西瓜谷氨酸脱羧酶GADs的克隆与表达分析

doi:10.7668/hbnxb.20194340

摘要/Abstract

摘要：

谷氨酸脱羧酶(GAD)是催化谷氨酸脱羧的裂解酶,对GABA合成通路起关键作用,并对植物抵御非生物胁迫有重要调控作用。前期通过代谢组和转录组关联分析筛选得到3个西瓜GAD基因家族成员(Cla97C01G007270、Cla97C01G007290和Cla97C04G079700)。在此基础上克隆了3个西瓜GAD家族基因,分别命名为ClGAD1、ClGAD2和ClGAD3,并对其进行生物信息学及表达模式分析。结果显示,ClGAD1、ClGAD2和ClGAD3的CDS分别为1 524,1 497,1 497 bp,其编码的氨基酸数量分别为507,498,498。3个蛋白均为亲水性蛋白;亚细胞定位于线粒体中;具有高度的保守性,保守结构域为谷氨酸脱羧酶;其启动子区域含有大量CAAT-box和TATA-box,以及与逆境胁迫相关的MYB、MYC等相应元件。3个GAD蛋白的二级结构均以α-螺旋、无规卷曲为主;三级结构均为同源六聚体结构。系统进化树显示,GAD基因家族成员与苦瓜、拟南芥的GAD亲缘关系最近。qRT-PCR分析显示,ClGAD1和ClGAD2在茎中表达量最高,而ClGAD3在花中表达量最高。ClGAD1、ClGAD2、ClGAD3分别在盐胁迫12,24,48 h表达量最高;冷和干旱胁迫下3个GAD基因的表达模式较为相似,均在胁迫早期发生大量表达,且ClGAD3在非生物胁迫下的表达量均高于其他2个家族成员。获得了西瓜3个GAD基因的cDNA全长,并对其进行生物信息学和表达模式分析,为丰富西瓜抗非生物胁迫基因资源提供了试验依据。

关键词: 西瓜, GADs, 非生物胁迫, 生物信息学分析, 表达分析

Abstract:

Glutamate decarboxylase(GAD)is a cleavage enzyme that catalyzes the decarboxylation of glutamic acid,plays a key role in GABA synthesis pathway and has an important influence on plants' resistance to abiotic stress.Three watermelon GAD gene family members(Cla97C01G007270,Cla97C01G007290 and Cla97C04G079700)were screened by the association analysis of metabolomics and transcriptomes.On this basis,three watermelon GAD family genes were cloned,named ClGAD1,ClGAD2 and ClGAD3 respectively,and their bioinformatics and expression patterns were analyzed.The results showed that the CDS of ClGAD1,ClGAD2 and ClGAD3 were 1 524,1 497 and 1 497 bp,respectively,and the number of amino acids encoded by them was 507,498 and 498,respectively.All three proteins were hydrophilic proteins and were located in mitochondria.It was highly conserved,and the conserved domain was glutamic acid decarboxylase.Its promoter region contained a large number of CAAT-box and TATA-box,as well as corresponding elements related to adversity stress such as MYB and MYC.The secondary structures of the three GAD proteins were mainly α-helix and random coil.The tertiary structures were all homohexamer structures.Phylogenetic tree showed that members of GAD gene family were closest to GAD of Momordica charantia and Arabidopsis thaliana.qRT-PCR analysis indicated that the highest expression levels of ClGAD1 and ClGAD2 were showed in stems,while that of ClGAD3 was showed in flowers.The expression levels of ClGAD1,ClGAD2 and ClGAD3 were the highest under salt stress for 12,24 and 48 h,respectively.Under cold and drought stress,the expression patterns of the three GAD genes were similar,and all of them were expressed in large quantities in the early stage of stress,and the expression of ClGAD3 under abiotic stress was higher than that of the other two family members.The full-length cDNA of three GAD genes in watermelon was obtained,and their bioinformatics and expression patterns were analyzed,which provided an experimental basis for enriching the abiotic stress resistance gene resources of watermelon.

