茶树CssHSP18.1基因克隆及表达分析

doi:10.13305/j.cnki.jts.2020.03.004

摘要/Abstract

摘要： sHSPs基因家族可编码一类小分子的热激蛋白,广泛分布于植物中,具有分子伴侣的功能,在植物抵抗逆境胁迫中起着重要作用。通过基因克隆的方法,获得1个茶树CssHSP18.1基因的开放阅读框（Open reading frame,ORF）,其全长480 bp,编码159个氨基酸。生物信息学分析表明,CssHSP18.1蛋白含有1个典型HSP20结构域,相对分子质量约为18.25 kDa,等电点为5.68,偏酸性,与栎和苹果亲缘关系最近,无信号肽与跨膜结构。RT-qPCR分析表明,CssHSP18.1在甘露醇（D-Mannitol）处理下表达量低于对照组;γ-氨基丁酸（GABA）能促进该基因的表达,在处理后1 h时表达量达到峰值;吲哚乙酸（IAA）和聚乙二醇（PEG 6000）处理后,CssHSP18.1在0.5 h时表达量最高,即GABA、IAA、PEG 6000均可诱导CssHSP18.1的表达。为获得CssHSP18.1可溶性蛋白,构建了pET-28a-CssHSP18.1重组质粒进行原核表达,并分别对表达菌株、诱导温度以及IPTG（异丙基- -D-硫代吡喃半乳糖苷）诱导浓度进行优化。结果显示,CssHSP18.1蛋白最佳表达菌株为BL21（DE3）,最佳诱导温度和IPTG浓度分别为30℃和1.2 mmol·L^-1。最后,采用Western blot对表达的CssHSP18.1蛋白进行验证。本研究为进一步揭示CssHSP18.1基因的生物学功能提供依据。

关键词: 茶树, CssHSP18.1, 基因克隆, 生物信息学分析, 胁迫, 表达分析

Abstract: The sHSPs gene family encodes a class of small molecular heat shock proteins, which are widely distributed in plants, functioned as molecular chaperones, and play an important role in plant resistance to stresses. In this study, the open reading frame (ORF) of CssHSP18.1 gene cDNA was obtained by gene cloning, which is 480 bp in length and encodes 159 amino acids. Bioinformatics analysis showed that CssHSP18.1 protein contained a typical HSP20 domain. Its molecular weight and isoelectric point are about 18.25 kDa and 5.68 respectively. Phylogenetic tree analysis showed that CssHSP18.1 has the closest relationship with quercus and apple. It was predicted that CssHSP18.1 protein was does not have signal peptide and transmembrane structure. RT-qPCR analysis showed that the expression of CssHSP18.1 under D-Mannitol treatment was lower than that in the control group. GABA could enhance the expression of CssHSP18.1 with its peak at 1 h after GABA treatment. The expression of CssHSP18.1 was upregulated upon IAA and PEG 6000 treatments, and reached the peaks at 0.5 h. Thus, GABA、IAA、PEG 6000 could induce the expression of CssHSP18.1. To obtain CssHSP18.1 soluble protein, a recombinant plasmid pET-28a-CssHSP18.1 was constructed and expressed in prokaryotic system. The expression strains, induction temperatures and induction concentrations of IPTG (isopropyl- -D-thiopyranogalactoside) were optimized. The results showed that the best expression strain of CssHSP18.1 protein was BL21 (DE3), and the best induction temperature and IPTG concentration were 30℃ and 1.2 mmol·L^-1 respectively. Finally, western blot was used to verify the expression of CssHSP18.1 protein. This study provided a basis for further study on the biological function of CssHSP18.1 gene.

Key words: Camellia sinensis, CssHSP18.1, gene cloning, bioinformatics analysis, stress, expression analysis

中图分类号:

S571.1
Q52

蒋君梅, 方远鹏, 宁娜, 陈美晴, 杨再福, 王勇, 李向阳, 谢鑫. 茶树CssHSP18.1基因克隆及表达分析[J]. 茶叶科学, 2020, 40(3): 328-340. doi: 10.13305/j.cnki.jts.2020.03.004.

