芍药<i>PlSPL1</i>基因克隆与表达特性分析

doi:10.7668/hbnxb.20194773

摘要/Abstract

摘要：

为研究芍药PlSPL1(SPL)基因的性质和功能,进一步阐明PlSPL1基因在不同物种中的共性与特征差异,探明PlSPL1在芍药茎秆挺直程度中的作用。以芍药红峰茎秆为材料,采用RACE技术获得了芍药PlSPL1基因全长序列,利用生物信息学软件分析预测PlSPL1的结构、理化性质和亲缘关系,随后利用qRT-PCR技术分析PlSPL1在芍药茎秆不同发育时期的表达量,并通过激光共聚焦扫描显微技术进行了蛋白质亚细胞定位分析。结果表明:PlSPL1基因开放阅读框为3 000 bp,编码999个氨基酸。蛋白分子式为C₄₈₆₉H₇₆₈₂N₁₄₀₆O₁₄₉₇S₄₃,分子量为111.25 ku,理论等电点为6.26,编码亲水性不稳定酸性蛋白,磷酸化修饰以丝氨酸为主,无信号肽,有跨膜结构,二级结构主要由无规则卷曲组成。系统进化树分析发现,PlSPL1蛋白与牡丹的亲缘关系最近,其次和葡萄有较近的亲缘关系;蛋白序列比对分析发现,PlSPL1蛋白具有SPL转录因子家族特有的SBP保守结构域。相对表达量分析发现,PlSPL1随着茎秆发育逐渐呈现下降趋势,表明PlSPL1负向调控芍药茎秆发育,推测其可能在茎秆挺直程度方面起重要作用;亚细胞定位显示,PlSPL1蛋白定位在细胞核中。上述结果表明,PlSPL1参与芍药茎秆发育过程。

关键词: 芍药, SPL, 基因克隆, 表达模式, 亚细胞定位

Abstract:

To investigate the nature and function of PlSPL1 (SPL)gene of Paeonia lactiflora,further elucidate the commonalities and characteristic differences of the PlSPL1 gene in different species,and explore the role of PlSPL1 in the degree of stem straightening in Paeonia lactiflora. The full-length sequence of the PlSPL1 gene was obtained by RACE technology using the stalk of herbaceous peony Hongfeng as the research material,and the structural,physicochemical properties,and phylogenetic relationships of PlSPL1 were analyzed and predicted using bioinformatics software,then,the expression level of PlSPL1 in different developmental stages of herbaceous peony stems was analyzed using qRT-PCR technology,and subcellular localization analysis of the protein was conducted using laser confocal microscopy technology.The results showed that the open reading frame of the PlSPL1 gene was 3 000 bp,encoding 999 amino acids.The protein had a molecular formula of C₄₈₆₉H₇₆₈₂N₁₄₀₆O₁₄₉₇S₄₃,a molecular weight of 111.25 ku,a theoretical isoelectric point of 6.26,encoding a hydrophilic unstable acidic protein,phosphorylation modification predominantly on serine,no signal peptide,with transmembrane structure,and had a secondary structure consisting mainly of random coil.Phylogenetic tree analysis revealed that the PlSPL1 protein was most closely related to peony,followed by a closer relationship with grape;protein sequence comparison analysis revealed that the PlSPL1 protein had a conserved SBP domain which was unique to SPL transcription factor family.The relative expression analysis found that PlSPL1 gradually showed a decreasing trend with stem development,indicating that PlSPL1 was negatively regulating the stem development of herbaceous peony and it was hypothesised that PlSPL1 played important roles in degree of stem straightness;and the subcellular localisation showed that the PlSPL1 protein was localised in the nucleus.The above results indicate that PlSPL1 is participated in the stalk development of herbaceous peony.

Key words: Paeonia lactiflora Pall., SPL, Gene cloning, Expression pattern, Subcellular location

许华杰, 卢莉莉, 汤寓涵, 赵大球, 孟家松, 陶俊. 芍药PlSPL1基因克隆与表达特性分析[J]. 华北农学报, 2024, 39(2): 55-61. doi: 10.7668/hbnxb.20194773.

