番茄液泡蔗糖转化酶抑制蛋白克隆、生物信息学及表达分析

doi:10.16178/j.issn.0528-9017.20230461

浙江农业科学 ›› 2024, Vol. 65 ›› Issue (1): 81-89.DOI: 10.16178/j.issn.0528-9017.20230461

番茄液泡蔗糖转化酶抑制蛋白克隆、生物信息学及表达分析

许洋¹(), 程远², 阮美颖², 王荣青², 叶青静², 姚祝平², 周国治², 刘乐承¹, 万红建²^,^*()

1.长江大学园艺园林学院,湖北荆州 434000
2.农产品质量安全危害因子与风险防控国家重点实验室浙江省农业科学院蔬菜研究所中澳作物改良中心,浙江杭州 310021

收稿日期:2023-05-05 出版日期:2024-01-11 发布日期:2024-01-17
通讯作者: 万红建(1980—),男,江苏涟水人,副研究员,博士,主要从事蔬菜遗传育种,E-mail:wanhongjian@sina.com。
作者简介:许洋(1998—),女,山东聊城人,硕士,主要从事蔬菜遗传育种,E-mail:15684178597@163.com。
基金资助:
浙江省重点研发计划项目(2021C02052);国家重点研发计划(2018YFD1000800);国家自然科学基金面上项目(32172555);国家自然科学基金面上项目(31772294);浙江省农业(蔬菜)新品种选育重大科技专项(2021C02065);国家现代农业产业技术体系资助项目(CARS-23-G44)

Molecular cloning, bioinformatics and expression analysis of vacuolar invertase inhibitor in tomato

XU Yang¹(), CHENG Yuan², RUAN Meiying², WANG Rongqing², YE Qingjing², YAO Zhuping², ZHOU Guozhi², LIU Lecheng¹, WAN Hongjian²^,^*()

1. College of Horticulture and Landscape Architecture, Yangtze University, Jingzhou 434000, Hubei
2. Institute of Vegetable, Zhejiang Academy of Agricultural Sciences/State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products/China-Australia Research Centre for Crop Improvement, Hangzhou 310021, Zhejiang

Received:2023-05-05 Online:2024-01-11 Published:2024-01-17

摘要/Abstract

摘要：

液泡转化酶抑制蛋白(vacuolar invertase inhibitor,VIF)在调节植物蔗糖代谢,调控生长发育及响应逆境等方面发挥重要作用。本试验从番茄Alisa中克隆到VIF基因(SlVIF)和cDNA序列。结果显示,SlVIF基因与cDNA序列全长均为528 bp,不含有内含子,共编码175个氨基酸。SlVIF蛋白分子量为19.92 ku,理论等电点为5.53,含有一个信号肽序列,保守结构域为PMEI。系统进化关系表明,茄科物种的VIF亲缘关系最近,且基序具有一致性。组织特异性表达结果显示,在不同的番茄材料中,SlVIF的表达模式存在差异,在Heinz番茄中,其在根部有高水平的表达;在LA1589番茄中,其在果实发育的各阶段,尤其是在成熟果实及根部有高水平的表达。说明SlVIF可能通过介导蔗糖代谢影响果实成熟并调控根系生长。SlVIF受到水分胁迫的诱导,响应非生物胁迫过程。

关键词: 液泡转化酶抑制蛋白, 番茄, 蔗糖代谢, 表达

Abstract:

Vacuolar invertase inhibitor (VIF) plays an important role in regulating higher plant sucrose metabolism and responding to abiotic stress. In this study, VIF gene and cDNA sequence were cloned from Alisa. The results indicated that the full length of SlVIF gene and cDNA was 528 bp, without intron region, encoding 175 amino acids. The molecular weight of SlVIF protein was 19.92 ku, and the theoretical isoelectric point was 5.53. It contained a signal peptide sequence, and the conservative domain was PMEI. The phylogenetic relationship exhibited that the VIF of Solanaceae species was the closest and the motifs were consistent. The tissue specific expression results showed that it had different expression patterns in various tomato materials. In Heinz, it had high transcription level in roots. In LA1589, it was highly expressed in all stages of fruit development, especially in mature fruits and roots, which indicated that SlVIF gene may play a role in influencing fruit ripening and regulating root growth by mediating sucrose metabolism. SlVIF was induced by water stress and responded to abiotic stress processes.

Key words: vacuolar invertase inhibitor, tomato, sucrose metabolism, expression

中图分类号:

S641.2

许洋, 程远, 阮美颖, 王荣青, 叶青静, 姚祝平, 周国治, 刘乐承, 万红建. 番茄液泡蔗糖转化酶抑制蛋白克隆、生物信息学及表达分析[J]. 浙江农业科学, 2024, 65(1): 81-89.

