陆地棉GhNAC1基因的克隆及抗黄萎病功能分析

doi:10.13304/j.nykjdb.2022.0280

摘要/Abstract

摘要：

黄萎病是造成棉花品质与产量下降的重要原因之一，探索棉花抗病机制对推动生态农业的可持续发展具有深远意义。在黄萎病菌胁迫的陆地棉ND601根组织全长cDNA文库中，筛选到1个与黄萎病胁迫相关的植物特异NAC转录因子基因，将其命名为GhNAC1。该基因cDNA序列全长1 213 bp，开放阅读框840 bp，编码279个氨基酸。GhNAC1没有信号肽和跨膜结构，定位在细胞核。qRT-PCR分析结果表明，GhNAC1受黄萎病菌胁迫后在根部特异表达，显著上调，表达量在抗性品种中显著高于感病品种。GhNAC1沉默后棉花对黄萎病抗性降低，水杨酸路径标志基因（PAD4、NDR1、NPR1和PR1）表达量降低，以上结果表明GhNAC1可能通过调控水杨酸信号通道参与棉花黄萎病抗性。

关键词: 棉花, NAC转录因子, 黄萎病抗性, 病毒诱导的基因沉默, 基因功能

Abstract:

Verticillium wilt is one of the important reasons for cotton quality and yield decline.It is of great significance to explore the resistance mechanism of cotton for promoting the sustainable development of ecological agriculture. A plant-specific NAC transcription factor gene related to Verticillium wilt resistance was identified and named as GhNAC1 in the full-length cDNA library of upland cotton ND601 root stressed by Verticillium dahliae. The cDNA sequence was 1 213 bp with an open reading frame of 840 bp， encoding 279 amino acids. GhNAC1 had no signal peptide and transmembrane structure， and was located in the nucleus. qRT-PCR analysis showed that GhNAC1 was specifically and significantly upregulated in root under V. dahliae， and the expression level in resistant cultivars was observably higher than in susceptible cultivars. Silencing GhNAC1 decreased cotton resistance to Verticillium wilt， and the expression levels of salicylic acid pathway marker genes （PAD4， NDR1， NPR1 and PR1） weakened. These results suggested that GhNAC1 might positively regulated Verticillium wilt resistance in cotton via salicylic acid signal pathway.

Key words: cotton, NAC transcription factor, Verticillium wilt resistance, VIGS, gene function

中图分类号:

S562

张曼, 张进, 张新雨, 王国宁, 王省芬, 张艳. 陆地棉GhNAC1基因的克隆及抗黄萎病功能分析[J]. 中国农业科技导报, 2023, 25(10): 35-44.

Man ZHANG, Jin ZHANG, Xinyu ZHANG, Guoning WANG, Xingfen WANG, Yan ZHANG. Cloning and Functional Analysis of GhNAC1 in Upland Cotton Involved in Verticillium Wilt Resistance[J]. Journal of Agricultural Science and Technology, 2023, 25(10): 35-44.

图/表 6

表1 试验所用引物序列

Table 1 Primers used in this experiment

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	用途 Utilization
NAC1-F	ATGAGCTACCAATCAAACC	基因克隆Gene cloning
NAC1-R	TTAAAAGTTGAGGATATTAGC	基因克隆Gene cloning
NAC1-RT-F	GAACACATCTCTTCCTTCATCATCTT	实时定量PCR Real-time PCR
NAC1-RT-R	AGTTGTCCCATATTTTCATTGCCTA	实时定量PCR Real-time PCR
NAC1-V-F	GAATTCGGTTGAACTTCCTGGCTTTA	载体构建Vector construction
NAC1-V-R	GGTACCGCAAAGTAGCATCAGGGAG	载体构建Vector construction
NAC1-G-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGA GCTACCAATCAAACC	载体构建Vector construction
NAC1-G-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGAAAGTT GAGGATATTAGC	载体构建Vector construction
GhUBQ14-F	CAACGCTCCATCTTGTCCTT	实时定量PCR Real-time PCR
GhUBQ14-R	TAGTCGTCTTTCCCGTAAGC	实时定量PCR Real-time PCR
GhNDR1-F	CCCGTAACCAAGGAGGCTGT	实时定量PCR Real-time PCR
GhNDR1-R	CTGCTAAGGGAAGGCAAGGATAG	实时定量PCR Real-time PCR
GhNPR1-F	GTCTGGCTGATGTCAATCTGCG	实时定量PCR Real-time PCR
GhNPR1-R	TCCTTCCCTTGCTCTGTCTTGG	实时定量PCR Real-time PCR
GhPR1-F	GGCACAGAACTACGCTAATCAACG	实时定量PCR Real-time PCR
GhPR1-R	GCTTTACCCTCTCACTAACCCACAT	实时定量PCR Real-time PCR
GhPAD4-F	GGATGGAAGAATGGAAAGAAATGAA	实时定量PCR Real-time PCR
GhPAD4-R	GAACTAGGAAAGCAGACTAAGGAACCA	实时定量PCR Real-time PCR

图2 GhNAC1蛋白结构分析A：GhNAC1信号肽预测结果；B：GhNAC1跨膜域预测结果；C：GhNAC1蛋白二级结构预测

Fig. 2 Structure analysis of GhNAC1 proteinA： Prediction result of GhNAC1 signal peptide； B： Prediction result of GhNAC1 transmembrane domain； C： Prediction of secondary structure of GhNAC1 protein

图2 GhNAC1亚细胞定位

Fig. 2 Subcellular localization of GhNAC1

图3 GhNAC1的表达模式A：农大601不同组织器官中GhNAC1的表达；B：黄萎病菌胁迫下GhNAC1在ND601根部的表达；C：黄萎病菌胁迫下GhNAC1在12个不同抗性品种根部的表达及病情指数；*和**分别表示与0 h相比在P < 0.05和P < 0.01水平差异显著

Fig. 3 Expression pattern of GhNAC1A： Expression analysis of GhNAC1 in different tissues and organs of ND601； B： Expression analysis of GhNAC1 in ND601 roots under Verticillium wilt stress； C： Expression analysis of GhNAC1 in roots of 12 different resistant cultivars under Verticillium wilt stress； * and ** indicate significant differences compared with 0 h at P < 0.05 and P < 0.01 levels， respectively

图4 沉默GhNAC1降低了棉花对大丽轮枝菌的抗性A：棉花CLA1基因沉默后的叶片白化现象作为阳性对照；B：侵染7 d后野生型对照和沉默植株GhNAC1表达水平；C：20 dpi对照植株与沉默植株病害表现；D：病情指数；E：茎部病菌侵染表型。CK表示对照组，VIGS表示沉默组；**表示与CK相比在 P < 0.01水平差异显著

Fig. 4 Silencing GhNAC1 reduced the resistance to V. dahliae in cottonA： CLA1 gene was used as a positive control with an albino phenotype on leave after VIGS in cotton； B： Relative expression levels of GhNAC1 in WT and GhNAC1-silenced plants 7 d after hand-infiltration； C： Disease manifestations of control plants and silent plants at 20 dpi； D： Disease index； E： Phenotype of stem infected by V. dahliae. CK stands for control group， VIGS stands for silent group； ** indicates significant difference compared with CK at P < 0.01 level

图5 GhNAC1沉默棉花接菌后水杨酸途径基因表达分析注：不同小写字母表示在P < 0.05水平差异显著。

Fig. 5 Analysis of gene expression of salicylic acid pathway in GhNAC1 silenced cotton after inoculationNote：Different lowercase letters represent significant differences at P < 0.05 level.

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