XU Huajie, LU Lili, TANG Yuhan, ZHAO Daqiu, MENG Jiasong, TAO Jun. Cloning and Expression Characteristic Analysis of PlSPL1 Gene in Paeonia lactiflora Pall.[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 55-61. doi: 10.7668/hbnxb.20194773.

http://www.hbnxb.net/CN/Y2024/V39/I2/55

图/表 10

图1 芍药PlSPL1基因PCR扩增产物检测 DNA Marker 5000;1.PCR扩增产物。

Fig.1 Detection of PCR amplification product of PlSPL1 gene in herbaceous peony DNA Marker 5000;1.PCR product.

图2 基于PlSPL1与拟南芥AtSPL1编码蛋白氨基酸序列构建的系统发育进化树

Fig.2 Phylogenetic tree constructed based on amino acid sequences of PlSPL1 and Arabidopsis thaliana AtSPL1 encoded protein

图3 PlSPL1蛋白磷酸化位点分析

Fig.3 Analysis of PlSPL1 protein phosphorylation sites

图4 PlSPL1蛋白信号肽预测

Fig.4 Signal peptide prediction of PlSPL1 protein in herbaceous peony

图5 PlSPL1蛋白跨膜结构预测

Fig.5 Transmembrane structure prediction of PlSPL1 protein in herbaceous peony

图6 PlSPL1蛋白二级结构预测

Fig.6 Prediction of secondary structure of PlSPL1 protein

图7 芍药等12种植物SPL蛋白的系统进化树分析

Fig.7 Phylogenetic tree analysis of SPL proteins in 12 plants including herbaceous peony

图8 芍药等12种植物SPL蛋白保守序列分析

Fig.8 Analysis of conserved sequence of SPL protein in 12 plants including herbaceous peony

图9 PlSPL1在茎秆不同发育时期的表达模式不同小写字母表示差异显著(P<0.05)。

Fig.9 Expression pattern of PlSPL1 at different developmental stages of the stem Different lowercase letters indicate significant differences(P<0.05).

