Cloning,Subcellular Localization and Expression Pattern Analysis of Tobacco Transcription Factor NtWRKY11 Gene

doi:10.7668/hbnxb.20194660

Abstract

Abstract:

The WRKY transcription factor family plays an important role in regulating abiotic stress.To systematically analyze the sequence characteristics and expression patterns of NtWRKY11 gene in tobacco,and explore the response mechanism of NtWRKY11 under abiotic stress such as low temperature and drought,the full-length cDNA sequence of NtWRKY11 gene was amplified by PCR using common tobacco cDNA as a template.The basic properties of NtWRKY11 protein were analyzed by bioinformatics software and the subcellular localization of NtWRKY11 protein was studied by constructing a plant expression vector.The expression of NtWRKY11 gene was detected by qRT-PCR technology in different tissues at the full flowering stage and under different abiotic stresses.The results showed that the full-length cDNA of NtWRKY11 gene in tobacco was 999 bp,encoding 332 amino acids,and it shared 54.23% similarity with ramie BnWRKY11.Its promoter region contained three kinds of cis-acting elements(one MBS,one MYB and three ARBE),which probably worked together to enhance the drought resistance of plants.It also contained three salicylic acid-responsive cis-acting elements(TCA-element),which could improve the low temperature tolerance of plants.Subcellular localization results indicated that the NtWRKY11 protein was located in the nucleus.The expression analysis of NtWRKY11 in different tissues at the full flowering stage showed that NtWRKY11 was highly expressed in old leaves,significantly higher than in roots.However,the expression in flowers was significantly lower than that in roots,and there was no significant difference between stems,new leaves and roots. The expression analysis under abiotic stress showed that the relative expression of the gene was significantly higher than that of normal (CK).The relative expression level under high salt stress was not significantly different from CK,while the relative expression level under high temperature stress was significantly lower than CK.All in all,NtWRKY11 is highly expressed in old leaves,and its expression level is enhanced under abiotic stresses such as drought stress and low temperature stress,indicating that this gene acts as a forward transcription factor to regulate drought and low temperature stress.

Key words: Tobacco, NtWRKY11, Drought, Low temperature, Transcription factor, Abiotic stress

WEN Haiyang, ZHU Zitong, ZHAN Jiawei, LI Chang, WU Bohan, YANG Yongxia, ZHANG Songtao, JIA Hongfang. Cloning,Subcellular Localization and Expression Pattern Analysis of Tobacco Transcription Factor NtWRKY11 Gene[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 60-66. doi: 10.7668/hbnxb.20194660.

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Figures/Tables 10

Tab.1 Primer sequences and uses

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	引物用途 Primer use
NtWRKY11 full length	F:CAGTGGTCTCACAACATGGCAGTTGATTTTATTGG	全长cDNA扩增
	R:CAGTGGTCTCATACATCACTCCTTCCTCTCTTCCG
NtWRKY11-YXB	F:ATTTGGAGAGAACACGGGGGACTTTGCAACATGGCAGTTGATTTTATTGGG	亚细胞定位
	R:GGAACCACTCCCTGAAGCGGCCGCTGTACAACTCCTTCCTCTCTTCCGTTGAC
qRT-NtWRKY11	F:GAGCACGAACACTCCGAAGA	qRT-PCR
	R:ACACGAGATTTCCTCTTTTTGCAG
NtL25	F: GGTTGACTCCTGACTATGAT	qRT-PCR
	R: ACACAAGGCACTTCCATT

Fig.1 Electrophoresis map of NtWRKY11 PCR product

Fig.2 Transmembrane domain analysis of NtWRKY11 protein

Fig.3 Hydrophilic/hydrophobic prediction of NtWRKY11 protein

Fig.4 Comparison of NtWRKY11 protein and homologous protein sequences NtWRKY11.XM_019405223.1;AtWRKY7.AEE84877.1;AtWRKY11.AEE85928.1;AtWRKY17.AEC07593.1;AtWRKY25.AEC08362.1;AtWRKY32.AEE85832.1;BnWRKY11.NP_001303197.1;GhWRKY11.AJT43299.1;LrWRKY11.QRX38908.1;OsWRKY6.DAA05071.1;OsWRKY7.ABC02807.1;OsWRKY11.DAA05076.1;ZmWRKY11.AIB05290.1。

Fig.5 Phylogenetic tree of NtWRKY11

Fig.6 Cis-acting elements of NtWRKY11 gene promoter region

Fig.7 Subcellular localization analysis results of NtWRKY11 protein

Fig.8 Expression analysis of NtWRKY11 gene in different tissues at full flowering stage Different capital letters on the column indicate significant differences(P<0.01).The same as Fig.9.

Fig.9 Expression analysis of NtWRKY11 gene under abiotic stress

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