Cloning and Functional Analysis of NtPRR37 Gene in Nicotiana tabacum L.

doi:10.13560/j.cnki.biotech.bull.1985.2024-0112

Abstract

Abstract:

【Objective】Pseudo response regulators（PRRs）are important genes that regulate the flowering pathway in higher plants. The aim of this study is to clone the NtPRR37 gene from Nicotiana tabacum L. and analyze its response to different photoperiods and its effect on flowering, providing a target gene for tobacco flowering regulation.【Method】We cloned NtPRR37 gene from N. tabacum L. by homologous cloning method. Then we performed bioinformatics analysis, tissue-specific expression analysis, and expression pattern analysis of NtPRR37 gene under different light duration treatments. Concurrently, we used Virus Induced Gene Silence（VIGS）to reduce the expression of NtPRR37 and observed the phenotype and detected the expression changes of flowering-related genes.【Result】The fulllength CDS of NtPRR37 was 2 472 bp, encoded a peptide of 823 amino acids with a relative molecular weight of 90.16 kD. It contained REC and CCT domains, which were typical conserved domains of the PRRs gene family. Through homology evolution analysis, it was found that tobacco NtPRR37 belonged to the same branch as PRR37 from Nicotiana tomentosiformis, Nicotiana sylvestris, and Nicotiana benthamiana in evolution. The quantitative reverse transcription-PCR（RT-qPCR）analysis showed that, the expressions of NtPRR37 varied in various tissues of N. tabacum L. at full-bloom stage, with the highest expression in pistils and the lowest expression in lateral roots. Under different photoperiod treatments, the expression of NtPRR37 increased with the prolongation of light exposure. In different light duration treatments, the expression of NtPRR37 increased with the extension of light time, with the lowest expression under darkness, and demonstratted circadian rhythmicity. In NtPRR37-silenced plants, the expression of NtPRR37 was significantly downregulated, and the flowering period was advanced, which may be related to the significant upregulation of flowering-related genes（NtFT4, NtAP1, NtCO, NtSOC1）. 【Conclusion】The expression of NtPRR37 is regulated by the photoperiod, and NtPRR37 acts as a flowering repressor during the flowering process of tobacco.

Key words: Nicotiana tabacum L., pseudo response regulators, NtPRR37, sequence analysis, expression analysis, virus-induced gene silencing, blooming stage

LI Yi-jun, YANG Xiao-bei, XIA Lin, LUO Zhao-peng, XU Xin, YANG Jun, NING Qian-ji, WU Ming-zhu. Cloning and Functional Analysis of NtPRR37 Gene in Nicotiana tabacum L.[J]. Biotechnology Bulletin, 2024, 40(8): 221-231.

Figures/Tables 9

References 36

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引物名称Primer name	碱基序列Base sequence（5'-3'）	用途Usage
NtPRR37-F	GAGGAAGATGAGTCAAGGAT	基因克隆 Cloning of gene
NtPRR37-R	TCTGTTCTGCGAGTCTCT	基因克隆 Cloning of gene
qNtPRR37-F	ACCATCATCACTACCATCAC	NtPRR37基因的RT-qPCR 检测 RT-qPCR detection of NtPRR37 gene
qNtPRR37-R	TGCTTCCATTGTTACTTCCT	NtPRR37基因的RT-qPCR 检测 RT-qPCR detection of NtPRR37 gene
L25-F	CCCCTCACCACAGAGTCTGC	内参基因的RT-qPCR检测 RT-qPCR detection of internal reference gene
L25-R	AAGGGTGTTGTTGTCCTCAATCTT	内参基因的RT-qPCR检测 RT-qPCR detection of internal reference gene
NtPRR37-VIGS-F	CACTTGTGCCCAGGTTGTC	VIGS载体的构建 Construction of VIGS vector
NtPRR37-VIGS-R	TCTAGGAGCGGCTACATCGT	VIGS载体的构建 Construction of VIGS vector
NtFT4-F	GTCACAGACATCCCAGCAACT	NtFT4基因的RT-qPCR检测 RT-qPCR detection of NtFT4 gene
NtFT4-R	CGAAACACTACGAAAACAAAGC	NtFT4基因的RT-qPCR检测 RT-qPCR detection of NtFT4 gene
NtAP1-F	CCTTACACCTTTTCTCAGACCAA	NtAP1基因的RT-qPCR检测 RT-qPCR detection of NtAP1gene
NtAP1-R	ATGTGCTTTCTTCGCTAAACCTC	NtAP1基因的RT-qPCR检测 RT-qPCR detection of NtAP1gene
NtCO-F	CAAATATGGCTCCTCAGGGA	NtCO基因的RT-qPCR检测 RT-qPCR detection of NtCO gene
NtCO-R	GGATGAAATGTATGCGTTATGG	NtCO基因的RT-qPCR检测 RT-qPCR detection of NtCO gene
NtSOC1-F	AAACAGTTGGAGCGGAGTG	NtSOC1基因的RT-qPCR检测 RT-qPCR detection of NtSOC1 gene
NtSOC1-R	GCATTTTCAGAAGCAAGGAT	NtSOC1基因的RT-qPCR检测 RT-qPCR detection of NtSOC1 gene

