草莓FaGH3.17基因的克隆及功能分析

doi:10.16420/j.issn.0513-353x.2023-1008

摘要/Abstract

摘要：

为了研究GH3.17在草莓中对盐胁迫的响应，克隆了FaGH3.17，分析其进化关系、编码蛋白的理化性质等，进行烟草亚细胞定位以及拟南芥异源过表达，验证其在盐胁迫中的功能。结果表明，FaGH3.17为酸性且不稳定的疏水性非分泌性蛋白，主要定位在细胞核和细胞膜。FaGH3.17与蕨麻的亲缘关系最近。盐胁迫下，异源过表达FaGH3.17拟南芥株系的电导率、MDA含量、H₂O₂含量均高于野生型,分别升高了15.43%、42.97%、18.86%，而Pro含量、POD、SOD及CAT活性均显著低于野生型，分别降低了13.28%、15.42%、14.22%、19.87%，并且盐响应相关基因的相对表达量均显著降低，表明异源过表达FaGH3.17显著降低了拟南芥的抗氧化酶活性和渗透调节物质含量，预示着GH3.17可能会减弱植株的耐盐性。

关键词: 草莓, 基因克隆, 盐胁迫, 功能验证

Abstract:

In order to study the response of GH3.17 gene to salt stress in strawberry，the evolutionary relationship and physicochemical properties of FaGH3.17 were analysed using bioinformatics，and the gene was cloned and subsequently subcellularly localised in tobacco and heterologously overexpressed in Arabidopsis to verify its function in salt stress. The results showed that FaGH3.17 is an acidic and unstable hydrophobic，non-secretory protein，and the analysis of the evolutionary relationship of species revealed that FaGH3.17 is the closest relative to Potentilla anserina. Subcellular localisation in tobacco revealed that FaGH3.17 was mainly localised in the nucleus and cell membrane. Heterologously overexpression analysis in Arabidopsis thaliana revealed that under salt stress，the conductivity，MDA and H₂O₂ content were higher than those of wild-type plants，which were elevated by 15.43%，42.97%，and 18.86%，respectively，whereas the Pro content，POD，SOD，and CAT activities were significantly lower than those of the wild-type plants，which were reduced by 13.28%，15.42%，14.22%，19.87%，respectively. And the relative expression of salt-responsive genes were all significantly reduced. It was shown that heterologous overexpression of FaGH3.17 significantly reduced the antioxidant enzyme activities and osmoregulatory substance contents in Arabidopsis，which predicts that GH3.17 may attenuate the salt tolerance of plants.

Key words: strawberry, gene cloning, salt stress, functional verification

杨娟博, 郭丽丽, 卢世雄, 苟惠敏, 王帅珽, 曾宝珍, 毛娟. 草莓FaGH3.17基因的克隆及功能分析[J]. 园艺学报, 2024, 51(11): 2483-2494.

YANG Juanbo, GUO Lili, LU Shixiong, GOU Huimin, WANG Shuaiting, ZENG Baozhen, MAO Juan. Cloning and Functional Analysis of FaGH3.17 Gene in Strawberry[J]. Acta Horticulturae Sinica, 2024, 51(11): 2483-2494.

图/表 9

表1 实时荧光定量引物序列

Table 1 Sequence of real-time fluorescence quantitative primers

基因 Gene	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
FaGH3.17	ACAGTTGATCTGGTGGATGTGAAGC	ACGGAACTGAGGAGCATTGTTGTG
AtHKT1	GTCTCTGCCATCACCGTCTCTTC	GCCACCGAGGAACATGAGGATAG
AtSOS1	TCATCTCCCGCCGCATTATCAC	GATTGCTCTTGCTCTCGTCTCAAC
AtNHX1	TTGACAAGTGGAGATCCGTGAGTG	TGCTCTTCCAACCATGACCAGAC
AtAPX1	CTCTGGGACGATGCCACAAG	CTCGACCAAAGGACGGAAAA
AtActin2	CTGGCCTACGTGGCACTTGACTT	AGCGATGGCTGGAACAGAAC

图1 草莓FaGH3.17的PCR检测（A）及其编码蛋白在烟草亚细胞中的定位（B）

Fig. 1 PCR detection of FaGH3.17 gene in strawberry（A）and subcellular mapping in tobacco（B）

图2 FaGH3.17与其他物种蛋白的系统进化分析

Fig. 2 Phylogenetic analysis of FaGH3.17 with proteins from other species

图3 FaGH3.17蛋白Motif分析

Fig. 3 Motif analysis of FaGH3.17 protein

图4 FaGH3.17异源过表达拟南芥的鉴定（A）及其盐胁迫15 d的表型（B） M：DL2000 marker；+：阳性对照；-：阴性对照；WT：野生型；OE：异源过表达株系。下同。

Fig. 4 Identification of FaGH3.17 heterologously overexpressing Arabidopsis thaliana（A）and phenotypic observation of overexpressing Arabidopsis thaliana under salt stress 15 days（B） M：DL2000 marker；+：Positive control；-：Negative control；WT：Wild type；OE：Heterologous overexpression strain. The same below.