XU Yang, CHENG Yuan, RUAN Meiying, WANG Rongqing, YE Qingjing, YAO Zhuping, ZHOU Guozhi, LIU Lecheng, WAN Hongjian. Molecular cloning, bioinformatics and expression analysis of vacuolar invertase inhibitor in tomato[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(1): 81-89.

图/表 8

表1 SlVIF基因引物信息

Table 1 SlVIF gene primer information

引物名称	序列(5'-3')
SlVIF-F	ATGAGAAATTTATTCCCCATATTGATG
SlVIF-R	TCATAATAACATTCTAATTATGGATTTAGC

图1 番茄SlVIF基因克隆 a表示SlVIF基因与cDNA序列PCR扩增产物电泳检测结果;b表示SlVIF核苷酸序列及氨基酸序列全长,其中标红字体为VIF蛋白保守位点;c表示SlVIF基因结构。

Fig.1 Cloning of tomato SlVIF gene

图2 SlVIF蛋白序列分析 a表示亲疏水性预测,b表示跨膜结构域预测,c表示信号肽预测。

Fig.2 SlVIF protein sequence analysis

图3 SlVIF蛋白结构预测 a表示二级结构,其中蓝色表示α-螺旋,红色表示延伸片段,绿色表示β-转角,紫色表示无规则卷曲;b表示三级结构

Fig.3 Structure prediction of SlVIF protein

图4 SlVIF蛋白保守结构域预测

Fig.4 Prediction of the conservative domain of SlVIF protein

图5 植物液泡蔗糖转化酶抑制蛋白系统发育关系及保守基序分析番茄:Solyc12g099200,Solyc12g099210[16];马铃薯:AIT42248,AIT42247[20];烟草:XP_016474343[19],CAA73333[14];辣椒:KAF3657138;拟南芥:AT1G47960,AT5G64620[20];茶:CSS0012414,CSS0020686,CSS0037733,CSS0047439[22];桃:Prupe_5G112600,Prupe_3G146800,Prupe_1G114500,Prupe_1G113800,Prupe_3G007700[13];梨:KAB2630300,KAB2632320[23];可可:EOY17136,EOY06785[24]。

Fig.5 Phylogenetic relationship and conserved motif analysis of plant vacuolar sucrose invertase inhibitory protein system

图6 番茄不同组织与器官中SlVIF基因的表达模式热图 a表示 Heinz番茄,b表示 LA1589番茄。

Fig.6 Heat map of the expression pattern of SlVIF gene in different tissues and organs of tomato

图7 SlVIF基因在不同非生物胁迫下的表达模式热图 a表示M82番茄,水分胁迫,40%、35%、30%、25%表示土壤含水量,20DPA表示果实生长天数,RR表示成熟果实(红熟);b表示Rio Grande番茄,不同DC3000菌株胁迫。

Fig.7 Heat map of expression patterns of SlVIF gene under different abiotic stresses