引物名称Primer name	碱基序列Base sequence（5'-3'）	用途Usage
NtPRR37-F	GAGGAAGATGAGTCAAGGAT	基因克隆 Cloning of gene
NtPRR37-R	TCTGTTCTGCGAGTCTCT	基因克隆 Cloning of gene
qNtPRR37-F	ACCATCATCACTACCATCAC	NtPRR37基因的RT-qPCR 检测 RT-qPCR detection of NtPRR37 gene
qNtPRR37-R	TGCTTCCATTGTTACTTCCT	NtPRR37基因的RT-qPCR 检测 RT-qPCR detection of NtPRR37 gene
L25-F	CCCCTCACCACAGAGTCTGC	内参基因的RT-qPCR检测 RT-qPCR detection of internal reference gene
L25-R	AAGGGTGTTGTTGTCCTCAATCTT	内参基因的RT-qPCR检测 RT-qPCR detection of internal reference gene
NtPRR37-VIGS-F	CACTTGTGCCCAGGTTGTC	VIGS载体的构建 Construction of VIGS vector
NtPRR37-VIGS-R	TCTAGGAGCGGCTACATCGT	VIGS载体的构建 Construction of VIGS vector
NtFT4-F	GTCACAGACATCCCAGCAACT	NtFT4基因的RT-qPCR检测 RT-qPCR detection of NtFT4 gene
NtFT4-R	CGAAACACTACGAAAACAAAGC	NtFT4基因的RT-qPCR检测 RT-qPCR detection of NtFT4 gene
NtAP1-F	CCTTACACCTTTTCTCAGACCAA	NtAP1基因的RT-qPCR检测 RT-qPCR detection of NtAP1gene
NtAP1-R	ATGTGCTTTCTTCGCTAAACCTC	NtAP1基因的RT-qPCR检测 RT-qPCR detection of NtAP1gene
NtCO-F	CAAATATGGCTCCTCAGGGA	NtCO基因的RT-qPCR检测 RT-qPCR detection of NtCO gene
NtCO-R	GGATGAAATGTATGCGTTATGG	NtCO基因的RT-qPCR检测 RT-qPCR detection of NtCO gene
NtSOC1-F	AAACAGTTGGAGCGGAGTG	NtSOC1基因的RT-qPCR检测 RT-qPCR detection of NtSOC1 gene
NtSOC1-R	GCATTTTCAGAAGCAAGGAT	NtSOC1基因的RT-qPCR检测 RT-qPCR detection of NtSOC1 gene

名称 Name	网址 Website	用途 Usage
Expasy	https://web.expasy.org/translate/	基因序列翻译成氨基酸序列Gene sequence translated into amino acid sequence
ProtParam	http://web.expasy.org/protparam/	分析氨基酸含量、分子量和等电点Analysis of amino acid content, molecular weight and isoelectric point
Protscale	https://web.expasy.org/protscale/	分析蛋白的亲水性及疏水性Analysis of hydrophilicity and hydrophobicity of proteins
SMART	http://smart.embl-heidelberg.de/	分析保守结构域Analysis of conserved domains
SOPMA	http://pbil.ibcp.fr/	预测二级结构Prediction of secondary structure
SWISS-MODEL	http://swissmodel.expasy.org/	预测三级结构Prediction of tertiary structure
SignalP-5.0	http://www.cbs.dtu.dk/services/SignalP/	分析信号肽Analysis of signal peptide

名称 Name	网址 Website	用途 Usage
Expasy	https://web.expasy.org/translate/	基因序列翻译成氨基酸序列Gene sequence translated into amino acid sequence
ProtParam	http://web.expasy.org/protparam/	分析氨基酸含量、分子量和等电点Analysis of amino acid content, molecular weight and isoelectric point
Protscale	https://web.expasy.org/protscale/	分析蛋白的亲水性及疏水性Analysis of hydrophilicity and hydrophobicity of proteins
SMART	http://smart.embl-heidelberg.de/	分析保守结构域Analysis of conserved domains
SOPMA	http://pbil.ibcp.fr/	预测二级结构Prediction of secondary structure
SWISS-MODEL	http://swissmodel.expasy.org/	预测三级结构Prediction of tertiary structure
SignalP-5.0	http://www.cbs.dtu.dk/services/SignalP/	分析信号肽Analysis of signal peptide