烟草NtPRR37基因克隆及功能分析

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

摘要/Abstract

摘要：

【目的】伪答应基因家族（pseudo response regulators, PRRs）是高等植物调控开花途径的重要基因。克隆烟草NtPRR37基因并分析其对不同光周期的应答及对开花的影响，为烟草开花调控提供靶标基因。【方法】利用同源克隆方法从普通烟草（Nicotiana tabacum L.）中克隆得到NtPRR37基因，并对其进行生物信息学分析，利用实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）分析其在不同组织中的表达及不同光照时长处理的表达模式。同时利用病毒诱导基因沉默（virus induced gene silence, VIGS）技术降低NtPRR37表达水平并观察表型变化及检测开花相关基因表达变化。【结果】 NtPRR37基因全长2 472 bp，编码823个氨基酸，相对分子质量90.16 kD，含有PRRs基因家族的典型保守结构域（REC和CCT结构域）。通过同源进化分析发现，烟草NtPRR37与绒毛烟草（Nicotiana tomentosiformis）、林烟草（Nicotiana sylvestris）及本氏烟草（Nicotiana benthamiana）的PRR37在进化上属于同一分支。采用RT-qPCR分析发现，该基因在盛花期烟草各个组织的表达特征存在差异性，在雌蕊中的表达量最高，在侧根的表达量最低；在不同光照时长处理下，NtPRR37随着光照时间的增加表达量呈上升趋势，全黑暗处理下表达量最低，且具有生物节律性；NtPRR37沉默植株中NtPRR37表达量明显下调且沉默植株开花期提前，这可能与诱导开花相关基因（NtFT4、NtAP1、NtCO、NtSOC1）表达量显著上调有关。【结论】NtPRR37的表达受到光周期的调控，且在烟草开花过程中NtPRR37作为开花抑制因子存在。

关键词: 烟草, 伪答应调控蛋白, NtPRR37, 序列分析, 表达分析, 基因沉默, 开花

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

李亦君, 杨小贝, 夏琳, 罗朝鹏, 徐馨, 杨军, 宁黔冀, 武明珠. 烟草NtPRR37基因克隆及功能分析[J]. 生物技术通报, 2024, 40(8): 221-231.

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.

图/表 9

表1 本实验所涉及引物及其名称

Table 1 Primers and their names involved in this experiment

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

表2 生物信息学在线工具

Table 2 Bioinformatics online tools

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

图1 NtPRR37基因的CDS序列PCR产物电泳图 M：Marker 5000；1：NtPRR37基因CDS序列扩增产物

Fig. 1 Electrophoretogram of PCR product of NtPRR37 gene CDS sequence M: Marker 5000. 1: Amplified product of CDS sequence of NtPRR37

图2 烟草与其他物种PRR37氨基酸序列多重比对图中红色方框内容为PRR37蛋白的结构域（REC和CCT结构域）

Fig. 2 Multiple sequence alignment of PRR37 between tobacco and other species The red box in the diagram contains the domains（REC and CCT domains）of PRR37 protein

图3 植物PRR37同源蛋白系统进化树

Fig. 3 Phylogenetic tree of plant PRR37 homologous protein

图4 烟草NtPRR37基因在烟草盛花期不同组织中的表达不同小写字母表示差异达到显著水平（P < 0.05），下同

Fig. 4 Expressions of NtPRR37 gene in different tissues of tobacco in blooming period Different lower letters indicate significant difference（P<0.05）

图5 光照对NtPRR37基因表达的影响 A：不同光周期对NtPRR37基因表达的影响；B：不同黑暗时长处理对NtPRR37基因表达的影响（黑暗处理为0 h/24 h，长光照处理为16 h/8 h）； C：连续长光照和短光照NtPRR37基因的表达情况分析（短光照处理为8 h/16 h，长光照为16 h/8 h）

Fig. 5 Effects of light on the expression of NtPRR37 gene A: The effect of different photoperiods on the expression of NtPRR37 gene. B: The effect of different darkness durations on the expression of NtPRR37 gene（Darkness treatment: 0 h/24 h, light treatment:16 h/8 h）. C : Analysis of the expression of NtPRR37 gene under continuous long light and short light（Short light treatment: 8 h/16 h, long light treatment: 16 h/8 h）

图6 农杆菌侵染本氏烟草后的烟草表型 A：空白对照（Con）与阳性对照（pTRV2-PDS）植株侵染7d后的表型对比；B：阴性对照（pTRV2）、空白对照（Con）及实验组（pTRV2-NtPRR37）植株侵染16 d后的表型对比；C：阴性对照（pTRV2）、空白对照（Con）及实验组（pTRV2-NtPRR37）植株开花时间对比

Fig. 6 Phenotype of Nicotiana benthamiana after Agrobacterium infection A: Phenotypic comparison of blank control（Con）and positive control（pTRV2-PDS）plants after 7 d of infection. B: Phenotypic comparison of negative control（pTRV2）, blank control（Con）and experimental group（pTRV2-NtPRR37）plants after 16 d of infection. C: Comparison of flowering time of negative control（pTRV2）, blank control（Con）and experimental group（pTRV2-NtPRR37）plants