JIANG Junmei, FANG Yuanpeng, NING Na, CHEN Meiqing, YANG Zaifu, WANG Yong, LI Xiangyang, XIE Xin. Cloning and Expression Analysis of CssHSP18.1 Gene in Camellia Sinensis[J]. Journal of Tea Science, 2020, 40(3): 328-340. doi: 10.13305/j.cnki.jts.2020.03.004.

参考文献 48

[1]	蒋会兵, 夏丽飞, 田易萍, 等. 基于转录组测序的紫芽茶树花青素合成相关基因分析[J]. 植物遗传资源学报, 2018, 19(5): 967-978.
	Jiang H B, Xia L F, Tian Y P, et al.Transcriptome analysis of anthocyanin synthesis related genes in purple bud tea plant[J]. Journal of Plant Genetic Resources, 2018, 19(5): 967-978.
[2]	Yue C, Cao H L, Lin H Z, et al.Expression patterns of alpha-amylase and beta-amylase genes provide insights into the molecular mechanisms underlying the responses of tea plants (Camellia sinensis) to stress and postharvest processing treatments[J]. Planta, 2019, 250(1): 281-298.
[3]	贾焱, 孙英杰, 何聪芬, 等. 分子内分子伴侣机制的研究进展[J]. 生物化学与生物物理进展, 2016, 43(5): 443-448.
	Jia Y, Sun Y J, He C F, et al.Research progress on the mechanism of intramolecular chaperone[J]. Progress in Biochemistry and Biophysics, 2016, 43(5): 443-448.
[4]	陈建南. 分子伴侣参与调控动、植物的发育和进化进程[J]. 遗传, 2010, 32(5): 443-447.
	Chen J N.Progress in molecular chaperones participating in regulations of plant and animal development and evolution[J]. Hereditas, 2010, 32(5): 443-447.
[5]	王佳丽. 辅助分子伴侣SlBAG蛋白在番茄抗病反应中的功能研究[D]. 杭州: 浙江大学, 2019.
	Wang J L.Functional analysis of the auxiliary molecular chaperone BAG proteins in disease resistance in tomato [D]. Hangzhou: Zhejiang University, 2019.
[6]	谷丰. 高温噬菌体TSP4分子伴侣CPN47对酶热稳定性的影响研究[D]. 昆明: 昆明理工大学, 2014.
	Gu F.Effect of chaperone CPN47 fromThermusphage TSP4 on the thermal stability of enzyme [D]. Kunming: Kunming University of Science and Technology, 2014.
[7]	陈成, 董爱武, 苏伟. 拟南芥组蛋白分子伴侣AtHIRA参与体细胞同源重组及盐胁迫响应[J]. 植物学报, 2018, 53(1): 42-50.
	Chen C, Dong A W, Su W.Histone chaperone AtHIRA is involved in somatic homologous recombination and salinity response inArabidopsis[J]. Chinese Bulletin of Botany, 2018, 53(1): 42-50.
[8]	万丽丽, 王转茸, 辛强, 等.BnA7HSP70分子伴侣结合蛋白超表达能够提高甘蓝型油菜耐旱性[J]. 作物学报, 2018, 44(4): 483-492.
	Wan L L, Wang Z R, Xin Q, et al.Enhanced accumulation ofBnA7HSP70molecular chaperone binding protein improves tolerance to drought stress in transgenicBrassica napus[J]. Acta Agronomica Sinica, 2018, 44(4): 483-492.
[9]	张美惠. 高温胁迫下小麦白粉病菌HSP基因表达研究及HIGS体系的建立[D]. 北京: 中国农业科学院, 2019.
	Zhang M H.HSPgenes experssion level ofBlumeria graminisf. sp.triticiunder heat stress and host-induced gene silencing (HIGS) system establishment [D]. Beijing: Chinese Academy of Agricultural Sciences, 2019.
[10]	李广隆, 刘思言, 鲁中爽, 等. 植物热激蛋白响应非生物胁迫研究进展[J]. 广东农业科学, 2019, 46(3): 24-30.
	Li G L, Liu S Y, Lu Z S, et al.Research progress of plant heat shock protein response to abiotic stress[J]. Guangdong Agricultural Sciences, 2019, 46(3): 24-30.
