绿盲蝽取食诱导赤霞珠冬芽全蛋白双向电泳体系的建立与优化

doi:10.7668/hbnxb.20192668

摘要/Abstract

摘要：

建立绿盲蝽取食胁迫下赤霞珠冬芽防御反应的蛋白质组学双向电泳技术体系,旨在为后续筛选和分析差异蛋白相关研究提供参考。选取赤霞珠葡萄冬芽,分别设置绿盲蝽取食胁迫和空白对照试验,于24 h 后采集样品用液氮带回,-80 ℃冻存;利用TCA-丙酮法、TCA-丙酮法+酚抽提法提取粗蛋白干粉;利用CBB G-250染色法进行蛋白质浓度测定;确定上样量及凝胶染色技术。扫描得到凝胶图像,进行对比分析。结果显示,利用不同蛋白质提取方法均可获得双向电泳所需粗蛋白,采用TCA-丙酮法获取的粗蛋白量明显多于TCA-丙酮法+酚抽提法;2种方法所提取的全蛋白浓度差异较大,TCA-丙酮法操作更为简便,蛋白丢失少。不同上样量对双向电泳图谱效果的影响差异显著,400 μg蛋白上样量比800 μg所得的凝胶图片上的蛋白点清晰,易于分离。考马斯亮蓝染色结果表明,R-350更适用于葡萄冬芽全蛋白双向凝胶电泳技术。利用TCA-丙酮法抽提蛋白,400 μg蛋白上样,pH值 4~7 NL 17 cm 的IPG胶条,G-350热染色双向电泳技术体系得到的赤霞珠冬芽全蛋白凝胶图谱,符合绿盲蝽取食诱导赤霞珠防御反应产生防御蛋白的检测要求。

关键词: 赤霞珠葡萄, 绿盲蝽, 蛋白质组学, 双向电泳体系, 诱导抗性

Abstract:

In order to establish a proteomic 2-DE system for the defense response of Cabernet sauvignon winter buds under Apolygus lucorum feeding stress,Cabernet sauvignon grape winter buds were used as materials to compare the effects of different protein extraction methods,sample loading and gel staining techniques on the 2-DE chromatogram.It provided reference for subsequent studies on screening and analyzing differential proteins.Winter buds of Cabernet sauvignon grape were selected,and feeding stress and blank control experiments were conducted,respectively.After 24 h,the samples were taken back with liquid nitrogen and frozen at -80 ℃.Crude protein powder was extracted by TCA-acetone and TCA-acetone+phenol extraction.Protein concentration was determined by CBB G-250 staining.Sample loading and gel staining techniques were determined.Gel images were obtained by scanning for comparative analysis.Crude protein for 2-DE could be obtained by different extraction methods.The amount of crude protein obtained by TCA-acetone method was significantly higher than that obtained by TCA-acetone+phenol extraction method.The concentrations of total protein extracted by the two methods were different,but both of them met the requirements of two-dimensional gel electrophoresis.TCA-acetone method was easier to operate and less protein loss.The influence of different loading amount on the effect of 2-DE was significant.Compared with 800 μg protein,400 μg protein showed clear protein spots and was easy to separate.The results of CBB staining showed that R-350 was more suitable for total protein two-dimensional gel electrophoresis of grape winter bud.Using TCA-acetone method,400 μg protein sample,17 cm pH value 4—7 IPG dry adhesive strip,G-350 thermal staining 2-DE system to obtain the total protein gel map of Cabernet sauvignon winter buds met the requirements of further test.

Key words: Cabernet sauvignon, Apolygus lucorum, Proteomics, Two-dimensional electrophoresis, Inducible resistance

高素红, 葛长虹, 周国娜, 姚淑娟, 路常宽, 高宝嘉. 绿盲蝽取食诱导赤霞珠冬芽全蛋白双向电泳体系的建立与优化[J]. 华北农学报, 2022, 37(1): 181-187. doi: 10.7668/hbnxb.20192668.

