甘蓝型油菜BnaA02CPSF6基因的克隆及功能分析

doi:10.11983/CBB24068

摘要/Abstract

摘要： 植物CPSF家族(cleavage and polyadenylation specificity factor)是体内mRNA前体中多聚腺苷酸化信号识别、剪切和添加poly(A)的重要蛋白因子, 对开花时间调控、环境响应和种子发育等具有重要作用。目前, 甘蓝型油菜CPSF家族功能尚不明确, 为探究甘蓝型油菜CPSF家族的功能和表达模式, 本研究从甘蓝型油菜品种‘中双11号’中克隆得到BnaA02.CPSF6, 并对其进行生物信息学、亚细胞定位、表达模式和基因功能分析。结果表明, BnaA02.CPSF6基因编码区全长1938 bp, 编码646个氨基酸, 无内含子结构, 在甘蓝型油菜中有6个同源基因; BnaA02.CPSF6基因启动子区域存在多个参与光反应的顺式作用元件和MYB结合位点; BnaA02.CPSF6在根、茎、叶、花和不同发育时期种子中均有表达, 特别是在15–35天的发育种子中显著高表达, 其编码的蛋白定位于细胞核; BnaA02.CPSF6基因受盐胁迫和干旱胁迫诱导上调表达; 在ABA、IAA, GA₃, SA, MeJA激素处理下, BnaA02.CPSF6基因表达先受到抑制再逐渐恢复至正常水平; 在正常条件下, BnaA02.CPSF6基因在拟南芥中过表达会出现提前抽薹开花的表型, 且莲座叶数量显著减少。综上所述, BnaA02.CPSF6参与非生物胁迫响应并受植物激素调控, 可能在开花调控中起促进作用。

关键词: 甘蓝型油菜, BnaA02CPSF6, 基因功能, 表达模式, 生长发育

Abstract: The CPSF family (cleavage and polyadenylation specificity factor) is a crucial protein factor that is responsible for polyadenylation signal recognition in mRNA precursors, cleavage and the addition of poly(A) tails to mRNA in plants, it plays a crucial role in the regulation of flowering time, environmental response, and seed development. Currently, the function of the CPSF family in Brassica napus is unclear. To explore the function and expression patterns of the CPSF gene family, this study cloned BnaA02.CPSF6 from B. napus variety 'Zhongshuang 11' and conducted bioinformatics analysis, subcellular localization, expression pattern analysis, and functional characterization of the gene. The results indicate that the coding region of the BnaA02.CPSF6 gene is 1938 bp, encoding 646 amino acids without intron structures. Its promoter region contains multiple cis-acting elements involved in light responses and MYB binding sites. Additionally, there are six homologous genes of BnaA02.CPSF6 in B. napus. The BnaA02.CPSF6 gene is expressed in root, stem, leaf, flower and different developmental seed of B. napus, especially significantly higher in 15-35d developmental seed, and its encoded protein is localized in the nucleus. The BnaA02.CPSF6 gene expression is upregulated under salt and drought stress. Under the treatment of hormones such as ABA, IAA, GA, SA, and MeJA, the expression of the BnaA02.CPSF6 gene is initially inhibited and then gradually recovers to normal levels. Under normal condition, overexpression of the BnaA02.CPSF6 gene in Arabidopsis thaliana results in an early bolting and flowering phenotype, along with a reduced number of rosette leaves. In summary, the above results indicate that the BnaA02.CPSF6 is involved in abiotic stress responses and regulated by phytohormones, may also play a promoting role in flowering regulation.

Key words: Brassica napus, BnaA02CPSF6, Gene function, Expression patterns, Growth and development

李青洋, 刘翠, 何李, 彭姗, 马嘉吟, 胡子祎, 刘宏波. 甘蓝型油菜BnaA02CPSF6基因的克隆及功能分析. 植物学报, DOI: 10.11983/CBB24068.

Qingyang Li, Cui Liu, Li He, Shan Peng, Jiayin Ma, Ziyi Hu, Hongbo Liu. Cloning and Functional Analysis of BnaA02CPSF6 gene from Brassica napus L.. Chinese Bulletin of Botany, DOI: 10.11983/CBB24068.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24068

