桃树枝条皮层响应低温的特异蛋白质2-DE分析

doi:10.7668/hbnxb.20191207

摘要/Abstract

摘要： 低温是限制桃树生长、发育、繁殖以及地理分布的一个重要环境因子。明确桃响应低温胁迫的分子调控机制有可能为培育和筛选出相对抗寒的桃品系提供研究基础。以抗寒性较强的秋燕桃为试材，将一年生休眠枝条置于-5（CK），-15，-25℃低温处理48 h，应用双向电泳技术（Two-dimensional electrophoresis，2-DE）测定枝条皮层蛋白质表达变化情况。结果显示：通过质谱鉴定发现，有150个蛋白质点的表达丰度在低温胁迫后发生明显变化，丰度显著上调的有97个，丰度显著下调的有53个，通过蛋白质组数据检索成功地鉴定了其中的20个蛋白，发现这些蛋白质主要参与氨基糖和核苷酸糖代谢、蔗糖和淀粉代谢、氨基酸代谢、MAPK信号通路、苯丙氨酸代谢和DNA的复制等代谢过程。利用RT-PCR验证了Alpha淀粉酶（M5VVU6）、叶绿素a/b结合蛋白（M5WUQ0）和环氧化物水解酶（D8L7V9）蛋白编码的基因，随着低温胁迫的增加而上调表达。研究结果暗示了这些代谢途径和3个特异蛋白可能在桃的抗寒机制中起重要作用。

关键词: 桃, 双向电泳(2-DE), 特异蛋白, 抗寒性

Abstract: Cold stress is a major environmental factor that limits peach growth,development reproduction and distribution. Clarifying the molecular mechanism of peach in response to low temperature stress could provide the research basis for cultivating and screening relatively cold resistant peach varieties. In cold-tolerant peach cultivar Qiuyan were selected as testing materials. 1 year dormant branches were exposed to-5(the control),-15,-25℃under controlled environment chamber for 48 h respectively. Two-dimensional electrophoresis(2-DE)were used to analyze proteomics of cortex of branches under 3 different low temperature treatments. The results showed that a total of 150 differentially expressed proteins were identified by mass spectrometry,97 significantly up-regulated proteins and 53 significantly down-regulated after cold stress. After further analysis of the protein spots by proteome data retrieval,20 proteins related to cold stress were identified,involving amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,amino acid metabolism,MAPK signaling pathway,phenylalanine metabolism and DNA replication. In addition,Real-time PCR (RT-PCR) indicated that three candidate protein-encoding genes Alpha-amylase(M5VVU6),Chlorophyll a/b binding protein(M5WUQ0)and Epoxide hydrolase 3(D8L7V9)were up-regulated with the increase of cold stress. It suggested that these metabolic pathways and three specific proteins might play an important role in the cold-resistant mechanism in peach.

Key words: Peach, Two-dimensional electrophoresis (2-DE), Specific proteins, Cold tolerance

中图分类号:

S662.1

李永红, 周语潮, 田启航, 常瑞丰, 刘国俭, 陈湖, 王召元. 桃树枝条皮层响应低温的特异蛋白质2-DE分析[J]. 华北农学报, 2020, 35(S1): 102-108. doi: 10.7668/hbnxb.20191207.

LI Yonghong, ZHOU Yuchao, TIAN Qihang, CHANG Ruifeng, LIU Guojian, CHEN Hu, WANG Zhaoyuan. 2-DE Analysis of Cold-response Specific Proteins in Cortex of Branches in Peach Tree[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(S1): 102-108. doi: 10.7668/hbnxb.20191207.