GAO Suhong, GE Changhong, ZHOU Guona, YAO Shujuan, LU Changkuan, GAO Baojia. Establishment and Optimization of Bidirectional Electrophoresis System of Total Protein from Cabernet sauvignon Buds Induced by Apolygus lucorum Feeding[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 181-187. doi: 10.7668/hbnxb.20192668.

http://www.hbnxb.net/CN/Y2022/V37/I1/181

图/表 5

表1 不同提取方法赤霞珠冬芽全蛋白质量及浓度比较

Tab.1 Comparison of total protein quality and concentration of winter buds of Cabernet sauvignon with different extraction methods

提取方法 Extraction method	粗蛋白干粉质量/g Crude protein dry powder weight	粗蛋白质产率/% Crude protein productivity	蛋白浓度/(μg/mL) Protein concentration
TCA-丙酮法 TCA-acetone method	0.034 2±0.004 8A	1.000 0±0.017 6A	89.450 0±7.254 0A
TCA-丙酮法+酚抽提	0.003 7±0.000 6B	0.100 0±0.001 9B	187.400 0±15.250 3B
TCA-acetone+phenol extraction

图1 pH值 3~10 的 24 cm 胶条绿盲蝽取食诱导赤霞珠冬芽全蛋白不同提取方法双向凝胶电泳图对比
A.TCA-丙酮法全蛋白2-DE图像;B.TCA-丙酮法+酚抽提全蛋白2-DE图像。

Fig.1 Comparison of different extraction methods of total protein from Cabernet sauvignon winter buds induced by Apolygus lucorum feeding with 24 cm rubber strips at pH 3—10 by two-dimensional gel electrophoresis
A.TCA-acetone total protein 2-DE image;B.TCA-acetone+phenol extraction total protein 2-DE image.

图2 pH值 4~7 的 17 cm 胶条不同上样量赤霞珠冬芽全蛋白双向凝胶电泳图对比
A.绿盲蝽取食诱导赤霞珠冬芽400 μg蛋白上样量的2-DE图像;B.绿盲蝽取食诱导赤霞珠冬芽800 μg蛋白上样量的2-DE图像;C.对照400 μg蛋白上样量的2-DE图像;D.对照800 μg蛋白上样量的2-DE图像。

Fig.2 Comparison of total protein of Cabernet sauvignon winter bud by two-dimensional gel electrophoresis of 17 cm rubber strips at pH 4—7 with different loading amount
A.2-DE image of 400 μg protein loading amount of Cabernet sauvignon winter bud induced by Apolygus lucorum feeding;B.2-DE image of 800 μg protein loading amount of Cabernet sauvignon winter bud induced by Apolygus lucorum feeding;C.2-DE image of 400 μg protein loading amount; D.2-DE image of 800 μg protein loading amount.

图3 pH值 4~7 的 17 cm 胶条不同染剂赤霞珠冬芽全蛋白双向凝胶电泳图对比
A.绿盲蝽取食诱导赤霞珠冬芽全蛋白R-350染剂2-DE图像;B.绿盲蝽取食诱导赤霞珠冬芽全蛋白R-250染剂的2-DE图像; C.绿盲蝽取食诱导赤霞珠冬芽全蛋白G-250染剂的2-DE图像。

Fig.3 Comparison of total protein of Cabernet sauvignon winter bud with 17 cm rubber strips at pH 4—7 with different dyes by two-dimensional gel electrophoresis
A.2-DE image of the total protein R-350 dye of Cabernet sauvignon winter buds induced by Apolygus lucorum feeding; B.2-DE image of the total protein R-250 dye of Cabernet sauvignon winter buds induced by Apolygus lucorum feeding; C.2-DE image of the total protein G-250 dye of Cabernet sauvignon winter buds induced by Apolygus lucorum feeding.

图4 pH值 4~7 的 17 cm 胶条赤霞珠冬芽全蛋白R-350冷热染色双向凝胶电泳图对比
A.绿盲蝽取食诱导赤霞珠冬芽全蛋白R-350热染色的2-DE图像;B.绿盲蝽取食诱导赤霞珠冬芽全蛋白R-350冷染色的2-DE图像。

Fig.4 Comparison on 2-DE image of R-350 cold and hot staining of Cabernet sauvignon winter buds total protein with 17 cm rubber strip at pH 4—7
A.2-DE image of R-350 hot staining of Cabernet sauvignon winter buds total protein induced by Apolygus lucorum feeding;B.2-DE image of R-350 cold staining of Cabernet sauvignon winter buds total protein induced by Apolygus lucorum feeding.

