茶叶科学 ›› 2020, Vol. 40 ›› Issue (3): 328-340.doi: 10.13305/j.cnki.jts.2020.03.004
蒋君梅1, 方远鹏1, 宁娜1, 陈美晴1, 杨再福1, 王勇1, 李向阳2,*, 谢鑫1,*
收稿日期:
2019-08-30
修回日期:
2020-01-07
出版日期:
2020-06-15
发布日期:
2020-06-09
通讯作者:
*xyli1@gzu.edu.cn;xiexin2097757@163.com
作者简介:
蒋君梅,女,主要从事茶树抗病基因功能方面的研究,jjmguangan@163.com。
基金资助:
JIANG Junmei1, FANG Yuanpeng1, NING Na1, CHEN Meiqing1, YANG Zaifu1, WANG Yong1, LI Xiangyang2,*, XIE Xin1,*
Received:
2019-08-30
Revised:
2020-01-07
Online:
2020-06-15
Published:
2020-06-09
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。
中图分类号:
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.
[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. |
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