[11]	Zhang K M, Ezemaduka A N. Wang Z, et al.A novel mechanism for small heat shock proteins to function as molecular chaperones[J]. Scientific Reports, 2015, 5: 8811. doi: 10.1038/srep08811.
[12]	Khan A, Ali M, Khattak A M, et al.Heat shock proteins: Dynamic biomolecules to counter plant biotic and abiotic stresses[J]. International Journal of Molecular Sciences, 2019, 20(21): 5321. doi: 10.3390/ijms20215321.
[13]	栗振义, 龙瑞才, 张铁军, 等. 植物热激蛋白研究进展[J]. 生物技术通报, 2016, 32(2): 7-13.
	Li Z Y, Long R C, Zhang T J, et al.Research progress on plant heat shock protein[J]. Biotechnology Bulletin, 2016, 32(2): 7-13.
[14]	张宁, 姜晶. 植物中小分子热激蛋白基因家族(sHSPs)研究进展[J]. 植物生理学报, 2017, 53(6): 943-948.
	Zhang N, Jiang J.Research advances of small heat shock protein gene family (sHSPs) in plants[J]. Plant Physiology Journal, 2017, 53(6): 943-948.
[15]	Lin Q, Xie Y J, Guan W Q, et al.Combined transcriptomic and proteomic analysis of cold stress induced sugar accumulation and heat shock proteins expression during postharvest potato tuber storage[J]. Food Chemistry, 2019, 297: 124991. doi: 10.1016/j.foodchem.2019.124991.
[16]	Peffer S, Gonçalves D, Morano K.Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast[J]. Journal of Biological Chemistry, 2019, 294(32): 12191-12202.
[17]	张莉. 小热休克蛋白26(sHSP26)在高温胁迫下保护玉米叶绿体的作用机制[D]. 郑州: 河南农业大学, 2012.
	Zhang L.The mechanism of small heat shock 26 (sHSP26) protecting maize chloroplast from heat stress [D]. Zhengzhou: Henan Agricultural University, 2012.
[18]	陈新海. 高温胁迫下水稻热激蛋白的作用机理研究[D]. 福州: 福建农林大学, 2011.
	Chen X H.Studies on heat shock proteins (HSPs) of rice (Oryza sativaL.) in response to heat stress [D]. Fuzhou: Fujian Agriculture and Forestry University, 2011.
[19]	Wu D, Vonk J J, Salles F, et al.The N terminus of the small heat shock protein HSPB7 drives its polyQ aggregation-suppressing activity[J]. Journal of Biological Chemistry, 2019, 294(25): 9985-9994.
[20]	俞佳虹. 番茄小热激蛋白SlHSP20基因家族的全基因组鉴定及表达分析[D]. 金华: 浙江师范大学, 2017.
	Yu J H.Genome-wide identification and expression profiling of theSlHSP20gene family in tomato [D]. Jinhua: Zhejiang Normal University, 2017.
[21]	梁潘霞, 黄杏, 李杨瑞. 甘蔗小分子量热激蛋白(sHSP)基因克隆及水分胁迫下的表达分析[J]. 生物技术通报, 2016, 32(10): 163-169.
	Liang P X, Huang X, Li Y R.Cloning of small heat-shock protein (HSP) gene from sugarcane and analysis of its expression under drought stress[J]. Biotechnology Bulletin, 2016, 32(10): 163-169.
[22]	张帅扬. 马铃薯小分子热激蛋白基因表达载体构建及胁迫诱导表达特性分析[D]. 长沙: 湖南农业大学, 2017.
	Zhang S Y.Construction of a plant experessing vector of small heat shock protein gene fromSolanum tuberosumand stress-induced experssion analysis [D]. Changsha: Hunan Agricultural University, 2017.
[23]	孙宇栋. 核桃sHSP家族基因筛选、响应模式及JrsHSP17.3基因的抗逆功能分析[D]. 杨凌: 西北农林科技大学, 2016.
	Sun Y D.WalnutsHSPfamily genetic screening, response pattern and resilience function analysis of geneJrsHSP17.3[D]. Yangling: Northwest A&F University, 2016.
[24]	李静婷, 赵旭耀, 刘超凡, 等. 热胁迫对转TasHSP16.9拟南芥幼苗生长生理特性的影响[J]. 江苏农业科学, 2016, 44(10): 113-116.