Key words: Watermelon, GADs, Abiotic stress, Bioinformatics analysis, Expression analysis

李明轩, 刘颖, 杨柏明, 张帆, 宋嘉欣, 粟柴静, 张卫华, 吴颖. 西瓜谷氨酸脱羧酶GADs的克隆与表达分析[J]. 华北农学报, 2023, 38(6): 18-25. doi: 10.7668/hbnxb.20194340.

LI Mingxuan, LIU Ying, YANG Baiming, ZHANG Fan, SONG Jiaxin, SU Chaijing, ZHANG Weihua, WU Ying. Cloning and Expression Analysis of Glutamate Decarboxylase GADs from Watermelon[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 18-25. doi: 10.7668/hbnxb.20194340.

http://www.hbnxb.net/CN/Y2023/V38/I6/18

图/表 9

表1 西瓜GAD蛋白的理化性质

Tab.1 Physicochemical properties of watermelon GAD proteins

基因名称 Gene name	氨基酸数量 Number of amino acid	分子量/ku Molecular weight	等电点 Isoelectric point	亲水性总平均值 GRAVY	亚细胞定位 Subcellular localization
ClGAD1	507	57.18	5.79	-0.237	线粒体
ClGAD2	498	56.24	5.51	-0.239	线粒体
ClGAD3	498	56.18	5.97	-0.219	线粒体

图1 ClGAD1(A)、ClGAD2(B)和ClGAD3(C)蛋白的保守结构域预测

Fig.1 The converse domain structure analysis of ClGAD1(A)、ClGAD2(B)and ClGAD3(C)protein

图2 ClGAD1磷酸化位点(A)、二级(B)和三级(C)结构预测

Fig.2 The phosphorylation site(A),secondary(B)and tertiary(C)structure models prediction of ClGAD1 protein

表2 西瓜GAD基因家族成员的二级结构预测

Tab.2 Secondary structure prediction of members of the watermelon GAD gene family %

蛋白名称 Protein name	α-螺旋 Alpha helix	β-转角 Beta turn	无规卷曲 Random coil	延伸链 Extended strand
ClGAD1	40.24	6.71	36.88	16.17
ClGAD2	40.16	6.63	37.75	15.46
ClGAD3	40.96	6.43	37.15	15.46

图3 西瓜GAD基因家族成员的基因结构

Fig.3 Genetic structure of members of the watermelon GAD gene family

图4 GAD基因家族进化树、motif和保守结构域分析

Fig.4 Phylogenetic tree,motif,and conserved domain analysis of GAD gene family

图5 ClGADs与其他植物GAD蛋白的系统进化树

Fig.5 Phylogenetic tree of ClGADs and GAD proteins in other plants

图6 GAD家族基因顺式作用元件预测

Fig.6 Prediction of cis-acting elements of GAD family genes

图7 ClGAD基因在不同组织(A)以及盐(B)、冷(C)和干旱胁迫(D)下的表达模式分析

Fig.7 Expression pattern analysis of ClGAD gene in different tissues(A)and under salt(B),cold(C)and drought stress(D)