图5 盐胁迫15 d后FaGH3.17在野生型和异源过表达拟南芥中的表达量（A）及过氧化氢含量（B）小写字母代表显著性水平（P < 0.05）。下同。

Fig. 5 Analysis of FaGH3.17 heterologously expression（A）and hydrogen peroxide content（B）in overexpressing Arabidopsis thaliana after salt stress 15 days Lowercase letters represent significance level at P < 0.05. The same below.

图6 FaGH3.17异源过表达拟南芥在盐胁迫下的细胞质膜渗透性分析

Fig. 6 Analysis of cytoplasmic membrane permeability under salt stress in FaGH3.17 heterologously overexpressing Arabidopsis thaliana

图7 盐胁迫下FaGH3.17异源过表达拟南芥中抗氧化酶的活性

Fig. 7 Determination of antioxidant enzyme activities of FaGH3.17 heterologously overexpressing Arabidopsis thaliana under salt stress

图8 盐胁迫下FaGH3.17异源过表达拟南芥中4个盐胁迫响应基因的表达水平

Fig. 8 Expression levels of 4 salt-responsive genes under salt in Arabidopsis

参考文献 43

[1]	Blom N, Gammeltoft S, Brunak S. 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of Molecular Biology, 294:1351-1362. doi: 10.1006/jmbi.1999.3310 pmid: 10600390
[2]	Du H, Wu N, Fu J, Wang S P, Li X H, Xiao J H, Xiong L Z. 2012. A GH 3 family member,OsGH3-2,modulates auxin and abscisic acid levels and differentially affects drought and and tolerance in rice. Journal of Experimental Botany, 63 (18):6467-6480.
[3]	Feng S G, Yue R Q, Tao S, Yang Y J, Zhang L, Xu M F, Wang H Z, Shen C J. 2015. Genome-wide identification,expression analysis of auxin-responsive GH3 family genes in maize(Zea mays L.)under abiotic stresses. Journal of Integrative Plant Biology, 57 (9):783-795.
[4]	Grimplet J, Pimentel D, Agudelo-Romero P, Martinez-Zapater J M, Fortes A M. 2017. The LATERAL ORGAN BOUNDARIES Domain gene family in grapevine:genome-wide characterization and expression analyses during developmental processes and stress responses. Scientific Reports, 7 (1):15968. doi: 10.1038/s41598-017-16240-5 pmid: 29162903
[5]	Guo Lili, Lu Shixiong, Nai Guojie, Ren Jiaxuan, Gou Huimin, Chen Baihong, Mao Juan. 2023. Identification and expression analysis of GH3 gene family in pineapple strawberry and woodland strawberry. Journal of Plant Physiology, 59 (3):579-598. (in Chinese)
	郭丽丽, 卢世雄, 乃国洁, 任家玄, 苟惠敏, 陈佰鸿, 毛娟. 2023. 凤梨草莓和森林草莓GH3基因家族的鉴定与表达分析. 植物生理学报, 59 (3):579-598.
[6]	Hagen G, Kleinschmidt A, Guilfoyle T. 1984. Auxin-regulated gene expression in intact soybean hypocotyl and excised hypocotyl sections. Planta. 162 (2):147-153. doi: 10.1007/BF00410211 pmid: 24254049
[7]	Jha R K, Mishra A. 2021. Introgression of SbERD4 gene encodes an early-responsive dehydration-stress protein that confers tolerance against different types of abiotic stresses in transgenic tobacco. Cells, 11 (1):62.
[8]	Jiang Lijuan. 2022. Characterization of MdMYB94 and its target gene MdGH3.6 in apple drought tolerance[Ph. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	姜丽娟. 2022. 苹果MdMYB94及其靶基因MdGH3.6的抗旱功能分析[博士论文]. 杨凌: 西北农林科技大学.
[9]	Kirungu J N, Magwanga R O, Lu P, Cai X Y, Zhou Z L, Wang X X, Peng R H, Wang K B, Liu F. 2019. Functional characterization of Gh_A08G1120(GH3.5)gene reveal their significant role in enhancing drought and salt stress tolerance in cotton. BMC Genetics, 20 (1):1-17.