	Li J T, Zhao X Y, Liu C F, et al.Effects of heat stress on growth physiology of transgenosisTasHSP16.9Arabidopsis seedlings[J]. Jiangsu Agricultural Sciences, 2016, 44(10): 113-116.
[25]	刘珊珊. 西瓜中与CGMMV结构蛋白互作因子的筛选及sHSP功能分析[D]. 北京: 中国农业科学院, 2019.
	Liu S S.Identifying factors interacted with CGMMV sturctual proteins and functional analysis of sHSP in wastermelon [D]. Beijing: Chinese Academy of Agricultural Sciences, 2019.
[26]	潘佳佳. 百合小热激蛋白的克隆及初步分析[D]. 兰州: 兰州大学, 2010.
	Pan J J.The identification of lily small heat shock protein gene and its preliminary research [D]. Lanzhou: Lanzhou University, 2010.
[27]	陈江飞, 高童, 万思卿, 等. 茶树小分子热激蛋白基因CsHSP22.4、CsHSP27.4、CsHSP17.5和CsHSP25.2的克隆与表达分析[J]. 园艺学报, 2018, 45(6): 1160-1172.
	Chen J F, Gao T, Wan S Q, et al.Cloning and expression analysis of small heat shock protein genesCsHSP22.4,CsHSP27.4,CsHSP17.5andCsHSP25.2inCamellia sinensis[J]. Acta Horticulturae Sinica, 2018, 45(6): 1160-1172.
[28]	Chen Y J, Yu P, Luo J C, et al.Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT[J]. Mammalian genome, 2003, 14(12): 859-865.
[29]	Hao X Y, Horvath D P, Chao W S, et al.Identification and evaluation of reliable reference genes for quantitative Real-Time PCR analysis in tea plant (Camellia sinensis(L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[30]	Chen X H, Lin S K, Liu Q L, et al.Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2014, 1844(4): 818-828.
[31]	Adão R, Zanphorlin L M, Lima T B, et al.Revealing the interaction mode of the highly flexibleSorghum bicolorHsp70/Hsp90 organizing protein (Hop): A conserved carboxylate clamp confers high affinity binding to Hsp90[J]. Journal of Proteomics, 2019, 191: 191-201.
[32]	Zhou Y L, Chen H H, Chu P, et al.NnHSP17.5, a cytosolic class Ⅱ small heat shock protein gene fromNelumbo nucifera, contributes to seed germination vigor and seedling thermotolerance in transgenicArabidopsis[J]. Plant Cell Reports, 2012, 31(2): 379-389.
[33]	李敏, 蒋昌华, 胡永红, 等. 月季Rchsp17.8基因转化烟草的非生物胁迫耐性研究[J]. 园艺学报, 2009, 36(8): 1191-1196.
	Li M, Jiang C H, Hu Y H, et al.Transformation of tobacco withRcHSP17.8from Chinese rose enhances tolerance to different abiotic stresses[J]. Acta Horticulturae Sinica, 2009, 36(8): 1191-1196.
[34]	Pla M, Huguet G, Verdaguer D, et al.Stress proteins co-expressed in suberized and lignified cells and in apical meristems[J]. Plant Science, 1998, 139(1): 49-57.
[35]	Kumar R R, Goswami S, Shamim M, et al.Exploring the heat-responsive chaperones and microsatellite markers associated with terminal heat stress tolerance in developing wheat[J]. Functional & Integrative Genomics, 2017, 17(6): 621-640.
[36]	Chauhan H, Khurana N, Nijhavan A, et al.The wheat chloroplastic small heat shock protein (sHSP26) is involved in seed maturation and germination and imparts tolerance to heat stress[J]. Plant, Cell & Environment, 2012, 35(11): 1912-1931.