参考文献 25

[1]	王晓君, 杨玉莹, 孙立新, 吴敬学, 毛世平. 新冠肺炎疫情对我国西瓜甜瓜产业的影响及后疫情时期发展建议[J]. 中国瓜菜, 2021, 34(5):125-131.doi:10.3969/j.issn.1673-2871.2021.05.025.
	Wang X J, Yang Y Y, Sun L X, Wu J X, Mao S P. Influence of the COVID-19 epidemic on China's watermelon and melon industry and its development countermeasures in the post-epidemic period[J]. China Cucurbits and Vegetables, 2021, 34(5):125-131.
[2]	Moustaka J, Moustakas M. Early-stage detection of biotic and abiotic stress on plants by chlorophyll fluorescence imaging analysis[J]. Biosensors, 2023, 13(8):796.doi:10.3390/bios13080796. URL
[3]	Ors S, Ekinci M, Yildirim E, Sahin U, Turan M, Dursun A. Interactive effects of salinity and drought stress on photosynthetic characteristics and physiology of tomato(Lycopersicon esculentum L.) seedlings[J]. South African Journal of Botan, 2021, 137:335-339.doi:10.1016/j.sajb.2020.10.031. URL
[4]	Lee H J, Lee J H, Wi S, Jang Y, An S, Choi C K, Jang S. Exogenously applied glutamic acid confers improved yield through increased photosynthesis efficiency and antioxidant defense system under chilling stress condition in Solanum lycopersicum L.cv.Dotaerang Dia[J]. Scientia Horticulturae, 2021, 277:109817.doi:10.1016/j.scienta.2020.109817. URL
[5]	肖杰文, 韩瑾, 乔郅钠, 张国栋, 黄武军, 钱凯, 徐美娟, 张显, 杨套伟, 饶志明. 植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产γ氨基丁酸[J]. 生物工程学报, 2023, 39(6):2108-2125.doi:10.13345/j.cjb.221012.
	Xiao J W, Han J, Qiao Z N, Zhang G D, Huang W J, Qian K, Xu M J, Zhang X, Yang T W, Rao Z M. Efficient biosynthesis of-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum[J]. Chinese Journal of Biotechnolog, 2023, 39(6):2108-2125.
[6]	Guo Z J, Gong J Q, Luo S T, Zuo Y X, Shen Y B. Role of gamma-aminobutyric acid in plant defense response[J]. Metabolites, 2023, 13(6):741.doi:10.3390/metabo13060741. URL
[7]	Bouché N, Fait A, Zik M, Fromm H. The root-specific glutamate decarboxylase(GAD1)is essential for sustaining GABA levels in Arabidopsis[J]. Plant Molecular Biolog, 2004, 55(3):315-325.doi:10.1007/s11103-004-0650-z.
[8]	Hyun T K, Eom S H, Jeun Y C, Han S H, Kim J S. Identification of glutamate decarboxylases as a-aminobutyric acid(GABA)biosynthetic enzyme in soybean[J]. Industrial Crops and Products, 2013, 49:864-870.doi:10.1016/j.indcrop.2013.06.046. URL
[9]	Wu X L, Jia Q Y, Ji S X, Gong B B, Li J R, Lü G Y, Gao H B. Gamma-aminobutyric acid(GABA)alleviates salt damage in tomato by modulating Na⁺ uptake,the GAD gene,amino acid synthesis and reactive oxygen species metabolism[J]. BMC Plant Biolog, 2020, 20(1):465.doi:10.1186/s12870-020-02669-w.
[10]	Liu X, Hu X M, Jin L F, Shi C Y, Liu Y Z, Peng S A. Identification and transcript analysis of two glutamate decarboxylase genes,CsGAD1 and CsGAD2,reveal the strong relationship between CsGAD1 and citrate utilization in citrus fruit[J]. Molecular Biology Reports, 2014, 41(9):6253-6262.doi:10.1007/s11033-014-3506-x. URL
[11]	喻锦莉. OsGAD2基因对水稻株高和籽粒的影响[D]. 南昌: 南昌大学, 2021.doi:10.27232/d.cnki.gnchu.2021.002923.
	Yu J L. Effects of OsGAD2 on plant height and grain of rice[D]. Nanchang: Nanchang University, 2021.
[12]	Ikuta S, Fukusaki E, Shimma S. Visualization of glutamate decarboxylase activity in barley seeds under salinity stress using mass microscope[J]. Metabolites, 2022, 12(12):1262.doi:10.3390/metabo12121262. URL