[10]	Koochak H, Ludwig-Müller J. 2021. Physcomitrium patens mutants in auxin conjugating GH 3 proteins show salt stress tolerance but auxin homeostasis is not involved in regulation of oxidative stress factors. Plants, 10 (7):1398.
[11]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33:1870-1874.
[12]	Lang M L, Zhang Y X, Chai T Y. 2005. Identification of genes up-regulated in response to Cd exposure in Brassica juncea L. Gene, 363 (1-2):151-158.
[13]	Letunic I, Doerks T, Bork P. 2011. SMART 7:recent updates to the protein domain annotation resource. Nucleic Acids Research, 40:D302-D305.
[14]	Liu Huijie. 2020. Functional study of OsbZIPX,a new transcription factor highly expressed in flower in rice[M. D. Dissertation]. Jinhua: Zhejiang Normal University. (in Chinese)
	刘慧洁. 2020. 水稻花期特异表达的转录因子OsbZIPX的功能研究[硕士论文]. 金华: 浙江师范大学.
[15]	Liu Xiaodong, Wang Ruozhong, Jiao Binbin, Dai Peihong, Li Yue. 2016. Indole acetic acid-amido synthetase GH3-6 negatively regulates response to drought and salt in Arabidopsis. Acta Botanica Sinica, 51 (5):586-593. (in Chinese)
	刘晓东, 王若仲, 焦彬彬, 代培红, 李月. 2016. 拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应. 植物学报, 51 (5):586-593. doi: 10.11983/CBB15223
[16]	Liu Yong, Wang Zeqiong, Gong Linzhong, Wang Furong, Wang Huiliang, Ai Xiaoyan, He Huaping. 2020. Cloning and functional analysis of ERF transcription factor gene PpERF1a in peach. Acta Horticulturae Sinica, 47 (6):1165-1171. (in Chinese)
	刘勇, 王泽琼, 龚林忠, 王富荣, 王会良, 艾小艳, 何华平. 2020. 桃乙烯应答因子PpERF1a的克隆与功能分析. 园艺学报, 47 (6):1165-1171. doi: 10.16420/j.issn.0513-353x.2019-0872
[17]	Liu Z B, Ulmasov T, Shi X Y, Hagen G, Guilfoyle T J. 1994. Soybean GH 3 promoter contains multiple auxin-inducible elements. Plant Cell, 6 (5):645-657. doi: 10.1105/tpc.6.5.645 pmid: 8038604
[18]	Lu Shixiong. 2021. Identification of Trihelix transcription factor and study on the function of grape VvTrihelix5in response to salt[M. D. Dissertation]. Lanzhou: Gansu Agricultural University. (in Chinese)
	卢世雄. 2021. 葡萄Trihelix转录因子家族鉴定及VvTrihelix5响应盐胁迫功能研究[硕士论文]. 兰州: 甘肃农业大学.
[19]	Lu S X, Wang P, Nai G J, Li Y M, Su Y L, Liang, G P, Chen B H, Mao J. 2022. Insight into VvGH3 genes evolutional relationship from monocotyledons and dicotyledons reveals that VvGH3-9 negatively regulates the drought tolerance in transgenic Arabidopsis. Plant Physiology and Biochemistry, 172:70-86.
[20]	Ma Mengnan, Liu Yuan, Ma Fengwang, Zou Yangjun. 2021. Function analysis of MdGH3-2/12 under salt stress in apple. Agricultural Research in the Arid Areas, 39 (6):39-52. (in Chinese)
	马梦楠, 刘媛, 马锋旺, 邹养军. 2021. 苹果MdGH3-2/12在盐胁迫下的功能分析. 干旱地区农业研究, 39 (6):39-52.
[21]	Nakazawa M, Yabe N, Ichikawa T, Yamamoto Y Y, Yoshizumi T, Hasunuma K, Matsui M. 2001. DFL1,an auxin-responsive GH3 gene homologue,negatively regulates shoot cell elongation and lateral root formation,and positively regulates the light response of hypocotyl length. The Plant Journal:for Cell and Molecular Biology, 25 (2):213-221.