参考文献 30

[1]	高素红,路常宽,赵春明,王硕,周雅茹.酿酒葡萄园区绿盲蝽种群动态与监测技术[J].应用昆虫学报,2015,52(5):1167-1173.doi:10.7679/j.issn.2095-1353.2015.139.
	Gao S H,Lu C K,Zhao C M,Wang S,Zhou Y R.The population dynamics and monitoring of Apolygus lucorum in vineyards[J].Chinese Journal of Applied Entomology,2015,52(5):1167-1173.
[2]	罗智捷.茶小绿叶蝉唾液介导茶树抗虫防御及其机理的研究[D].福州:福建农林大学,2020.doi:10.27018/d.cnki.gfjnu.2020.000327.
	Luo Z J.The mechanism of tea leafhopper saliva mediating defense response of tea plants[D].Fuzhou:Fujian Agriculture and Forestry University,2020.
[3]	韩月雯,吴瑞,马超锋,李园园.病毒-宿主蛋白相互作用组学研究进展[J].病毒学报,2021,37(4):997-1003.doi:10.13242/j.cnki.bingduxuebao.004015.
	Han Y W,Wu R,Ma C F,Li Y Y.Research progress in viral-host protein interactomics[J].Chinese Journal of Virology,2021,37(4):997-1003.
[4]	O'Farrell P.High resolution two-dimensional electrophoresis of proteins[J].Journal of Biological Chemistry,1975,250(10):4007-4021.doi:10.1016/S0021-9258(19)41496-8.
[5]	Bjellqvist B,Ek K,Giorgio Righetti P,Gianazza E,Görg A,Westermeier R,Postel W.Isoelectric focusing in immobilized pH gradients:Principle,methodology and some applications[J].Journal of Biochemical and Biophysical Methods,1982,6(4):317-339.doi:10.1016/0165-022X(82)90013-6.
[6]	左惠心,殷元虎,韩玲,马君义,宋仁德,余群力.宰后牦牛肉保水性变化与差异蛋白的生物信息学分析[J].农业机械学报,2017,48(7):325-331,300.doi:10.6041/j.issn.1000-1298.2017.07.041.
	Zuo H X,Yin Y H,Han L,Ma J Y,Song R D,Yu Q L.Changes of postmortem water-holding capacity in yak muscle and bioinformatic analysis of differentially abundant proteins[J].Transactions of the Chinese Society for Agricultural Machinery,2017,48(7):325-331,300.
[7]	Weiland F,Zammit C M,Reith F,Hoffmann P.High resolution two-dimensional electrophoresis of native proteins[J].ELECTROPHORESIS,2014,35(12/13):1893-1902.doi:10.1002/elps.201400060.
[8]	刘莹洁,王瑛丽,王琴,杨方云,周彦,周常勇.褐色橘蚜全蛋白双向电泳体系条件优化[J].植物保护学报,2018,45(4):689-696.doi:10.13802/j.cnki.zwbhxb.2018.2017191.
	Liu Y J,Wang Y L,Wang Q,Yang F Y,Zhou Y,Zhou C Y.Optimization of two-dimensional gel electrophoresis technology system for total protein from brown Citrus aphid Toxoptera citricida[J].Journal of Plant Protection,2018,45(4):689-696.
[9]	张辉,白晨,王华忠,张惠忠,李晓东,付增娟,赵尚敏,鄂圆圆,张自强,王良.甜菜双向电泳体系条件的优化[J].华北农学报,2017,32(5):112-116.doi:10.7668/hbnxb.2017.05.017.
	Zhang H,Bai C,Wang H Z,Zhang H Z,Li X D,Fu Z J,Zhao S M,E Y Y,Zhang Z Q,Wang L.Optimization of two dimensional electrophoresis system of sugar beet[J].Acta Agriculturae Boreali-Sinica,2017,32(5):112-116.
[10]	于亚文,朱新荣,张建.高白鲑肌肉蛋白质组双向电泳技术体系的优化[J].中国食品学报,2019,19(7):168-174.doi:10.16429/j.1009-7848.2019.07.022.
	Yu Y W,Zhu X R,Zhang J.Optimization of two-dimensional gel electrophoresis for proteome from Coregonus peled muscle[J].Journal of Chinese Institute of Food Science and Technology,2019,19(7):168-174.