[37]	左丽萍, 张瑞华, 金桂秀, 等.OsHSP18.0-CI调控水稻对白叶枯病的抗性[J]. 植物病理学报, 2019, 49(1): 90-100.
	Zuo L P, Zhang R H, Jin G X, et al.OsHsp18.0-CIregulates disease resistance to bacterial blight in rice[J]. Acta Phytopathologica Sinica, 2019, 49(1): 90-100.
[38]	Mota T M, Oshiquiri L H, Lopes É C V, et al. Hsp genes are differentially expressed duringTrichoderma asperellumself-recognition, mycoparasitism and thermal stress[J]. Microbiological Research, 2019(227): 126296. doi: 10.1016/j.micres.2019.126296.
[39]	Jiang S S, Wu B, Jiang L L, et al.Triticum aestivumheat shock protein 23.6 interacts with the coat protein of wheat yellow mosaic virus[J]. Virus Genes, 2019, 55(2): 209-217.
[40]	王明乐, 朱旭君, 王伟东, 等. 茶树小分子量热激蛋白基因CsHSP17.2的克隆与表达分析[J]. 南京农业大学学报, 2015, 38(3): 389-394.
	Wang M L, Zhu X J, Wang W D, et al.Molecular cloning and expression analysis of low molecular weight heat shock protein geneCsHSP17.2fromCamellia sinensis[J]. Journal of Nanjing Agricultural University, 2015, 38(3): 389-394.
[41]	Wang M L, Zou Z W, Li Q H, et al.Heterologous expression of threeCamellia sinensissmall heat shock protein genes confers temperature stress tolerance in yeast andArabidopsis thaliana[J]. Plant Cell Reports, 2017, 36(7): 1125-1135.
[42]	张胜. 侧柏对干旱与自然低温胁迫响应的分子机制研究[D]. 杨凌: 西北农林科技大学, 2017.
	Zhang S.Studies on mechanisms of molecular response to drought and natural low temperature stress inPlatycladus orientalis(L.) [D]. Yangling: Northwest A&F University, 2017.
[43]	Ding G B, Wu G F, Li B C, et al.High-yield expression inEscherichia coli, biophysical characterization, and biological evaluation of plant toxin gelonin[J]. 3 Biotech, 2019, 9: 19. doi: 10.1007/s13205-018-1559-6.
[44]	Sørensen H P, Mortensen K K.Advanced genetic strategies for recombinant protein expression inEscherichia coli[J]. Journal of Biotechnology, 2005, 115(2): 113-128.
[45]	Rosano G L, Ceccarelli E A.Recombinant protein expression inEscherichia coli: advances and challenges[J]. Frontiers in Microbiology, 2014, 5: 172. doi: 10.3389/fmicb.2014.00172.
[46]	樊佳, 王毅, 徐莺, 等. 麻疯树小热激蛋白基因JcHSP15.9的原核表达及耐热胁迫[J]. 应用与环境生物学报, 2013, 19(1): 74-78.
	Fan J, Wang Y, Xu Y, et al.Expression, purification and heat stress tolerance ofJatropha curcasL.JcHSP15.9gene in prokaryotic cells[J]. Chinese Journal of Applied and Environmental Biology, 2013, 19(1): 74-78.
[47]	胡雨晴. 蜡梅热激蛋白基因CpHSP1的分子特征、原核表达及其转录的实时荧光定量分析[D]. 重庆: 西南大学, 2011.
	Hu Y Q.Molecular characteristics, prokaryotic expression and transcriptional expression analysis of a heat shock protein geneCpHSP1fromChimonanthus praecox[D]. Chongqing: Southwest University, 2011.
[48]	郭会娜. 巴西橡胶树小热激蛋白基因克隆、表达及功能研究[D]. 海口: 海南大学, 2014.
	Guo H N.Cloning, expression and functional characterizations of small heat shock protein genes fromHevea brasiliensis[D]. Haikou: Hainan University, 2014.