图7 NtPRR37基因沉默后开花相关基因表达分析阴性对照（pTRV2）、空白对照（Con）及实验组（pTRV2-NtPRR37）植株侵染15 d后的相关基因表达分析

Fig. 7 Expression analysis of genes related to flowering after NtPRR37 gene silenced Expression analysis of related genes after 15 d of infection in negative control (pTRV2), blank control (Con) and experimental group (pTRV2-NtPRR37) plants

参考文献 36

[1]	杨甲甲, 杨米连, 胡彦如. 生物钟PRR蛋白促进拟南芥幼苗中花青素的合成[J]. 广西植物, 2023, 43(4): 676-687.
	Yang JJ, Yang ML, Hu YR. Circadian clock PRR proteins stimulate anthocyanin synthesis in Arabidopsis thaliana seedlings[J]. Guihaia, 2023, 43(4): 676-687.
[2]	Hargreaves JK, Oakenfull RJ, Davis AM, et al. Multiple metals influence distinct properties of the Arabidopsis circadian clock[J]. PLoS One, 2022, 17(4): e0258374.
[3]	莫伟亮. 拟南芥蓝光受体CRY2介导相分离调控生物钟机制的研究[D]. 长春: 吉林大学, 2022.
	Mo WL. Study on the mechanism of Arabidopsis blue light receptor CRY2-mediated phase separation to regulate the circadian clock[D]. Changchun: Jilin University, 2022.
[4]	Mizuno T, Nakamichi N. Pseudo-response regulators(PRRs)or true oscillator components(TOCs)[J]. Plant Cell Physiol, 2005, 46(5): 677-685.
[5]	Koo BH, Yoo SC, Park JW, et al. Natural variation in OsPRR37 regulates heading date and contributes to rice cultivation at a wide range of latitudes[J]. Mol Plant, 2013, 6(6): 1877-1888.
[6]	Salomé PA, McClung CR. PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock[J]. Plant Cell, 2005, 17(3): 791-803.
[7]	Zhang L, Li QP, Dong HJ, et al. Three CCT domain-containing genes were identified to regulate heading date by candidate gene-based association mapping and transformation in rice[J]. Sci Rep, 2015, 5: 7663. doi: 10.1038/srep07663 pmid: 25563494
[8]	Cockram J, Thiel T, Steuernagel B, et al. Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae[J]. PLoS One, 2012, 7(9): e45307.
[9]	Nimmo HG, Laird J. Arabidopsis thaliana PRR7 provides circadian input to the CCA1 promoter in shoots but not roots[J]. Front Plant Sci, 2021, 12: 750367.
[10]	Yuan L, Hu Y, Li SL, et al. PRR9 and PRR7 negatively regulate the expression of EC components under warm temperature in roots[J]. Plant Signal Behav, 2021, 16(2): 1855384.
[11]	He YQ, Yu YJ, Wang XL, et al. Aschoff's rule on circadian rhythms orchestrated by blue light sensor CRY2 and clock component PRR9[J]. Nat Commun, 2022, 13(1): 5869. doi: 10.1038/s41467-022-33568-3 pmid: 36198686
[12]	Wang L, Kim J, Somers DE. Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription[J]. Proc Natl Acad Sci USA, 2013, 110(2): 761-766. doi: 10.1073/pnas.1215010110 pmid: 23267111
[13]	Zhang B, Liu HY, Qi FX, et al. Genetic interactions among Ghd7, Ghd8, OsPRR37 and Hd1 contribute to large variation in heading date in rice[J]. Rice, 2019, 12(1): 48. doi: 10.1186/s12284-019-0314-x pmid: 31309345
[14]	Klein RR, Miller FR, Dugas DV, et al. Allelic variants in the PRR37 gene and the human-mediated dispersal and diversification of sorghum[J]. Theor Appl Genet, 2015, 128(9): 1669-1683.
[15]	Murphy RL, Klein RR, Morishige DT, et al. Coincident light and clock regulation of pseudoresponse regulator protein 37(PRR37)controls photoperiodic flowering in sorghum[J]. Proc Natl Acad Sci USA, 2011, 108(39): 16469-16474.
[16]	Kamioka M, Takao SR, Suzuki T, et al. Direct repression of evening genes by CIRCADIAN CLOCK-ASSOCIATED1 in the Arabidopsis circadian clock[J]. Plant Cell, 2016, 28(3): 696-711.
[17]	Mizoguchi T, Wright L, Fujiwara S, et al. Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis[J]. Plant Cell, 2005, 17(8): 2255-2270. doi: 10.1105/tpc.105.033464 pmid: 16006578
[18]	Blázquez M. Flower development pathways[J]. J Cell Sci, 2000, 113(Pt 20): 3547-3548.
[19]	Komeda Y. Genetic regulation of time to flower in Arabidopsis thaliana[J]. Annu Rev Plant Biol, 2004, 55: 521-535.