http://www.hbnxb.net/CN/Y2020/V35/IS1/102

参考文献

[1] 常瑞丰,王召元,张立莎,李永红,陈湖,韩继成,刘国俭.晚熟桃新品种秋燕[J].园艺学报,2016,43(11):2281-2282.doi:10.16420/j.issn.0513-353x.2016-0330.
Chang R F,Wang Z Y,Zhang L S,Li Y H,Chen H,Han J C,Liu G J. A new late-ripening peach cultivar Qiuyan'[J]. Acta Horticulturae Sinica,2016,43(11):2281-2282.
[2] Mamonea G,Stasioa L D,Caro S D,Picariello G,Nicolai M A,Ferranti P. Comprehensive analysis of the peanut allergome combining 2-DE gel-based and gel-free proteomics[J]. Food Research International,2019,116:1059-1065.doi:10.1016/j.foodres.2018.09.045.
[3] Li L J,Lu X C,Ma H Y,Lyu D G. Comparative proteomic analysis reveals the roots response to low root-zone temperature in Malus baccata[J]. Journal of Plant Research,2018,131:865-878. doi:10.1007/s10265-018-1045-6.
[4] Zhou Y,Wu X X,Zhang Z,Gao Z H. Comparative proteomic analysis of floral color variegation in peach[J]. Biochemical and Biophsical Research Communications,2015,464(4):1101-1106. doi:10.1016/j.bbrc.2015.07.084.
[5] Zeng G Q,Zhang P F,Li C,Peng F,Li M Y,Xu Y,Yu F L,Chen M J,Yi H,Li G Q,Chen Z C,Xiao Z Q. Comparative proteome analysis of human lung squamous carcinoma using two different methods:two-dimensional gel electrophoresis and iTRAQ analysis[J]. Technology in Cancer Research and Treatment,2012,11(4):395-408. doi:10.7785/tcrt.2012.500287.
[6] Strohkamp S,Gemoll T,Habermann J K. Possibilities and limitations of 2DE-based analyses for identifying low-abundant tumor markers in human serum and plasma[J]. Proteomics,2016,16(19):2519-2532. doi:10.1002/pmic.201600154.
[7] Swatton J E,Prabakaran S,Karp N A,Lilley K S,Bahn S. Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis(2-D DIGE)[J]. Molecular Psychiatry,2004,9(2):128-143. doi:10.1038/sj.mp.4001475.
[8] Marouf K,Mohammad R N,Yousefzadeh S.Protein pattern analysis in tolerant and susceptible wheat cultivars undersalinity stress conditions[J]. Acta Agriculturae Slovenica,2018,111(3):545-558. doi:10.14720/aas.2018.111.3.03.
[9] Koehler G,Wilson R C,Goodpaster J V,Sonsteby A,Lai X Y,Witzmann F A,You J S,Rohloff J,Randall S K,Alsheikh M. Proteomic study of low-temperature responses in strawberry cultivars (Fragaria ananassa) that differ in cold tolerance[J]. Plant Physiology,2012,159:1787-1805. doi:10.1104/pp.112.198267.
[10] 陈奇,袁金海,刘自刚,孙万仓,方彦,赵新旺,马骊,蒲媛媛,赵艳宁,曾秀存.白菜型冬油菜‘陇油7号’叶片响应低温胁迫的差异蛋白鉴定与分析[J].中国生态农业学报,2017,25(3):381-390. doi:10.13930/j.cnki.cjea.160581.
Chen Q,Yuan J H,Liu Z G,Sun W C,Fang Y,Zhao X W,Ma L,Pu Y Y,Zhao Y N,Zeng X C. Differentially expressed proteins in response to low temperature in Brassica campestris Longyou No. 7 seedlings[J]. Chinese Journal of Eco-Agriculture,2017,25(3):381-390.
[11] 滕云龙,李春敏,赵永波,曾广娟,陈东玫,董文轩,张新忠.苹果实生树韧皮部中阶段转变特异蛋白质的SDS-PAGE分析[J].华北农学报,2009,24(1):112-114.doi:10.7668/hbnxb.2009.01.025.
Teng Y L,Li C M,Zhao Y B,Zeng G J,Chen D M,Dong W X,Zhang X Z. SDS-PAGE analysis of phase change-related proteins in phloem in apple(Malus domestica Borhk.)[J]. Acta Agriculturae Boreali-Sinica,2009,24(1):112-114.
[12] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72(1-2):248-254. doi:10.1016/0003-2697(76)90527-3.
[13] 李陈.人脐血间充质干细胞源性的类许旺细胞与人胎儿坐骨神经许旺细胞差异蛋白组学的研究[D].蚌埠:蚌埠医学院,2014.
Li C.Analysis of different proteomics between human umbilical cord blood mesenchymal stem cells derived schwann-like cells and fetal sciatic nerve's schwann cells[D]. Bengbu:Bengbu Medical College,2014.
[14] Shi X F,Wang X B,Cheng F,Cao H S,Liang H,Lu J Y,Kong Q S,Bie Z L. iTRAQ-based quantitative proteomics analysis of cold stress-induced mechanisms in grafted watermelon seedlings[J]. Journal of Proteomics,2019,192:311-320. doi:10.1016/j.jprot.2018.09.012.
[15] Li Q Y,Chang R,Sun Y J,Li B S. iTRAQ-based quantitative proteomic analysis of Spirulina platensis in response to low temperature stress[J]. PLoS One,2016,13(4):e0196442.doi:10.1371/joumal.pone.0196442.
[16] Zhao N,Hou M,Wang T,Chen Y F,Lü Y,Li Z R,Zhang R,Xin W T,Zou X Y,Hou L. Cloning and expression patterns of the brine shrimp(Artemia sinica) glycogen phosphorylase (GPase) gene during development and in response to temperature stress[J]. Molecular Biology Reports,2014,41:9-18. doi:10.1007/s11033-013-2764-3.