参考文献

[1]崔国新, 侯杰, 佟玲, 许志茹(2010).植物基因光反应元件及其结合蛋白.植物生理学通讯, 46:991-1000.
[2]黄郢, 赵培森, 谢伶俐, 徐劲松, 程勇, 张学昆, 徐本波( (2024)).长江流域冬油菜品种产量构成及育种策略分析. .中国油料作物学报, 46:13-18.
[3]冷博峰, 李先容, 陈雪婷, 唐晶, 陈卓尔, 张青, 王涛, 田裕, 魏梦升, 闫华, 刘成, 冯中朝 ((2021)).2008-2019年中国油菜生产性状变化趋势. .中国油料作物学报, 43:171-185.
[4]李谷成, 牛秋纯, 冷博峰, 丁逸飞, 童婷, 范丽霞 ((2024)).新时代十年: 我国油菜产业发展与路径选择. .中国油料作物学报, 46:228-235.
[5]李巧峡, 张丽, 王玉, 黄小霞( (2019)).赤霉素调控植物开花及花器官发育的研究进展. .中国细胞生物学学报, 41:746-758.
[6]张小玲, 尹显明, 朱骞, 董陈文华, 郭效琼, 孙明姬, 陈丽娟, 李东宣( (2018)).水稻新基因 OsCPSF7 启动子克隆及其时空表达特性分析. .分子植物育种, 16:9-15.
[7]Bao S, Hua C, Shen L and Yu H(2020).New insights into gibberellin signaling in regulating flowering in Arabidopsis.Journal of Integrative Plant Biology, 62:118-131.
[8]Chan SL, Huppertz I, Yao C, Weng L, Moresco JJ, Yates JR, Ule J, Manley JL, Shi Y(2014).CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3′ processing.Genes & Development, 28:2370-2380.
[9]Dai Y, Luo L, Zhao Z( (2023)).Genetic robustness control of auxin output in priming organ initiation. .Proceedings of the National Academy of Sciences, 120:e2221606120-.
[10]Eckardt NA(2002).Alternative splicing and the control of flowering time.The Plant Cell, 14:743-747.
[11]Edwalds-Gilbert G, Milcarek C(1995).The binding of a subunit of the general polyadenylation factor cleavage-polyadenylation specificity factor (CPSF) to polyadenylation sites changes during B cell development.Nucleic Acids Symposium Series, 33:229-233.
[12]Feng W, Jacob Y, Veley KM, Ding L, Yu X, Choe G, Michaels SD(2011).Hypomorphic alleles reveal FCA-independent roles for FY in the regulation of FLOWERING LOCUS C.Plant Physiology, 155:1425-1434.
[13]Hao S, Zhang L, Zhao D, Zhou J, Ye C, Qu H, Li Q(2023).Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE and POLYADENYLATION SPECIFICITY FACTOR 73.Plant Physiology, 193:537-554.
[14]Henderson IR, Liu F, Drea S, Simpson GG, Dean C(2005).An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.Development, 132:3597-3607.
[15]Herr AJ, Molnàr A, Jones A, Baulcombe DC(2006).Defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis.Proceedings of the National Academy of Sciences, 103:14994-15001.
[16]Hornyik C, Terzi LC, Simpson GG(2010).The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.Developmental Cell, 18:203-213.
[17]Hou Y, Sun J, Wu B, Gao Y, Nie H, Nie Z, Quan S, Wang Y, Cao X, Li S(2021).CPSF30-L-mediated recognition of mRNA m6A modification controls alternative polyadenylation of nitrate signaling-related gene transcripts in Arabidopsis.Molecular Plant, 14:688-699.
[18]Hunt AG, Xing D, Li Q(2012).Plant polyadenylation factors: conservation and variety in the polyadenylation complex in plants.BMC genomics, 13:641-653.
[19]Hunt AG, Xu R, Addepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhyay A, Dampanaboina L, Marion A, Lanken CV, Li Q(2008).Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling.BMC genomics, 9:220-235.
[20]Lin J, Xu R, Wu X, Shen Y, Li Q(2017).Role of cleavage and polyadenylation specificity factor 100: anchoring poly (A) sites and modulating transcription termination.The Plant Journal, 91:829-839.
[21]Liu F, Marquardt S, Lister C, Swiezewski S, Dean C(2010).Targeted 3′ processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing.Science, 327:94-97.
[22]Ma L, Guo C, Li Q(2014).Role of alternative polyadenylation in epigenetic silencing and antisilencing.Proceedings of the National Academy of Sciences, 111:9-10.
[23]Mandel CR, Bai Y, Tong L(2008).Protein factors in pre-mRNA 3′-end processing.Cellular and Molecular Life Sciences, 65:1099-1122.
[24]Mouradov A, Cremer F, Coupland G ((2002)).Control of flowering time: interacting pathways as a basis for diversity. .The Plant Cell, 14:S111-S130.
[25]Proudfoot N(2004).New perspectives on connecting messenger RNA 3′ end formation to transcription.Current opinion in cell biology, 16:272-278.
[26]Quesada V, Macknight R, Dean C, Simpson GG(2003).Autoregulation of FCA pre‐mRNA processing controls Arabidopsis flowering time.The EMBO Journal, 22:3142-3152.
[27]Schul W, Groenhout B, Koberna K, Takagaki Y, Jenny A, Manders E, Raska I, Driel RV, Jong LD(1996).The RNA 3′ cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.The EMBO journal, 15:2883-2892.
[28]Simpson G, Quesada V, Henderson I, Dijkwel P, Macknight R, Dean C(2004).RNA processing and Arabidopsis flowering time control.Biochem Soc Trans, 32:565-566.
[29]Song P, Yang J, Wang C, Lu Q, Shi L, Tayier S, Jia G(2021).Arabidopsis N6-methyladenosine reader CPSF30-L recognizes FUE signals to control polyadenylation site choice in liquid-like nuclear bodies.Molecular Plant, 14:571-587.
[30]Thomas PE, Wu X, Liu M, Gaffney B, Ji G, Li Q, Hunt AG(2012).Genome-wide control of polyadenylation site choice by CPSF30 in Arabidopsis.The Plant cell, 24:4376-4388.
[31]Tian B, Manley JL(2017).Alternative polyadenylation of mRNA precursors.Nature Reviews Molecular Cell biology, 18:18-30.
[32]Tzafrir I, Pena-Muralla R, Dickerman A, Berg M, Rogers R, Hutchens S, Sweeney TC, McElver J, Aux G, Patton D, Meinke D(2004).Identification of genes required for embryo development in Arabidopsis.The Plant Journal, 135:1206-1220.
[33]Yao P, Potdar AA, Arif A, Ray PS, Mukhopadhyay R, Willard B, Xu Y, Yan J, Saidel GM, Fox PL(2012).Coding region polyadenylation generates a truncated tRNA synthetase that counters translation repression.Cell, 149:88-100.
[34]Yu Z, Lin J, Li Q(2019).Transcriptome analyses of FY mutants reveal its role in mRNA alternative polyadenylation.The Plant Cell, 31:2332-2352.