[20]	Harig L, Beinecke FA, Oltmanns J, et al. Proteins from the FLOWERING LOCUS T-like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco[J]. Plant J, 2012, 72(6): 908-921.
[21]	Schmidt GW, Delaney SK. Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abiotic stress[J]. Mol Genet Genomics, 2010, 283(3): 233-241. doi: 10.1007/s00438-010-0511-1 pmid: 20098998
[22]	武明珠, 刘瑞霞, 王中, 等. 利用VIGS技术研究NtbHLH93基因在烟草甾醇代谢中的功能[J]. 烟草科技, 2019, 52(6): 16-22.
	Wu MZ, Liu RX, Wang Z, et al. Study on function of NtbHLH93 in sterol metabolism of tobacco by VIGS[J]. Tob Sci Technol, 2019, 52(6): 16-22.
[23]	李剑峰, 李婷, 贾小平. PRRs家族功能基因的研究进展[J]. 植物遗传资源学报, 2019, 20(6): 1399-1407. doi: 10.13430/j.cnki.jpgr.20190403001
	Li JF, Li T, Jia XP. Advances on unlocking the functional basis of PRRs family genes[J]. J Plant Genet Resour, 2019, 20(6): 1399-1407.
[24]	Nakamichi N, Takao SR, Kudo T, et al. Improvement of Arabidopsis biomass and cold, drought and salinity stress tolerance by modified circadian clock-associated PSEUDO-RESPONSE REGULATORs[J]. Plant Cell Physiol, 2016, 57(5): 1085-1097. doi: 10.1093/pcp/pcw057 pmid: 27012548
[25]	向芬, 黄帅, 赵小英, 等. 拟南芥prr5突变体对ABA 的响应[J]. 激光生物学报, 2013, 22(6): 546-550.
	Xiang F, Huang S, Zhao XY, et al. The response of the prr5 mutant to ABA in Arabidopsis thaliana[J]. Acta Laser Biology Sinica, 2013, 22(6): 546-550.
[26]	Nakamichi N, Kita M, Niinuma K, et al. Arabidopsis clock-associated pseudo-response regulators PRR9, PRR7 and PRR5 coordinately and positively regulate flowering time through the canonical CONSTANS-dependent photoperiodic pathway[J]. Plant Cell Physiol, 2007, 48(6): 822-832. doi: 10.1093/pcp/pcm056 pmid: 17504813
[27]	Hayama R, Sarid-Krebs L, Richter R, et al. PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length[J]. EMBO J, 2017, 36(7): 904-918. doi: 10.15252/embj.201693907 pmid: 28270524
[28]	贾小平, 李剑峰, 张博, 等. 谷子SiPRR37基因对光温、非生物胁迫的响应特点及其有利等位变异鉴定[J]. 作物学报, 2021, 47(4): 638-649. doi: 10.3724/SP.J.1006.2021.04139
	Jia XP, Li JF, Zhang B, et al. Responsive features of SiPRR37 to photoperiod and temperature, abiotic stress and identification of its favourable allelic variations in foxtail millet(Setaria italica L.)[J]. Acta Agron Sin, 2021, 47(4): 638-649.
[29]	王玉岚. 生物钟基因OsPRR37耐旱功能的研究及其编辑在水稻育种中的应用[D]. 武汉: 华中农业大学, 2022.
	Wang YL. Study on drought resistance function of biological clock gene OsPRR37 and application of gene editing in rice breeding[D]. Wuhan: Huazhong Agricultural University, 2022.
[30]	Gao H, Jin MN, Zheng XM, et al. Days to heading 7, a major quantitative locus determining photoperiod sensitivity and regional adaptation in rice[J]. Proc Natl Acad Sci USA, 2014, 111(46): 16337-16342. doi: 10.1073/pnas.1418204111 pmid: 25378698
[31]	Li CC, Ma J, Wang GP, et al. Exploring the SiCCT gene family and its role in heading date in foxtail millet[J]. Front Plant Sci, 2022, 13: 863298.
[32]	Campoli C, Shtaya M, Davis SJ, et al. Expression conservation within the circadian clock of a monocot: natural variation at barley Ppd-H1 affects circadian expression of flowering time genes, but not clock orthologs[J]. BMC Plant Biol, 2012, 12: 97. doi: 10.1186/1471-2229-12-97 pmid: 22720803
[33]	Yamamoto Y, Sato E, Shimizu T, et al. Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis[J]. Plant Cell Physiol, 2003, 44(11): 1119-1130. pmid: 14634148
[34]	Ito S, Kawamura H, Niwa Y, et al. A genetic study of the Arabidopsis circadian clock with reference to the TIMING OF CAB EXPRESSION 1(TOC1)gene[J]. Plant Cell Physiol, 2009, 50(2): 290-303.
[35]	Pin PA, Nilsson O. The multifaceted roles of FLOWERING LOCUS T in plant development[J]. Plant Cell Environ, 2012, 35(10): 1742-1755.
[36]	Wang LW, Sun S, Wu TT, et al. Natural variation and CRISPR/Cas9-mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean[J]. Plant Biotechnol J, 2020, 18(9): 1869-1881. doi: 10.1111/pbi.13346 pmid: 31981443