[17] Nam M H,Huh S M,Kim K M,Park W W,Seo J B,Cho K,Kim D Y,Kim B G,Yoon I I. Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice[J]. Proteome Science,2012,10:25. doi:10.1186/1477-5956-10-25.
[18] Xu J,Li Y,Sun J,Du L,Zhang Y,Yu Q,Liu X. Comparative physiological and proteomic response to abrupt low temperature stress between two winter wheat cultivars differing in low temperature tolerance[J]. Plant Biol,2013,15(2):292-303. doi:10.1111/j.1438-8677.2012.00639.x.
[19] 徐艳红.拟南芥捕光叶绿素a/b结合蛋白参与脱落酸信号转导[D].北京:中国农业大学,2011.
Xu Y H. Light-harvesting ChlorophⅡ a/b binding proteins are involved in ABA signaling in Arabidopsis[D].Beijing:China Agricultural University,2011.
[20] Xu Y Z,Zeng X C,Wu J,Zhang F Q,Li C X,Jiang J J,Wang Y P,Sun W C. iTRAQ-based quantitative proteome revealed metabolic changes in winter turnip rape (Brassica rapa L.) under cold stress[J]. Int J Mol Sci,2018,19(11):3346. doi:10.3390/ijms19113346.
[21] Zeng X C,Xu Y Z,Jiang J J,Zhang F Q,Ma L,Wu D W,Wang Y P,Sun W C. Identification of cold stress responsive microRNAs in two winter turnip rape (Brassica rapa L.) by high throughput sequencing[J]. BMC Plant Biol,2018,18:52-64. doi:10.1186/s12870-018-1242-4.
[22] Mansour M M F. Nitrogen containing compounds and adaptation of plants to salinity stress[J]. Biol Plant,2000,43:491-500. doi:10.1023/a:1002873531707.
[23] Szabados L,Savoure A. Proline:a multifunctional amino acid[J]. Trends Plant Sci,2010,15(2):89-97. doi:10.1016/j.tplants.2009.11.009.
[24] Claussen W. Proline as a measure of stress in tomato plants[J]. Plant Science,2005,168:241-248. doi:10.1016/j.plantsci.2004.07.039.
[25] Deng Y X,Lu S F. Biosynthesis and regulation of phenylpropanoids in plants[J]. Critical Reviews in Plant Sciences,2017,36(4):257-290.doi:10.1080/07352689.2017.1402852.
[26] Bonawitz N D,Chapple C. The genetics of lignin biosynthesis:connecting genotype to phenotype[J]. Annual Review of Genetics,2010,44:337-363. doi:10.1146/annurev-genet-102209-163508.
[27] Vanholme R,Storme V,Vanholme B,Sundin L,Cristensen J H,Goeminne G,Halpin C,Rohde A,Morreel K,Boerjan W. A systems biology view of responses to lignin biosynthesis perturbations in Arabidopsis[J]. Plant Cell,2012,24:3506-3529.doi:10.2307/41692822.
[28] Vanholme R,Cesarino I,Rataj K,Xiao Y G,Sundin L,Goeminne G,Kim H,Cross J,Morreel K,Araujo P,Welsh L,Haustraete J,McClellan C,Vanholme B,Ralph J,Simpson G G,Halpin C,Boerjan W. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis[J]. Science,2013,341(6150):1103-1106. doi:10.1126/science.1241602.
[29] Zhu J K. Abiotic stress signaling and responses in plants[J]. Cell,2016,167(5):313-324. doi:10.1016/j.cell.2016.08.029.
[30] 张亨. 逆境胁迫下LEA3蛋白亲水结构域的类分子伴侣功能研究[D].绵阳:西南科技大学,2015.
Zhang H. Investigationg of chaperone-like function of the LEA3 hydrophilic domain under stress conditions[D].Mianyang:Southwest University of Science and Technology,2015.
[31] 刘士霞. 逆境胁迫相关蛋白和含有Jmic结构域的组蛋白去甲基化酶在番茄抗病中的功能研究[D].杭州:浙江大学,2018.
Liu S X. Functional analysis of stress-associated proteins and Jmic domain-containing histone demethylases in disease resistance against Botrytis cinerea and pst DC3000 in tomato[D]. Hangzhou:Zhejiang Uiniversity,2018.
[32] Panigrahy D,Kalish B T,Huang S,Bielenberg D R,Le H D,Yang J,Edin M L, Lee C R,Benny O, Mudge D K,Butterfield C E,Mammoto A,Mammoto T,Inceoglu B,Jenkins R L, Simpson M A, Akino T, Lih F B, Tomer K B, Ingber D E,Hammock B D, Falck J R,Manthati V L, Kaipainen A, D'Amore P A, Puder M, Zeldin D C, Kieran M W.Epoxyeicosanoids promote organ and tissue regeneration[J]. Proc Natl Acad Sci USA,2013,110(33):13528-13533.doi:10.1073/pnas.1311565110.
[33] 郑柳城,朱宏波.水稻水溶性环氧化合物水解酶的生物信息学分析[J].生物信息学,2009,7(2):108-112.doi:10.3969/j.issn.1672-5565.2009.02.007.
Zheng L C,Zhu H P. Bioinformatics analysis on epoxide hydrolase of rice (Oryza saliva)[J]. China Journal of Bioinformaties,2009,7(2):108-112.

选择文件类型/文献管理软件名称

选择包含的内容