图10 芍药PlSPL1蛋白在烟草表皮中亚细胞定位

Fig.10 Subcellular localization of PlSPL1 protein in tobacco epidermis

参考文献 29

[1]	Zheng W, Shi J, Zhu Z Y, Jin P, Chen J H, Zhang L, Zhang E, Lin T, Zhu Z J, Zang Y X, Wu J G. Transcriptomic analysis of succulent stem development of Chinese kale(Brassica oleracea var.alboglabra Bailey)and its synthetic allotetraploid via RNA sequencing[J]. Frontiers in Plant Science, 2022,13:1004590.doi:10.3389/fpls.2022.1004590.
[2]	夏星. 芍药花茎木质素特征观察与转录组分析[D]. 扬州: 扬州大学, 2019.doi:10.27441/d.cnki.gyzdu.2019.000965.
	Xia X. Lignin characteristic observation and transcriptome analysis of P. lactiflora inflorsecence stem[D]. Yangzhou: Yangzhou University,2019.
[3]	Liu X J, Wu C Y, Su D D, Yang Y, Xian Z Q, Yu C Y, Li Z G, Hao Y W, Chen R Y. The SlHB8 acts as a negative regulator in stem development and lignin biosynthesis[J]. International Journal of Molecular Sciences, 2021, 22(24):13343.doi:10.3390/ijms222413343. URL
[4]	Tang Y H, Lu L L, Sheng Z P, Zhao D Q, Tao J. An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony[J]. The Plant Journal, 2023, 113(6):1237-1258.doi:10.1111/tpj.16107. URL
[5]	Zhong R Q, Lee C H, Ye Z H. Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis[J]. Molecular Plant, 2010, 3(6):1087-1103.doi:10.1093/mp/ssq062. URL
[6]	冉书瑶. 甘蓝型油菜BnaA9.NST3调控木质部发育及开花的功能研究[D]. 重庆: 西南大学, 2022.doi:10.27684/d.cnki.gxndx.2022.004433.
	Ran S Y. Functional study on the regulation of xylem development and flowering by BnaA9.NST3 in Brassica napus[D]. Chongqing: Southwest University, 2022.
[7]	Huijser P, Klein J, Lönnig W E, Meijer H, Saedler H, Sommer H. Bracteomania,an inflorescence anomaly,is caused by the loss of function of the MADS-box gene SQUAMOSA in Antirrhinum majus[J]. The EMBO Journal, 1992, 11(4):1239-1249.doi:10.1002/j.1460-2075.1992.tb05168.x.
[8]	Cardon G, Höhmann S, Klein J, Nettesheim K, Saedler H, Huijser P. Molecular characterisation of the Arabidopsis SBP-box genes[J]. Gene, 1999, 237(1):91-104.doi:10.1016/s0378-1119(99)00308-x. pmid: 10524240
[9]	Xie K B, Wu C Q, Xiong L Z. Genomic organization,differential expression,and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice[J]. Plant Physiology, 2006, 142(1):280-293.doi:10.1104/pp.106.084475. URL
[10]	Yang Z F, Wang X F, Gu S L, Hu Z Q, Xu H, Xu C W. Comparative study of SBP-box gene family in Arabidopsis and rice[J]. Gene, 2008, 407(1/2):1-11.doi:10.1016/j.gene.2007.02.034. URL
[11]	Cheng H T, Hao M Y, Wang W X, Mei D S, Tong C B, Wang H, Liu J, Fu L, Hu Q. Genomic identification,characterization and differential expression analysis of SBP-box gene family in Brassica napus[J]. BMC Plant Biology, 2016, 16(1):196.doi:10.1186/s12870-016-0852-y. URL
[12]	Hou H M, Li J, Gao M, Singer S D, Wang H, Mao L Y, Fei Z J, Wang X P. Genomic organization,phylogenetic comparison and differential expression of the SBP-box family genes in grape[J]. PLoS One, 2013, 8(3):e59358.doi:10.1371/journal.pone.0059358. URL
[13]	Jiao Y Q, Wang Y H, Xue D W, Wang J, Yan M X, Liu G F, Dong G J, Zeng D L, Lu Z F, Zhu X D, Qian Q, Li J Y. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice[J]. Nature Genetics, 2010, 42(6):541-544.doi:10.1038/ng.591. pmid: 20495565
[14]	Miura K, Ikeda M, Matsubara A, Song X J, Ito M, Asano K, Matsuoka M, Kitano H, Ashikari M. OsSPL14 promotes panicle branching and higher grain productivity in rice[J]. Nature Genetics, 2010, 42(6):545-549.doi:10.1038/ng.592. pmid: 20495564
[15]	张铁怀. 小麦株型调控基因 TaIPA1 的克隆与其功能的初步研究[D]. 咸阳: 西北农林科技大学, 2015.
	Zhang T H. Cloning and preliminary functional analysis of TaIPA1 gene regulating plant architecture in wheat(Triticum aestivum)[D]. Xianyang: Northwest A&F University, 2015.