[22]	Park J E, Park J Y, Kim Y S, Kim Y S, Staswick Paul E, Jeon J, Yun J, Kim S Y, Kim J, Lee Y H, Park C M. 2007. GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. Journal of Biological Chemistry, 282 (13):10036-10046.
[23]	Petersen T N, Brunak S, Von Heijne G, Nielsen H. 2011. SignalP 4.0:discriminating signal peptides from transmembrane regions. Nature Methods, 8 (10):785-786. doi: 10.1038/nmeth.1701 pmid: 21959131
[24]	Song Q, He F, Kong L F, Yang J R, Wang X J, Zhao Z J, Zhang Y Q, Xu C Z, Fan C F, Luo K M. 2024. The IAA17. 1/HSFA5a module enhances salt tolerance in Populus tomentosa by regulating flavonol biosynthesis and ROS levels in lateral roots. New Phytologist, 241 (2):592-606.
[25]	Staswick P E, Tiryaki I, Rowe M L. 2002. Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic,salicylic,and indole-3-acetic acids in an assay for adenylation. The Plant Cell, 14 (6):1405-1415.
[26]	Staswick P E, Serban B, Rowe M, Tiryaki I, Maldonado M T, Maldonado M C, Suza W. 2005. Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell, 17 (2):616-627. doi: 10.1105/tpc.104.026690 pmid: 15659623
[27]	Su Daifa, Tong Jiangyun, Yang Junyu, Chen Shanyan, Luo Zhiwei, Shen Xuemei, Lai Yonghong, Arslan Jamil, Wei Shijie, Cui Xiaolong. 2018. Advances in research,exploitation and utilization of Fragaria spp. germplasm resources in China. Journal of Yunnan University (Natural Science Edition), 40 (6):1261-1276. (in Chinese)
	苏代发, 童江云, 杨俊誉, 陈杉艳, 罗志伟, 沈雪梅, 赖泳红, Arslan Jamil, 魏世杰, 崔晓龙. 2018. 中国草莓属植物种质资源的研究、开发与利用进展. 云南大学学报(自然科学版), 40 (6):1261-1276.
[28]	Sun Tao,Chai Tuanyao,Liu Geyu,Zhang Yuxiu. 2008. Progress in the plant GH3 gene family. Chinese Journal of Bioengineering,(11):1860-1866. (in Chinese)
	孙涛, 柴团耀, 刘戈宇, 张玉秀. 2008. 植物GH3基因家族研究进展. 生物工程学报,(11):1860-1866.
[29]	Sun Xueli, Liu Fan, Tian Na, Xiang Leilei, Hao Xiangyang, Wang Yun, Peng Liyun, Wang Tianchi, Cheng Chunzhen, Lai Zhongxiong. 2019. Genome-wide identification and expression analysis of AUX/IAA gene family in banana. Acta Horticulturae Sinica, 46 (10):1919-1935. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0743
	孙雪丽, 刘范, 田娜, 项蕾蕾, 郝向阳, 王云, 彭丽云, 王天池, 程春振, 赖钟雄. 2019. 香蕉Aux/IAA基因家族的全基因组鉴定及表达分析. 园艺学报, 46 (10):1919-1935. doi: 10.16420/j.issn.0513-353x.2018-0743
[30]	Swarup R, Parry G, Graham N, Allen T, Bennett M. 2002. Auxin cross-talk:integration of signalling pathways to control plant development. Plant Molecular Biology, 49 (3-4):411-426.
[31]	Urban J, Maria K, Ingela J, Sofia G, Sara S, Laure F, Alfons R W, Per K. 2001. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiology, 126 (4):1358-1369.
[32]	Wang Chuqiao, Huang Yimei, Luo Xian, Jia Yongxia, Tang Shanshan. 2023. Effects of exogenous ABA on ROS metabolism of pitaya seedling under cold stress. Acta Botanica Sinica, 43 (8):1344-1351. (in Chinese)
	王楚侨, 黄一梅, 罗弦, 贾永霞, 唐姗姗. 2023. 外源ABA对低温胁迫下火龙果苗活性氧代谢的影响. 西北植物学报, 43 (8):1344-1351.
[33]	Xie Xiaofang, Huang Qinyi, Wu Weiren. 2010. Bioinformatic analysis of the GH 3 gene family in plant. Genomics and Applied Biology, 29 (5):829-837. (in Chinese)