参考文献 35

[1]	RUAN Y L, JIN Y, YANG Y J, et al. Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat[J]. Molecular Plant, 2010, 3(6): 942-955.
[2]	赵杰堂. 蔗糖转化酶在高等植物生长发育及胁迫响应中的功能研究进展[J]. 热带亚热带植物学报, 2016, 24(3):352-358.
[3]	RUAN Y L. Sucrose metabolism: gateway to diverse carbon use and sugar signaling[J]. Annual Review of Plant Biology, 2014, 65: 33-67.
[4]	WAN H J, WU L M, YANG Y J, et al. Evolution of sucrose metabolism: the dichotomy of invertases and beyond[J]. Trends in Plant Science, 2018, 23(2): 163-177.
[5]	GREINER S, KRAUSGRILL S, RAUSCH T. Cloning of a tobacco apoplasmic invertase inhibitor, Proof of function of the recombinant protein and expression analysis during plant development[J]. Plant Physiology, 1998, 116(2): 733-742.
[6]	LINK M, RAUSCH T, GREINER S. In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles[J]. FEBS Letters, 2004, 573(1/2/3): 105-109.
[7]	BATE N J, NIU X P, WANG Y W, et al. An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development[J]. Plant Physiology, 2004, 134(1): 246-254.
[8]	RECA I B, BRUTUS A, D'AVINO R, et al. Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato (Solanum lycopersicum) and its use to purify a vacuolar invertase[J]. Biochimie, 2008, 90(11/12): 1611-1623.
[9]	JIN Y, NI D, RUAN Y L. Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level[J]. The Plant Cell, 2009, 21(7): 2072-2089.
[10]	BRUMMELL D A, CHEN R K Y, HARRIS J C, et al. Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants[J]. Journal of Experimental Botany, 2011, 62(10): 3519-3534.
[11]	SHEN L B, QIN Y L, QI Z Q, et al. Genome-wide analysis, expression profile, and characterization of the acid invertase gene family in pepper[J]. International Journal of Molecular Sciences, 2018, 20(1): 15.
[12]	MA M, WANG L B, ZHANG S L, et al. Acid vacuolar invertase 1 (PbrAc-Inv1) and invertase inhibitor 5 (PbrII5) were involved in sucrose hydrolysis during postharvest pear storage[J]. Food Chemistry, 2020, 320: 126635.
[13]	WANG X X, CHEN Y, JIANG S, et al. PpINH1, an invertase inhibitor, interacts with vacuolar invertase PpVIN2 in regulating the chilling tolerance of peach fruit[J]. Horticulture Research, 2020, 7: 168.
[14]	钟荣. 萱草转化酶抑制蛋白的克隆和表达[D]. 上海: 上海应用技术大学, 2021.
[15]	RAUSCH T, GREINER S. Plant protein inhibitors of invertases[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2004, 1696(2): 253-261.
[16]	QIN G Z, ZHU Z, WANG W H, et al. A tomato vacuolar invertase inhibitor mediates sucrose metabolism and influences fruit ripening[J]. Plant Physiology, 2016, 172(3): 1596-1611.
[17]	KANAYAMA Y. Sugar metabolism and fruit development in the tomato[J]. The Horticulture Journal, 2017, 86(4): 417-425.
[18]	QUINET M, ANGOSTO T, YUSTE-LISBONA F J, et al. Tomato fruit development and metabolism[J]. Frontiers in Plant Science, 2019, 10: 1554.
[19]	DATIR S, GHOSH P. In silico analysis of the structural diversity and interactions between invertases and invertase inhibitors from potato (Solanum tuberosum L.)[J]. 3 Biotech, 2020, 10(4): 178.
[20]	FISCHER M, KUCKENBERG M, KASTILAN R, et al. Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors[J]. Molecular Genetics and Genomics, 2015, 290(1): 387-398.
[21]	苏涛, 周怀烨, 周碧瑶, 等. 杨树根特异性表达β-果糖苷酶抑制子的功能性验证[J]. 南京林业大学学报(自然科学版), 2020, 44(6):169-174.
[22]	HE S, LI B, WANG H, et al. Characterization of invertase inhibitors (InvInhs) in tea plant, and their potential roles in participating in growth, development and cold response[J]. Scientia Horticulturae, 2023, 308: 111580.
[23]	OU C Q, WANG F, WANG J H, et al. A de novo genome assembly of the dwarfing pear rootstock Zhongai 1[J]. Scientific Data, 2019, 6: 281.
[24]	MOTAMAYOR J C, MOCKAITIS K, SCHMUTZ J, et al. The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color[J]. Genome Biology, 2013, 14(6): r53.
[25]	FENG Z L, ZHENG F H, WU S L, et al. Functional characterization of a cucumber (Cucumis sativus L.) vacuolar invertase, CsVI1, involved in hexose accumulation and response to low temperature stress[J]. International Journal of Molecular Sciences, 2021, 22(17): 9365.
[26]	CHEN S F, LIANG K, YIN D M, et al. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis[J]. Journal of Enzyme Inhibition and Medicinal Chemistry, 2016, 31(6): 1381-1385.
[27]	OHYAMA A, ITO H, SATO T, et al. Suppression of acid invertase activity by antisense RNA modifies the sugar composition of tomato fruit[J]. Plant and Cell Physiology, 1995, 36(2): 369-376.
[28]	SCHWIMMER S, MAKOWER R U, ROREM E S. Invertase & invertase inhibitor in potato[J]. Plant Physiology, 1961, 36(3): 313-316.
[29]	SERGEEVA L I, KEURENTJES J J B, BENTSINK L, et al. Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(8): 2994-2999.
[30]	WANG L, LI X R, LIAN H, et al. Evidence that high activity of vacuolar invertase is required for cotton fiber and Arabidopsis root elongation through osmotic dependent and independent pathways, respectively[J]. Plant Physiology, 2010, 154(2): 744-756.
[31]	CHEN L, ZHENG F H, FENG Z L, et al. A vacuolar invertase CsVI₂ regulates sucrose metabolism and increases drought tolerance in Cucumis sativus L[J]. International Journal of Molecular Sciences, 2021, 23(1): 176.
[32]	MOLLAH M D A, ZHANG X, ZHAO L, et al. Two vacuolar invertase inhibitors PpINHa and PpINH3 display opposite effects on fruit sugar accumulation in peach[J]. Frontiers in Plant Science, 2022, 13: 1033805.
[33]	MCKENZIE M J, CHEN R K Y, HARRIS J C, et al. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers[J]. Plant, Cell & Environment, 2013, 36(1): 176-185.
[34]	SHIVALINGAMURTHY S G, ANANGI R, KALAIPANDIAN S, et al. Identification and functional characterization of sugarcane invertase inhibitor (ShINH1): a potential candidate for reducing pre-and post-harvest loss of sucrose in sugarcane[J]. Frontiers in Plant Science, 2018, 9: 598.
[35]	刘腾飞. SbSnRK1对马铃薯液泡转化酶活性调控的初步研究[D]. 武汉: 华中农业大学, 2015.

