Prokaryotic Expression,Antibody Preparation and Application of Rice Caffeoyl Coenzyme A-O-methyltransferase Gene

doi:10.13560/j.cnki.biotech.bull.1985.2021-1509

Abstract

Abstract:

Caffeoyl-coenzyme A-O-methyltransferase（CCoAOMT）plays an important role in the biosynthesis of plant lignin. At present,the research on rice CCoAOMT mainly focuses on the gene level,only few researches on its function at the protein level. In this paper,OsCCoAOMT1 gene was cloned by PCR amplification technology,and prokaryotic expression vector pET30a（+）-OsCCoAOMT1,then transformed into Escherichia coli,and the recombinant protein was expressed. Purified protein immunized New Zealand male rabbits of polyclonal antibodies were successfully prepared. Western blot analysis showed that the antibody specifically bound to the protein in different tissues of rice. Immunofluoresent labeling assay revealed that the signal of this protein can be marked in Ubi:OsCCoAOMT1-GFP transgenic rice and wild-type rice protoplasts via laser confocal microscopy. Furthermore,the GFP signal of the transgenic rice fusion protein can be co-localized with the fluorescent signal of the protein antibody. Combined with the Western blot analysis of rice nucleoplasm protein isolation,OsCCoAOMT1 protein is mainly distributed in the cytoplasm. This indicated that the prepared antibody can be used for the specific detection of the protein and laid a good foundation for the subsequent functional research of rice CCoAOMT.

Key words: caffeoyl-coenzyme A-O-methyltransferase, prokaryotic expression, polyclonal antibody, Western blot, immunofluorescence labelling

SUO Qing-qing, WU Nan, YANG Hui, LI Li, WANG Xi-feng. Prokaryotic Expression,Antibody Preparation and Application of Rice Caffeoyl Coenzyme A-O-methyltransferase Gene[J]. Biotechnology Bulletin, 2022, 38(8): 135-141.

Figures/Tables 9

Fig.1 PCR amplifying product of OsCCoAOMT1 gene M：DL 2000 DNA marker;1：PCR product of OsCCoAOMT1

Fig.2 Restriction identification of recombinant plasmid pET30a（+）-OsCCoAOMT1 M：DL 5000 DNA marker;1：pET30a（+）-OsCCoAOMT1 digested by Nde I and Hind III;2：pET30a（+）plasmid

Fig.3 SDS-PAGE analysis of recombinant plasmid pET30a（+）-OsCCoAOMT1 under the different inducing condition M：Protein marker;PC1：BSA（1 μg）;PC2：BSA（2 μg）. NC：Cell lysate without induction. 1：Cell lysate with induction for 16 h at 15℃. 2：Cell lysate with induction for 4 h at 37℃. NC1：Supernatant of cell lysate without induction. 3：Supernatant of cell lysate with induction for 16 h at 15℃. 4：Supernatant of cell lysate with induction for 4 h at 37℃. NC2：Pellet of cell lysate without induction. 5：Pellet of cell lysate with induction for 16 h at 15℃. 6：Pellet of cell lysate with induction for 4 h at 37℃

Fig.4 Western blot analysis of recombinant plasmid pET30a（+）-OsCCoAOMT1 under the different inducing condition M：Western blot marker. 1：Supernatant of cell lysate induced at 15℃ for 16 h. 2：Supernatant of cell lysate induced at 37℃ for 4 h

Fig.5 Purification of OsCCoAOMT1 protein M：Western blot marker. 1：BSA. 2：Purified OsCCoAOMT1 protein

Fig.6 Western blot analysis of OsCCoAOMT1 in rice stem and leaf M：Western blot marker. A：Rice control antibody（1：rice stem protein;2：rice leaf protein）. B：OsCCoAOMT1 antibody（1：rice stem protein;2：rice leaf protein）

Fig.7 Western blot analysis of different protein concentra-tions in rice M：Western blot marker;A：rice control antibody（1：100 μg;2：50 μg;3：25 μg;4：12.5 μg）. B：OsCCoAOMT1 antibody（1：100 μg;2：50 μg;3：25 μg;4：12.5 μg）

Fig.8 Location analysis of OsCCoAOMT1 protein in the wild-type（A）and transgenic（B）rice stem protoplasts

Fig. 9 Expression level analysis the OsCCoAOMT1 protein in rice cytoplasm and nucleus M：Western blot marker;1：rice stem cytoplasmic protein;2：rice stem nucleoprotein