[11]	张鹏飞,黄愉淋,耿双双,陈富美,付强,黄凤玲,陆阳清,张明.水牛睾丸曲精细管蛋白质组双向电泳方法的建立及优化[J].基因组学与应用生物学,2019,38(4):1474-1479.doi:10.13417/j.gab.038.001474.
	Zhang P F,Huang Y L,Geng S S,Chen F M,Fu Q,Huang F L,Lu Y Q,Zhang M.Establishment and optimization of two-dimensional electrophoresis for proteome of buffalo testicular seminiferous tubules[J].Genomics and Applied Biology,2019,38(4):1474-1479.
[12]	高佳敏,高素红,高宝嘉.昆虫取食和机械损伤对葡萄叶片代谢物的影响[J].西北农业学报,2019,28(9):1543-1551.doi:10.7606/j.issn.1004-1389.2019.09.021.
	Gao J M,Gao S H,Gao B J.Effects of insect feeding and mechanical damages on metabolites in leaves of grape[J].Acta Agriculturae Boreali-occidentalis Sinica,2019,28(9):1543-1551.
[13]	李林懋,门兴元,叶保华,于毅,张安盛.绿盲蝽对冬枣不同生长期的为害[J].植物保护学报,2013,40(6):545-550.doi:10.13802/j.cnki.zwbhxb.2013.06.010.
	Li L M,Men X Y,Ye B H,Yu Y,Zhang A S.Damage of mirid bug Apolygus lucorum to winter jujube at different growth stages[J].Acta Phytophylacica Sinica,2013,40(6):545-550.
[14]	王银翠,周国娜,张斌,陈明叶,高宝嘉.油松毛虫取食和剪叶刺激胁迫下油松的蛋白质表达差异分析[J].林业科学,2016,52(8):68-75.doi:10.11707/j.1001-7488.20160809.
	Wang Y C,Zhou G N,Zhang B,Chen M Y,Gao B J.Difference in protein expression of Pinus tabulaeformis induced by Dendrolimus tabulaeformis feeding and leaf-cutting stimulation[J].Scientia Silvae Sinicae,2016,52(8):68-75.
[15]	王瑞璞,崔蕾,张小莹,王茜,张朝红,李琰.葡萄种子蛋白质双向电泳体系的建立[J].北方园艺,2015(20):86-91.doi:10.11937/bfyy.201520021.
	Wang R P,Cui L,Zhang X Y,Wang Q,Zhang C H,Li Y.Establishment of two-dimensional electrophoresis system of grape seed[J].Northern Horticulture,2015(20):86-91.
[16]	倪瑞涓,曹洪祥,杨传平,李海英,王柏臣.一种聚丙烯酰胺凝胶的快速染色方法——热考染法[J].黑龙江大学自然科学学报,2008,25(3):393-395,400.doi:10.3969/j.issn.1001-7011.2008.03.028.
	Ni R J,Cao H X,Yang C P,Li H Y,Wang B C.A rapid staining method of polyacrylamide gel electrophoresis—heated CBB staining[J].Journal of Natural Science of Heilongjiang University,2008,25(3):393-395,400.
[17]	周琳,申加枝,段玉,王婷,王玉花,朱旭君,马媛春,房婉萍.茶树叶片蛋白双向电泳体系建立与应用[J].亚热带植物科学,2019,48(3):220-226.doi:10.3969/j.issn.1009-7791.2019.03.003.
	Zhou L,Shen J Z,Duan Y,Wang T,Wang Y H,Zhu X J,Ma Y C,Fang W P.Establishment and application of two-dimensional gel electrophoresis technology system for total protein of Camellia sinensis leaf[J].Subtropical Plant Science,2019,48(3):220-226.
[18]	宋归华,张迎新,侯泽豪,孙坤坤,方正武,马东方,张改生,王书平.小麦小花细胞核蛋白质双向电泳体系的优化[J].麦类作物学报,2018,38(6):686-692.doi:10.7606/j.issn.1009-1041.2018.06.08.
	Song G H,Zhang Y X,Hou Z H,Sun K K,Fang Z W,Ma D F,Zhang G S,Wang S P.Optimization of two-dimensional electrophoresis for nucleus from wheat floret[J].Journal of Triticeae Crops,2018,38(6):686-692.
[19]	焦竹青,许培磊,王振兴,艾军,刘迎雪,秦红艳,杨义明.山葡萄花序蛋白质双向电泳技术体系的建立[J].果树学报,2012,29(5):945-951.doi:10.13925/j.cnki.gsxb.2012.05.039.
