中国南瓜 CmoAux/IAA 基因的克隆和表达特征分析

Cloning and expression analysis of CmoAux/IAA gene in Cucurbita mos- chata

摘要

在前期对中国南瓜败育雌蕊和正常雌蕊进行转录组分析发现， CmoAux/IAA 基因可能参与了雌花发育，为进一步探究其生物学功能，利用同源序列克隆法从中国南瓜中克隆得到全长为 600 bp 的 CmoAux/IAA 基因，该基因编码 199 个氨基酸；保守结构域分析结果表明，其属于 Aux/IAA 家族，包含结构域Ⅰ、Ⅲ、Ⅳ；该蛋白无信号肽和跨膜结构。多序列比对和系统进化树分析结果显示，CmoAux/IAA 氨基酸序列与印度南瓜 XP_022986212.1 的氨基酸序列、美洲南瓜 XP_023513171.1 的氨基酸序列亲缘关系较近，相似度高达 98.01%、96.50%。拟南芥原生质体亚细胞定位结果显示，CmoAux/IAA 蛋白定位于细胞核和细胞质。实时荧光定量结果显示，该基因具有组织表达特异性，主要在南瓜植株生长点及雌花柱头中表达，柱头中的相对表达量是雄蕊的 137 倍，在叶和根中不表达，其可能在植株生长点幼嫩组织分化或雌花发育过程中发挥着重要作用。

Abstract

In the previous study, transcriptomic analysis of abortive pistil and normal pistil of Cucurbita moschata found that CmoAux/IAA gene might be involved in female flower development. In order to further explore its biological function, a 600 bp gene (CmoAux/IAA) was cloned from Cucurbita moschata by homologous sequence cloning method and it encodes 199 amino acids. The results of conserved domain analysis showed that it belongs to the Aux/IAA family and includes domains Ⅰ, Ⅲ, and Ⅳ. The protein has no signal peptide and transmembrane structure. Multiple sequence alignment and phylogenetic tree results showed that the amino acid sequence of CmoAux/IAA were closely related to XP_022986212.1 of Cucurbita maxima and XP_023513171.1 of Cucurbita pepo, and the similarity was 98.01% and 96.50%, respectively. The results of subcellular localization of Arabidopsis protoplasts showed that the protein was located in nucleus and cytoplasm. Real-time fluorescence quantitative results showed that CmoAux/IAA has tissue expression specificity, mainly expressed in the growing points and stigma of female flowers in pumpkin plants, and the expression level of CmoAux/IAA in stigma was 137 fold that of in stamen, but not expressed in leaves and roots and it may play an important role in the differentiation of young tissue or the development of female flowers.

关键词

中国南瓜 / Aux/IAA / 基因克隆 / 生物信息学分析 / 亚细胞定位

Key words

Cucurbita moschata / Aux/IAA / Gene cloning / Bioinformatics analysis / Subcellular localization

参考文献

[1] 赵一鹏, 李新峥, 周俊国. 世界南瓜生产现状及其种群多样性特征[J]. 内蒙古农业大学学报(自然科学版), 2004, 25(3): 112-115.

[2] SU Y L, HE H H, WANG P, et al. Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid (Aux/ IAA) gene family in apple (Malus domestica)[J]. Gene, 2021, 768: 145302.

[3] XU H W, LIU Y, ZHANG S M, et al. Genome-wide identification and expression analysis of the Aux/ IAA gene family in turnip (Brassica rapa ssp. rapa)[J]. BMC Plant Biology, 2023, 23(1), 342.

[4] ZHU H Y, LI H J, YU J Y, et al. Regulatory mechanisms of ArAux/IAA13 and ArAux/IAA16 in the rooting process of Acer rubrum[J]. Genes, 2023, 14(6): 1206.

[5] HU Q, YANG J J, MENG L H, et al. Genome-wide identification of Aux/IAA genes in watermelon reveals a crucial role for ClIAA16 during fruit ripening[J]. Horticulturae, 2023, 9(11): 1167.

[6] IQBAL S, HAYAT F, MUSHTAQ N, et al. Bioinformatics study of Aux/IAA family genes and their expression in response to different hormones treatments during Japanese apricot fruit development and ripening[J]. Plants-Basel, 2022, 11(15): 1898.

[7] LUO J, ZHOU J J, ZHANG J Z. Aux/IAA gene family in plants:Molecular structure, regulation, and function[J]. International Journal of Molecular Sciences, 2018, 19(1): 259.

[8] WANG J, XUE L, ZHANG X, et al. A new function of MbI-AA19 identified to modulate Malus plants dwarfing growth[J]. Plants-Basel, 2023, 12(17): 3097.

[9] REDDY V A, SAJU J M, NADIMUTHU K, et al. A non-canonical Aux/IAA gene MsIAA32 regulates peltate glandular trichome development in spearmint[J]. Frontiers in Plant Science, 2024, 15: 1284125.

[10] FAN J M, DENG M J, LI B B, et al. Genome-wide identification of the Paulownia fortunei Aux/ IAA gene family and its response to witches' broom caused by phytoplasma[J]. International Journal of Molecular Sciences, 2024, 25(4): 2260.

[11] CHENG W H, ZHANG M, CHENG T R, et al. Genome-wide identification of Aux/ IAA gene family and their expression analysis in Prunus mume[J]. Frontiers in Genetics, 2023, 14: 1208488.

[12] OUELLET F, OVERVOORDE P J, THEOLOGIS A. IAA17/AXR3:Biochemical insight into an auxin mutant phenotype[J]. Plant Cell, 2001, 13(4): 829-841.

[13] GUIFOYLE T J, HAGEN G. Getting a grasp on domain III/IV responsible for auxin response factor-IAA protein interactions[J]. Plant Science, 2012, 190: 82-88.