[16]	Wilkins M R, Gasteiger E, Bairoch A, Sanchez J C, Williams K L, Appel R D, Hochstrasser D F. Protein identification and analysis tools in the ExPASy server[J]. Methods in Molecular Biology, 1999,112:531-552.doi:10.1385/1-59259-584-7:531.
[17]	Blom N, Gammeltoft S, Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[J]. Journal of Molecular Biology, 1999, 294(5):1351-1362.doi:10.1006/jmbi.1999.3310. pmid: 10600390
[18]	Nakai K, Kanehisa M. Expert system for predicting protein localization sites in gram-negative bacteria[J]. Proteins, 1991, 11(2):95-110.doi:10.1002/prot.340110203. pmid: 1946347
[19]	Chou K C, Shen H B. Cell-PLoc:a package of Web servers for predicting subcellular localization of proteins in various organisms[J]. Nature Protocols, 2008, 3(2):153-162.doi:10.1038/nprot.2007.494.
[20]	Geourjon C, Deléage G. SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Computer Applications in the Biosciences, 1995, 11(6):681-684.doi:10.1093/bioinformatics/11.6.681. pmid: 8808585
[21]	Kumar S, Stecher G, Tamura K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7):1870-1874.doi:10.1093/molbev/msw054. URL
[22]	Crooks G E, Hon G, Chandonia J M, Brenner S E. WebLogo:a sequence logo generator[J]. Genome Research, 2004, 14(6):1188-1190.doi:10.1101/gr.849004. pmid: 15173120
[23]	Apweiler R, Attwood T K, Bairoch A, Bateman A, Birney E, Biswas M, Bucher P, Cerutti L, Corpet F, Croning M D R, Durbin R, Falquet L, Fleischmann W, Gouzy J, Hermjakob H, Hulo N, Jonassen I, Kahn D, Kanapin A, Karavidopoulou Y, Lopez R, Marx B, Mulder N J, Oinn T M, Pagni M, Servant F, Sigrist C J A, Zdobnov E M. InterPro—an integrated documentation resource for protein families,domains and functional sites[J]. Bioinformatics, 2000, 16(12):1145-1150.doi:10.1093/bioinformatics/16.12.1145. pmid: 11159333
[24]	Lu L L, Tang Y H, Xu H J, Qian Y, Tao J, Zhao D Q. Selection and verification of reliable internal reference genes in stem development of herbaceous peony(Paeonia lactiflora Pall.)[J]. Physiology and Molecular Biology of Plants, 2023, 29(6):773-782.doi:10.1007/s12298-023-01325-5.
[25]	王俊文, 石晶. 枸杞Lb_SPL12基因的克隆及表达分析[J]. 基因组学与应用生物学, 2021, 40(2):766-774.doi:10.13417/j.gab.040.000766.
	Wang J W, Shi J. Cloning and expression analysis of Lb_SPL12 Gene in Lycium barbarum L.[J]. Genomics and Applied Biology, 2021, 40(2):766-774.
[26]	Zhang L L, Huang Y Y, Zheng Y P, Liu X X, Zhou S X, Yang X M, Liu S L, Li Y, Li J L, Zhao S L, Wang H, Ji Y P, Zhang J W, Pu M, Zhao Z X, Fan J, Wang W M. Osa-miR535 targets SQUAMOSA promoter binding protein-like 4 to regulate blast disease resistance in rice[J]. The Plant Journal, 2022, 110(1):166-178.doi:10.1111/tpj.15663. URL
[27]	Cardon G H, Höhmann S, Nettesheim K, Saedler H, Huijser P. Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3:a novel gene involved in the floral transition[J]. The Plant Journal, 1997, 12(2):367-377.doi:10.1046/j.1365-313x.1997.12020367.x.
[28]	Sun Z X, Su C, Yun J X, Jiang Q, Wang L X, Wang Y N, Cao D, Zhao F, Zhao Q S, Zhang M C, Zhou B, Zhang L, Kong F J, Liu B H, Tong Y P, Li X. Genetic improvement of the shoot architecture and yield in soya bean plants via the manipulation of GmmiR156b[J]. Plant Biotechnology Journal, 2019, 17(1):50-62.doi:10.1111/pbi.12946. pmid: 29729214
[29]	Li B B, Zhao Y J, Wang S, Zhang X H, Wang Y W, Shen Y, Yuan Z H. Genome-wide identification,gene cloning,subcellular location and expression analysis of SPL gene family in P.granatum L.[J]. BMC Plant Biology, 2021, 21(1):400.doi:10.1186/s12870-021-03171-7.