番茄液泡蔗糖转化酶抑制蛋白克隆、生物信息学及表达分析

Molecular cloning, bioinformatics and expression analysis of vacuolar invertase inhibitor in tomato

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 35

相关文章 15

编辑推荐

Metrics

本文评价

[1]	余慧, 陈磊, 杜然, 姚红梅, 罗金燕. 新德里番茄曲叶病毒的发生危害及阻截防控措施[J]. 浙江农业科学, 2024, 65(2): 375-381.
[2]	陈柳, 倪征, 刘可姝, 叶伟成, 华炯钢, 云涛, 朱寅初, 张存. 慢病毒介导的稳定表达鸭干扰素-γ细胞株的构建[J]. 浙江农业科学, 2024, 65(1): 208-212.
[3]	郑剑超, 李明, 董飞. 和田地区设施番茄抗黄化曲叶病毒病的比较试验[J]. 浙江农业科学, 2024, 65(1): 144-147.
[4]	李波, 陈小翔, 张文军, 翟争光, 刘化冰, 江智敏, 徐志强. 外源GA₃和ABA对烤烟内源激素调控效应和常规化学成分的影响[J]. 浙江农业科学, 2024, 65(1): 181-189.
[5]	杨忠浩, 范仲卿, 郭新送, 尹静, 孟庆羽, 王建新, 丁秀红, 马学文. 有机物料处理方式对番茄土壤理化性状、生物学性状及产量的影响[J]. 浙江农业科学, 2023, 64(8): 1913-1919.
[6]	庄黎波, 李勇, 刘强, 章伟江. 基于改进Densenet的番茄病害识别[J]. 浙江农业科学, 2023, 64(8): 1939-1944.
[7]	姚祝平, 肖文铎, 关思慧, 阮美颖, 周国治, 王荣青, 叶青静, 万红建, 叶璐, 程远. 番茄浙樱粉1号高品质栽培模式效益分析及关键技术示范[J]. 浙江农业科学, 2023, 64(7): 1679-1683.
[8]	贾利强, 刘洋, 杨胜美, 杨雨娇, 杨秀万. 玉米bZIP亚家族基因的表达模式分析[J]. 浙江农业科学, 2023, 64(6): 1429-1437.
[9]	张占平, 孙嘉莹, 陆佳欣, 孔令阳, 徐姣, 马伟, 刘秀波. 植物特有的YABBY基因特征与功能研究现状[J]. 浙江农业科学, 2023, 64(6): 1545-1552.
[10]	苏秀敏, 王佼, 韩文清, 李鹏, 王秋兰, 刘永忠. CRITIC 法分析化肥减量配施有机肥对旱地番茄产量和品质的影响[J]. 浙江农业科学, 2023, 64(5): 1037-1041.
[11]	胡玉林, 姜俊杰, 罗健琛, 周斌. 基于多因素方差分析探究灌水施肥量对番茄产量的影响[J]. 浙江农业科学, 2023, 64(5): 1042-1045.
[12]	李佳, 黄灵丹, 张丽娟, 朱倩楠, 曲继松. 不同基质栽培对温室番茄生长、品质和产量的影响[J]. 浙江农业科学, 2023, 64(5): 1046-1049.
[13]	朱明义, 林晨曦, 何蓓蓓, 姚冬冬. 樱桃番茄不同授粉方式试验[J]. 浙江农业科学, 2023, 64(4): 873-875.
[14]	陈利民, 吴全聪, 缪叶旻子, 潘逸明. 7种药剂对番茄病毒病的防效比较[J]. 浙江农业科学, 2023, 64(2): 418-420.
[15]	泮张彬, 陈剑, 何玲玲, 陈真, 项玉英. 茭白秸秆堆肥与化肥减量配施对番茄生长、产量及土壤容重的影响[J]. 浙江农业科学, 2023, 64(2): 359-362.