References 22

[1]	Bonawitz ND, Chapple C. The genetics of lignin biosynthesis:connecting genotype to phenotype[J]. Annu Rev Genet, 2010, 44:337-363. doi: 10.1146/annurev-genet-102209-163508 URL
[2]	Engelhardt J. Sources, industrial derivatives and commercial application of cellulose[J]. Carbohydrate Eur, 1995, 12:5-14.
[3]	Ma QH, Luo HR. Biochemical characterization of caffeoyl coenzyme A 3-O-methyltransferase from wheat[J]. Planta, 2015, 242(1):113-122. doi: 10.1007/s00425-015-2295-3 URL
[4]	Ralph J, Lundquist K, Brunow G, et al. Lignins:Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids[J]. Phytochem Rev, 2004, 3(1/2):29-60. doi: 10.1023/B:PHYT.0000047809.65444.a4 URL
[5]	魏建华, 宋艳茹. 木质素生物合成途径及调控的研究进展[J]. 植物学报, 2001, 43(8):771-779.
	Wei JH, Song YR. Recent advances in study of lignin biosynthesis and manipulation[J]. Acta Bot Sin, 2001, 43(8):771-779.
[6]	陈冰玉, 邸明伟. 木质素解聚研究新进展[J]. 高分子材料科学与工程, 2019, 35(6):157-164.
	Chen BY, Di MW. Progress on research of depolymerization of lignin[J]. Polym Mater Sci Eng, 2019, 35(6):157-164.
[7]	Ye ZH, Kneusel RE, Matern U, et al. An alternative methylation pathway in lignin biosynthesis in Zinnia[J]. Plant Cell, 1994, 6(10):1427-1439. pmid: 7994176
[8]	Pakusch AE, Kneusel RE, Matern U. S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures[J]. Arch Biochem Biophys, 1989, 271(2):488-494. pmid: 2499260
[9]	Pinçon G, Maury S, Hoffmann L, et al. Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth[J]. Phytochemistry, 2001, 57(7):1167-1176. pmid: 11430989
[10]	林楠, 王彦珍, 李桢, 等. 抑制咖啡酰-辅酶A甲基转移酶(CCoAOMT)表达的转基因多年生杨树检测与分析[J]. 农业生物技术学报, 2009, 17(6):1121-1122.
	Lin N, Wang YZ, Li Z, et al. Analysis and evaluation of multiple-year-old transgenic poplar with antisense caffeoyl-CoA-O-methyltransferase(CCoAOMT)[J]. J Agric Biotechnol, 2009, 17(6):1121-1122.
[11]	Zhong R, Morrison WH 3rd, Himmelsbach DS, et al. Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants[J]. Plant Physiol, 2000, 124(2):563-578. pmid: 11027707
[12]	Bhuiyan NH, Selvaraj G, Wei YD, et al. Role of lignification in plant defense[J]. Plant Signal Behav, 2009, 4(2):158-159. doi: 10.4161/psb.4.2.7688 pmid: 19649200
[13]	宫世龙. 甜菜M14品系咖啡酰辅酶A-O-甲基转移酶基因功能的研究[D]. 哈尔滨: 黑龙江大学, 2013.
	Gong SL. Study on the function of caffeoyl-CoA-O-methyltransferase gene in sugar beet M14[D]. Harbin: Helongjiang University, 2013.
[14]	Chun HJ, Lim LH, Cheong MS, et al. Arabidopsis CCoAOMT1 plays a role in drought stress response via ROS- and ABA-dependent manners[J]. Plants(Basel), 2021, 10(5):831.
[15]	赵华燕, 沈庆喜, 吕世友, 等. 水稻咖啡酰辅酶A-O-甲基转移酶基因(CCoAOMT)表达特性分析[J]. 科学通报, 2004, 49(14):1390-1394.
	Zhao HY, Shen QX, Lv SY, et al. Analysis of expression characteristics of rice caffeoyl-CoA-O-methyltransferase gene(CCoAOMT)[J]. Chin Sci Bull, 2004, 49(14):1390-1394. doi: 10.1360/csb2004-49-14-1390 URL
[16]	Wang YJ, Mao QZ, Liu WW, et al. Localization and distribution of wheat dwarf virus in its vector leafhopper, Psammotettix alienus[J]. Phytopathology, 2014, 104(8):897-904. doi: 10.1094/PHYTO-09-13-0251-R URL
[17]	程彦伟, 李亮, 沈嵘, 等. 水稻LRR型类受体蛋白激酶胞外区的原核表达及多克隆抗体制备[J]. 生物化学与生物物理进展, 2008, 35(9):1077-1083.
	Cheng YW, Li L, Shen R, et al. Prokaryotic expression and polyclonal antibody preparation of the extracellular domain about rice LRR receptor-like protein kinase[J]. Prog Biochem Biophys, 2008, 35(9):1077-1083.
[18]	王增, 代茹, 张江巍, 等. 拟南芥WUSCHEL蛋白的原核表达、亲和纯化和多克隆抗体制备[J]. 生物工程学报, 2009, 25(9):1409-1416.
	Wang Z, Dai R, Zhang JW, et al. Induced expression of Arabidopsis thaliana WUSCHEL in Escherichia coli, affinity protein purification and polyclonal antibody preparation[J]. Chin J Biotechnol, 2009, 25(9):1409-1416.
[19]	秦伟, 黄昆仑, 贺晓云, 等. 水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化[J]. 食品科学, 2008, 29(7):267-271.
	Qin W, Huang KL, He XY, et al. Expression of rice Codon optimized cry2A* gene in Escherichia coli and purification of its expressed proteins[J]. Food Sci, 2008, 29(7):267-271.
[20]	Chai HZ, Wu SB, Deng JF, et al. Preparation and identification of polyclonal antibody against human Cytomegalovirus encoding protein UL23[J]. Protein Expr Purif, 2019, 161:78-83. doi: 10.1016/j.pep.2019.04.008 URL
[21]	Yang Q, He YJ, Kabahuma M, et al. A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens[J]. Nat Genet, 2017, 49(9):1364-1372. doi: 10.1038/ng.3919 pmid: 28740263
[22]	Wang YG, Zhan YN, Wu C, et al. Cloning of a cystatin gene from sugar beet M14 that can enhance plant salt tolerance[J]. Plant Sci, 2012, 191/192:93-99. doi: 10.1016/j.plantsci.2012.05.001 URL