	Jiao Z Q,Xu P L,Wang Z X,Ai J,Liu Y X,Qin H Y,Yang Y M.Establishment of two-dimensional electrophoresis platform for Vitis amurensis flower[J].Journal of Fruit Science,2012,29(5):945-951.
[20]	张玉洁,张军科,罗世杏.葡萄叶片蛋白质双向电泳技术体系的建立[J].北方园艺,2010(13):147-151.doi:10.11937/bfyy.2010013054.
	Zhang Y J,Zhang J K,Luo S X.Study on the conditions of 2-DE analysis of proteome in grape leaveas[J].Northern Horticulture,2010(13):147-151.
[21]	索慧英,郑密,卢晗,曲冠证,李莹.杨树叶片蛋白质双向电泳图谱的建立[J].林业科学研究,2020,33(2):128-137.doi:10.13275/j.cnki.lykxyj.2020.02.016.
	Suo H Y,Zheng M,Lu H,Qu G Z,Li Y.Two-dimensional electrophoresis profiles for proteins of poplar leaf[J].Forest Research,2020,33(2):128-137.
[22]	张微,刘迪,于婷婷,刘晓羽.苹果梨芽变果实蛋白质荧光差异双向凝胶电泳体系建立[J].分子植物育种,2019,17(9):2996-3000.doi:10.13271/j.mpb.017.002996.
	Zhang W,Liu D,Yu T T,Liu X Y.Establishment of 2D-DIGE for the protein of pingguoli bud mutation fruit[J].Molecular Plant Breeding,2019,17(9):2996-3000.
[23]	Bhattacharjee M,Dhar S,Handique P J,Acharjee S,Sarmah B K.Defense response in chickpea pod wall due to simulated herbivory unfolds differential proteome profile[J].The Protein Journal,2020,39(3):240-257.doi:10.1007/s10930-020-09899-9.
[24]	Hajian Z,Ghasemi M F E,Alikhani F E.The study of stress conditions on growth and proteome of Raoultella planticola:A new emerging pathogen[J].Archives of Microbiology,2021,203(6):3269-3278.doi:10.1007/s00203-021-02312-8.
[25]	Mishra S,Verma S K.Differential expression of proteins in Pseudomonas mendocina SMSKVR-3 under arsenate stress[J].Journal of Basic Microbiology,2021,61(4):351-361.doi:10.1002/jobm.202000671.
[26]	Jian M Y,Zhang D Z,Wang X Y,Wei S W,Zhao Y,Ding Q,Han Y C,Ma L J.Differential expression pattern of the proteome in response to cadmium stress based on proteomics analysis of wheat roots[J].BMC Genomics,2020,21(1):343.doi:10.1186/s12864-020-6716-8.
[27]	Scali M,Moscatelli A,Bini L C,Onelli E,Vignani R,Wang W.Protein analysis of pollen tubes after the treatments of membrane trafficking inhibitors gains insights on molecular mechanism underlying pollen tube polar growth[J].The Protein Journal,2021,40(2):205-222.doi:10.1007/s10930-021-09972-x.
[28]	Altenbach S B,Chang H C,Simon-Buss A.Deciphering the immunogenic potential of wheat flour:A reference map of the salt-soluble proteome from the US wheat Butte 86[J].Proteome Science,2020,18(1):1-13.doi:10.1186/s12953-020-00164-6.
[29]	Zhang P,Zhu Y Q,Shen C J,Zhou S J.Proteome analysis of cucumber responses to Pseudoperonospora cubensis infection[J].Journal of Plant Pathology,2019,101(4):917-925.doi:10.1007/s42161-019-00290-x.
[30]	Yamada E,Sasai K,Higa R,Mizuguchi H,Fuse Y.Characterization and dynamic analysis of dissolved proteins in lake Biwa using two-dimensional electrophoresis[J].Analytical Sciences,2020,36(6):753-759.doi:10.2116/analsci.19P450.

选择文件类型/文献管理软件名称

选择包含的内容