选择文件类型/文献管理软件名称

选择包含的内容

芍药PlSPL1基因克隆与表达特性分析

Cloning and Expression Characteristic Analysis of PlSPL1 Gene in Paeonia lactiflora Pall.

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 29

相关文章 15

编辑推荐

Metrics

本文评价

[1]	曹丽茹, 叶飞宇, 李伟亚, 马晨晨, 庞芸芸, 梁小菡, 张新, 鲁晓民. *玉米生长素响应因子基因ZmARF10在干旱胁迫中的功能分析*[J]. 华北农学报, 2024, 39(2): 1-10.
[2]	吴茵茵, 温思怡, 韩卓然, 孙敬锋. *半滑舌鳎突触体相关蛋白29基因(SNAP29)克隆及其组织分布和时序表达分析*[J]. 华北农学报, 2024, 39(2): 231-238.
[3]	胡莲, 刘益丽, 赵迪, 王泽宁, 江明锋. *麦洼牦牛MFSD4A基因克隆及组织表达分析*[J]. 华北农学报, 2024, 39(2): 174-183.
[4]	高泽仁, 潘鹏丞, 徐文文, 陈宝剑, 刘明君, 关志惠, 谢炳坤, 覃兆鲜. *陆川猪MyoD1基因克隆及组织表达分析*[J]. 华北农学报, 2024, 39(2): 168-173.
[5]	王慧珍, 张朝政, 黄一鸣, 黎耀心, 程子洋, 乐超银. *高粱抗病相关基因SbRPM1的克隆及表达分析*[J]. 华北农学报, 2024, 39(2): 153-160.
[6]	曹正林, 陈福源, 黄玉兰, 李有真, 丁洪霞, 夏凯旋, 魏文琦. *防风SAUR32/23基因生物信息学分析及编码蛋白的亚细胞定位*[J]. 华北农学报, 2024, 39(2): 71-78.
[7]	贺彩霞, 李长忠, 保长虹, 王丽楠, 严青春, 金文杰, 赵娟, 王国杰, 简生龙, 王振吉, 陈艳霞. *花斑裸鲤MSTN-1基因克隆及表达特性分析*[J]. 华北农学报, 2024, 39(1): 228-238.
[8]	张自阳, 周谦, 王依, 王志伟, 朱启迪, 茹振钢, 刘明久. *小麦倒春寒响应基因TaJAZ6的克隆、表达模式及亚细胞定位分析*[J]. 华北农学报, 2024, 39(1): 27-36.
[9]	李春阳, 侯占铭. *尖孢镰刀菌亚麻专化型Folcdc15基因参与调控分生孢子发生和菌丝生长*[J]. 华北农学报, 2023, 38(6): 175-183.
[10]	陈娜, 詹文文, 刘兴雨, 石磊鑫, 李若楠, 谢镕, 却志群. *番茄WRKY转录因子SlWRKY75的克隆、表达及功能分析*[J]. 华北农学报, 2023, 38(6): 1-10.
[11]	张元臣, 胡梦杰, 薛爽, 郭文军, 沈红, 王景顺. *黏虫新型抗菌肽Mysdiap基因克隆、序列分析及原核表达*[J]. 华北农学报, 2023, 38(6): 184-191.
[12]	柳婷, 李敏敏, 赖章龙, 刘彦斌, 刘凯, 肖伟, 赛清云, 田永华, 杨立强, 阮超岭, 杨瑞兰, 刘哲, 连总强. *兰州鲇Dmrta1基因克隆及其组织时空表达规律*[J]. 华北农学报, 2023, 38(6): 228-238.
[13]	袁惠君, 张瑞艳, 关玉晨, 余诗曼, 徐琰莹, 马倩国, 鲍婧婷. *扁果枸杞表皮蜡质转运相关基因LbABCG11的克隆及其表达特征分析*[J]. 华北农学报, 2023, 38(6): 45-54.
[14]	黄渺, 罗悠悠, 倪芮, 肇瑾. *观赏辣椒ARP基因克隆及表达分析*[J]. 华北农学报, 2023, 38(5): 69-76.
[15]	张晓红, 许佩阳, 闫绪, 张贝贝, 于婵婵. *棉花GhFUL1基因及其启动子的克隆和功能分析*[J]. 华北农学报, 2023, 38(5): 60-68.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

芍药PlSPL1基因克隆与表达特性分析

Cloning and Expression Characteristic Analysis of PlSPL1 Gene in Paeonia lactiflora Pall.

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 29

相关文章 15

编辑推荐

Metrics

本文评价