, ZHANG Jianhua, ZHAO Zengqiang, CAI Shudong, ZHANG Wei. Functional Analysis of Fusarium wilt Resistance Gene GhERF14 in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 133-140. doi: 10.7668/hbnxb.20195360.

http://www.hbnxb.net/CN/Y2025/V40/I3/133

图/表 7

表1 实时荧光定量引物

Tab.1 qRT-PCR primer

基因名称 Gene name	正向引物序列(5'—3') Forward primer sequence(5'—3')	反向引物序列(5'—3') Reverse primer sequence(5'—3')
GhPR1	TTTAGTAAGGTTTTTGACCGACGAA	GACTCTGTCCATCTTGGTTGTGCTA
GhPR2	TTCAAGGTTTGCACTCGGAAGA	CCACCAGCAGCAGAAGTTATCG
GhPR3	GATGACTCCACAATCACCGAAGC	GCGGTCTTCTACCTGGGCATT)
GhPR5	TTGGCTCTTACTTCCGACCATCT	GCCGTGATTCATACAGTTATCCTCA
GhICS1	AAGAATGCCAGAGGTAAGAGGAGGA	ATGGATGAATGGGTGCGAAGG
GhEDS1	GCAGCAACAGCTCCTCTACCTCAA	GGCAGACCAAGACGCTACAGATACA
GhNPR1	CACGTGGTGCTGTTGTTGTTACTG	GCGAATCGTTGCTTTCTTCTTCA
GhPAD4	GGATGGAAGAATGGAAAGAAATGAA	GAACTAGGAAAGCAGACTAAGGAACCA
GhWRKY70	TGGCAACATACGAAGGACAACACA	CTTCGCTCGTCCCGGAAAATATCGC
GhPDF1.2	AAAATCGACTGAAATGGCTA	TGTCGGTTACAAAGTAAATTGC
GhJAZ1	CTTTGGTTCCACTGCTGCTGATT	GACCAAATCTTGTGGCATCTACCTC
GhAOS	CGATGTCCCAACTTCAATCTCAAAC	ATCCGACGGTGGAGAATAAACAGT
GhAOC4	CAAAAATGCCAGACCCACCAGTA	CAAAAATGCCAGACCCACCAGTA
GhEIN2	CCCGAGCTGTGGGGAAAATACACT	GGGATTTGAAGGCAGAAGCAGGAT
GhEIN3	CCACCAATCTTCGTTCCCGGTA	AAACGGAAACCCATGTAGCAG
GhACO	TGACCTCAAGTTCCAAGCCAA	AGAATTTATCCATCAGCCGACT
GhPAL	TGGTGGCTGAGTTTAGGAAA	TGAGTGAGGCAATGTGTGA
GhC4H1	CCGAACCCGACACCCATAAGC	GCAGGGATGTCATACCCACCAAG
GhERF1	CTTTAGATCACAACTCGCTTC	TTTTCTTTCGTCGGGCTT
GhF5H2	GGCAGATGCGAAAGATTTGTGTA	ACATCCATCATTTCTTGCTGCTC
Gh4CL	ATTCAAAAGGGAGATGCC	GAGAAGGGCAAAGCAACA
GhCCR1	GCACTGTAAGGAACCCAGACGAT	CATTTGTTCAGGATCATCAGTCACA
GhCCoAOMT	TACGACAACACGCTGTGGAATGGG	CATCGCCGACAGGAAACATACAGA
GhSOD	CATCTCACGCACTCTGTC	CCTTAGCCATTTCTGTCTGTG
GhPOD	ATATCCTTGTTCTGTCTGCTA	CTCCTTCTACCGTCTCTTC
GhCAT	TGATAAGTTGCTCCAGACTCG	CTTCGTGGTGATTGTTGTGA
GhUBQ7	GAAGACCTACACCAAGCCCAA	CGGACTCTACTCAATCCCCACC
qPCRERF14	GGAGTATCCAATGGGTAGCGGC	CCATCGACGACGACGAAGAAGA

图1 干扰片段GhERF14的扩增(A)和干扰载体双酶切 (Kpn Ⅰ和BamH Ⅰ)的验证(B) A:M.DL2000,1.阴性对照,2—7.GhERF14的PCR扩增;B:1.阴性对照,2.pTRV2-GhERF14酶切。

Fig.1 Amplification of interference fragment GhERF14(A)and interference carrier double enzyme digestion(Kpn Ⅰ and BamH Ⅰ)(B) A:M.DL2000,1.Negative control,2—7.PCR amplification of GhERF14;B:1.Negative control,2.pTRV2-GhERF14 enzyme digestion.

图2 GhERF14基因沉默效率检测 A.注射pTRV2-GhCLA1 14 d后植株的表型;B.GhERF14在沉默株系和对照中的表达水平。pTRV2-00.对照组植株;pTRV2-GhERF14.沉默植株。不同小写字母代表0.05水平上差异显著。图3同。

Fig.2 Detection of GhERF14 gene silencing efficiency A.Phenotype of pTRV2-GhCLA1 plants after 14 days;B.The expression levels of GhERF14 in silent lines and controls.pTRV2-00.Control group plants;pTRV2-GhERF14.Silent plants.Different lowercase letters indicate significant differences at 0.05 level.The same as Fig.3.

图3 病情指数统计及枯萎病菌恢复培养 A.接菌12 d的表型;B.接菌24 d的表型;C.病原菌的恢复培养;D.病情指数统计。

Fig.3 Statistics of disease index and recovery culture of Fusarium wilt pathogens A.Phenotype after 12 days of inoculation;B.Phenotype after 24 days of inoculation;C.Recovery culture of pathogenic bacteria;D.Disease index statistics.

图4 不同株系中防卫基因的表达分析以转化空载植株(pTRV2-00)为对照组,以沉默植株(pTRV2-GhERF14)为试验组,分别比较抗病相关基因表达情况。不同小写字母代表对照组与试验组在0.05水平上差异显著。图5同。

Fig.4 Expression analysis of defense genes in different lines The expression of disease resistance related genes was compared using plants transformed with an empty vetor(pTRV2-00)as the control group and silenced plants(pTRV2-GhERF14)as the experimental group.Different lowercase letters indicate that the control and experimental group are significant differences at 0.05 level.The same as Fig.5.

图5 GhERF14基因沉默对棉花植株生理生化的影响

Fig.5 The effect of GhERF14 gene silencing on the physiology and biochemistry of cotton plants

图6 台盼蓝组织化学染色前后的叶片表型 A.沉默植株和对照组植株接菌的表型;B.台盼蓝染色后沉默植株和对照组的表型。

Fig.6 The phenotype of leaf before and after histochemical staining of trypan blue A.The phenotypes of silenced plants and control plants after Fusarium oxysporum treatment;B.The phenotypes of silenced plants and controls after trypan blue staining.

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棉花抗枯萎病基因GhERF14的功能分析

Functional Analysis of Fusarium wilt Resistance Gene GhERF14 in Cotton

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