[14] WU J, PENG Z, LIU S Y, et al. Genome-wide analysis of Aux/IAA gene family in solanaceae species using tomato as a model[J]. Molecular Genetics and Genomics, 2012, 287(4): 295-311.

[15] KORASICK D A, WESTFALL C S, LEE S G, et al. Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(14): 5427-5432.

[16] LV B S, YU Q Q, LIU J J, et al. Non-canonical Aux/IAA protein IAA33 competes with canonical Aux/IAA repressor IAA5 to negatively regulate auxin signaling[J]. Embo Journal, 2020, 39(1): e101515.

[17] GUILFOYLE T J. The PB1 domain in auxin response factor and Aux/IAA proteins:A versatile protein interaction module in the auxin response[J]. Plant Cell, 2015, 27(1): 33-43.

[18] WANG S C. Light promotes protein stability of Auxin Response Factor 7[J]. Phyton-International Journal of Experimental Botany, 2023, 92(4): 1153-1160.

[19] GALLI M, KHAKHAR A, LU Z F, et al. The DNA binding landscape of the maize auxin response factor family[J]. Nature Communications, 2018, 9(1): 4526.

[20] WEI S Y, CHEN Y N, HOU J, et al. Aux/IAA and ARF gene families in Salix suchowensis:Identification, evolution, and dynamic transcriptome profiling during the plant growth process[J]. Frontiers in Plant Science, 2021, 12: 666310.

[21] 王婧. 拟南芥 Aux/ IAA 家族基因 IAA8 参与花器官发育的研究[D]. 武汉: 武汉大学, 2018.

[22] ISHIGURO S, KAWAI-ODA A, UEDA J, et al. The DEFECTIVE IN ANTHER DEHISCIENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis[J]. Plant Cell, 2001, 13(10): 2191-2209.

[23] NAGPAL P, ELLIS C M, WEBER H, et al. Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation[J]. Development, 2005, 132(18): 4107-4118.

[24] 李艳艳, 齐艳华. 植物 Aux/ IAA 基因家族生物学功能研究进展[J]. 植物学报, 2022, 57(1): 30-41.

[25] RINALDI M A, LIU J, ENDERS T A, et al. A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility[J]. Plant Molecular Biology, 2012, 79(4/5): 359-373.

[26] YOO S D, CHO Y H, SHEEN J. Arabidopsis mesophyll protoplasts:A versatile cell system for transient gene expression analysis[J]. Nature Protocols, 2007, 2(7): 1565-1572.

[27] 陈培雯, 张立, 陈学进, 等. 中国南瓜 CmNPR1 基因的克隆和表达分析[J]. 西北植物学报, 2021, 41(12): 2038-2045.

[28] 李庆飞, 王梦梦, 郭卫丽, 等. 南瓜转录因子基因 CmRAV 的克隆与表达分析[J]. 植物生理学报, 2021, 57(12): 2337-2345.

[29] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 - △△C T Method[J]. Methods, 2001, 25(4): 402-408.

[30] JIANG L, LI Z N, YU X M, et al. Bioinformatics analysis of Aux/IAA gene family in maize[J]. Agronomy Journal, 2021, 113(2): 932-942.

[31] BREITEL D A, CHAPPELL-MAOR L, MEIR S, et al. Auxin response factor 2 intersects hormonal signals in the regulation of tomato fruit ripening[J]. PLoS Genetics, 2016, 12(3): e1005903.

[32] JIA Y C, CHEN C X, GONG F F, et al. An Aux/IAA family member, RhIAA14,involved in ethylene-inhibited petal expansion in Rose (Rosa hybrida)[J]. Genes, 2022, 13(6): 1041.

[33] 刘湘楠, 胡祥祥, 乔梦燚, 等. 红花 Aux/ IAA 基因家族鉴定及盐胁迫响应分析[J]. 江苏农业科学, 2024, 52(6): 68-76.

[34] 冯曦, 豆浩, 孙佳佳, 等. 桑树 Aux/ IAA 基因家族鉴定与 IBA 处理下表达模式分析[J]. 河南农业大学学报, 2023, 57(6): 936-948.

[35] 方智振, 姜翠翠, 周丹蓉 等. 三月李及其红肉突变体 Aux/ IAA 家族基因鉴定及分析[J]. 核农学报, 2020, 34(2): 247-255.

[36] AUDRAN-DELALANDE C, BASSA C, MILA I, et al. Genome-wide identification, functional analysis and expression profiling of the Aux/ IAA gene family in tomato[J]. Plant and Cell Physiology, 2012, 53(4): 659-672.

[37] MA C H, DANG K T, XIE Q K, et al. Over-expression of ZmIAA29,an Aux/ IAA transcription factor, improved maize flowering time[J]. Agronomy-Basel, 2023, 13(8): 2028.

[38] WANG J, YAN D W, YUAN T T, et al. A gain-of-function mutation in IAA8 alters Arabidopsis floral organ development by change of jasmonic acid level[J]. Plant Molecular Biology, 2013, 82(1/2): 71-83.

[39] SI C, ZENG D Q, TEIXEIRA DA SILVA J A, et al. Genome-wide identification of Aux/ IAA and ARF gene families reveal their potential roles in flower opening of Dendrobium officinale[J]. BMC Genomics, 2023, 24(1): 199.

[40] ABEL S, OELLER P W, THEOLOGIS A. Early auxin-induced genes encode short-lived nuclear proteins[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(1): 326-330.

基金资助

国家自然科学基金(32302559)

河南省科技攻关项目(242102110312)

河南省高等学校青年骨干教师培养计划(2024GGJS102)

河南省重大科技专项(241100110200)