	谢小芳, 黄勤怡, 吴为人. 2010. 植物GH3基因家族的生物信息学分析. 基因组学与应用生物学, 29 (5):829-837.
[34]	Yang G, Chen S, Wang S, Liu G F, Li H Y, Huang H J, Jiang J. 2015. BpGH3.5,an early auxin-response gene,regulates root elongation in Betula platyphylla × Betula pendula. Plant Cell,Tissue & Organ Culture, 120 (1):239-250.
[35]	Yang Jianyu. 2022. Research on signal transduction and transcriptional regulation function in saline-alkali stress resistance enhanced by exogenous spermidine in tomato[Ph. D. Dissertation]. Yangling: Northwest Agriculture and Forestry University. (in Chinese)
	杨建宇. 2022. 外源亚精胺在增强番茄盐碱胁迫耐性中信号转导和转录调控功能研究[博士论文]. 杨凌: 西北农林科技大学.
[36]	Yuan H Z, Zhao K, Lei H J, Shen X J, Liu Y, Liao Y, Li T H. 2013. Genome-wide analysis of the GH3 family in apple (Malus × domestica). BMC Genomics, 14:1-14.
[37]	Yuan Xin, Xu Yunhe, Zhang Yupei, Shan Nan,Chen Chuying,Wan Chunpeng,Kai Wenbin,Zhai Xiawan,Chen Jinyin,Gan Zengyu. 2023. Studies on AcAREB 1 regulating the expression of AcGH3.1 during postharvest ripening of kiwifruit. Acta Horticulturae Sinica, 50 (1):53-64. (in Chinese)
	袁馨, 徐云鹤, 张雨培, 单楠, 陈楚英, 万春鹏, 开文斌, 翟夏琬, 陈金印, 甘增宇. 2023. 猕猴桃后熟过程中ABA响应结合因子AcAREB1调控AcGH3.1的表达. 园艺学报, 50 (1):53-64. doi: 10.16420/j.issn.0513-353x.2021-1086
[38]	Zeng Hongxue. 2015. Physiological and ecological performance of strawberry under salt stress and the ways to improve the salt tolerance of strawberry. Heilongjiang Agricultural Sciences,(3):43-46. (in Chinese)
	曾洪学. 2015. 盐胁迫条件下草莓的生理生态表现及提高草莓耐盐性的途径. 黑龙江农业科学,(3):43-46.
[39]	Zeng Wenfang, Wang Xiaobei, Pan Lei, Niu Liang, Lu Zhenhua, Cui Guochao, Wang Zhiqiang. 2017. Identification and expression profiling of Aux/IAA family gene during peach fruit ripening. Acta Horticulturae Sinica, 44 (2):233-244. (in Chinese) doi: 10.16420/j.issn.0513-353x.2016-0388
	曾文芳, 王小贝, 潘磊, 牛良, 鲁振华, 崔国朝, 王志强. 2017. 桃Aux/IAA家族基因鉴定及在果实成熟过程中的表达分析. 园艺学报, 44 (2):233-244. doi: 10.16420/j.issn.0513-353x.2016-0388
[40]	Zeng Ya,Ding Xinhua,Shen Xiangling,Li Xianghua,Wang Shiping. 2008. Analysis of protein expression profiling in rice disease resistance gene-mediated resistance to Xanthomonas oryzae pv. oryzae. Chinese Journal of Rice Science,(3):234-242. (in Chinese)
	曾亚, 丁新华, 沈祥陵, 李香花, 王石平. 2008. 水稻抗病基因介导的抗白叶枯病反应中蛋白质表达谱的比较分析. 中国水稻科学,(3):234-242.
[41]	Zhai Hanhan, Zhai Yujie, Tian Yi, Zhang Ye, Yang Li, Wen Zhiliang, Chen Haijiang. 2023. Genome-wide identification of peach SAUR gene family and characterization of PpSAUR5 gene. Acta Horticulturae Sinica, 50 (1):1-14. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0932
	翟含含, 翟宇杰, 田义, 张叶, 杨丽, 温陟良, 陈海江. 2023. 桃SAUR家族基因分析及PpSAUR5功能鉴定. 园艺学报, 50 (1):1-14. doi: 10.16420/j.issn.0513-353x.2021-0932
[42]	Zhang Z Q, Li Q, Li Z M, Staswick P E, Wang W Y, Zhu Y, He Z H. 2007. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction. Plant Physiology, 145 (2):450-464.
[43]	Zhu Yubin, Kong Yingying, Wang Junhui. 2014. Research advances in auxin-responsive SAUR genes. Chinese Bulletin of Life Sciences, 26 (4):407-413. (in Chinese)
	朱宇斌, 孔莹莹, 王君晖. 2014. 植物生长素响应基因SAUR的研究进展. 生命科学, 26 (4):407-413.