[13]	Huang H, He Y X, Cui A H, Sun L Q, Han M G, Wang J, Rui C, Lei Y Q, Liu X Y, Xu N, Zhang H, Zhang Y X, Fan Y P, Feng X X, Ni K S, Jiang J, Zhang X P, Chen C, Wang S, Chen X G, Lu X K, Wang D L, Wang J J, Yin Z J, Qaraevna B Z, Guo L X, Zhao L J, Ye W W. Genome-wide identification of GAD family genes suggests GhGAD6 functionally respond to Cd²⁺ stress in cotton[J]. Frontiers in Genetics, 2022, 13:965058.doi:10.3389/fgene.2022.965058. URL
[14]	Hyun T K, Eom S H, Han X, Kim J S. Evolution and expression analysis of the soybean glutamate decarboxylase gene family[J]. Journal of Biosciences, 2014, 39(5):899-907.doi:10.1007/s12038-014-9484-2. pmid: 25431418
[15]	Mazzucotelli E, Tartari A, Cattivelli L, Forlani G. Metabolism of γ-aminobutyric acid during cold acclimation and freezing and its relationship to frost tolerance in barley and wheat[J]. Journal of Experimental Botan, 2006, 57(14):3755-3766.doi:10.1093/jxb/erl141. URL
[16]	AL-Quraan N A, Sartawe F A B, Qaryouti M M. Characterization of-aminobutyric acid metabolism and oxidative damage in wheat(Triticum aestivum L.) seedlings under salt and osmotic stress[J]. Journal of Plant Physiolog, 2013, 170(11):1003-1009.doi:10.1016/j.jplph.2013.02.010.
[17]	Hu X H, Xu Z R, Xu W N, Li J M, Zhao N, Zhou Y. Application of-aminobutyric acid demonstrates a protective role of polyamine and GABA metabolism in muskmelon seedlings under Ca(NO₃)₂ stress[J]. Plant Physiology and Biochemistr, 2015, 92:1-10.doi:10.1016/j.plaphy.2015.04.006.
[18]	贾斌, 高龙飞, 张卫华, 勾峰, 李天杰, 刘颖, 靳丹丹, 吴颖, 刘海学. 西瓜苗期干旱胁迫下的代谢组学分析[J]. 分子植物育种, 2023, 21(21):7161-7170.doi:10.13271/j.mpb.021.007161.
	Jia B, Gao L F, Zhang W H, Gou F, Li T J, Liu Y, Jin D D, Wu Y, Liu H X. Metabolomics analysis of watermelon seedling under drought stress[J]. Molecular Plant Breeding, 2023, 21(21):7161-7170.
[19]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-^ΔΔCT ethod[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. pmid: 11846609
[20]	李承龙. 拟南芥GAD家族基因的时空表达和突变体表型分析[D]. 武汉: 中南民族大学, 2021.doi:10.27710/d.cnki.gznmc.2021.000485.
	Li C L. Temporal and spatial expression and mutant phenotype analysis of GAD family genes in Arabidopsis thaliana[D]. Wuhan: South-central University for Nationalities, 2021.
[21]	Sita K, Kumar V. Role of gamma amino butyric acid(GABA)against abiotic stress tolerance in legumes:a review[J]. Plant Physiology Reports, 2020, 25(4):654-663.doi:10.1007/s40502-020-00553-1.
[22]	Lee J H, Kim Y J, Jeong D Y, Sathiyaraj G, Pulla R K, Shim J S, In J G, Yang D C. Isolation and characterization of a Glutamate decarboxylase(GAD)gene and their differential expression in response to abiotic stresses from Panax ginseng C.A.Meyer[J]. Molecular Biology Reports, 2010, 37(7):3455-3463.doi:10.1007/s11033-009-9937-0. URL
[23]	A gene expression map of Arabidopsis thaliana development[J]. Nature Genetics, 2005, 37(5):501-506.doi:10.1038/ng1543.
[24]	陈炜, 成铁龙, 纪敬, 武妍妍, 谢田田, 江泽平, 史胜青. 杨树GABA支路3个基因家族的鉴定和表达分析[J]. 南京林业大学学报(自然科学版), 2020, 44(5):67-77.doi:10.3969/j.issn.1000-2006.201912029.
	Chen W, Cheng T L, Ji J, Wu Y Y, Xie T T, Jiang Z P, Shi S Q. Identification of three gene families in the GABA shunt and their expression analysis in poplar[J]. Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(5):67-77.
[25]	Zhuang Y L, Ren G J, He C M, Li X Y, Meng Q M, Zhu C F, Wang R C, Zhang J R. Cloning and characterization of a maize cDNA encoding glutamate decarboxylase[J]. Plant Molecular Biology Reporter, 2010, 28(4):620-626.doi:10.1007/s11105-010-0191-3. URL

选择文件类型/文献管理软件名称

选择包含的内容