水稻咖啡酰辅酶A-O-甲基转移酶基因的原核表达、抗体制备和应用

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

摘要/Abstract

摘要：

咖啡酰辅酶A-O-甲基转移酶（caffeoyl coenzyme A-O-methyltransferase,CCoAOMT）在植物木质素生物合成过程中发挥重要作用。目前关于水稻咖啡酰辅酶A-O-甲基转移酶的研究主要集中在基因水平,对其在蛋白水平上的功能研究却相当少。通过PCR扩增技术克隆得到OsCCoAOMT1基因,并成功构建了原核表达载体pET30a（+）-OsCCoAOMT1,转化大肠杆菌,表达出了重组蛋白。蛋白经纯化后,注射新西兰公兔,成功制备了多克隆抗体。Western blot检测表明该抗体能特异性地结合水稻不同组织的该蛋白。利用制备的抗体对水稻茎原生质体进行免疫荧光标记,激光共聚焦显微镜观察发现在Ubi：OsCCoAOMT1-GFP转基因水稻和野生型水稻原生质体里均可标记到该蛋白的信号,且转基因水稻融合蛋白的GFP信号可以与该蛋白抗体的荧光信号共定位,同时结合水稻核质蛋白分离的Western blot检测结果可知OsCCoAOMT1蛋白在主要分布在细胞质中。表明制备的抗体能用于该蛋白的特异性检测,为后续对水稻咖啡酰辅酶A-O-甲基转移酶的功能研究奠定了良好基础。

关键词: 咖啡酰辅酶A-O-甲基转移酶, 原核表达, 多克隆抗体, Western blot, 免疫荧光标记

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

索青青, 吴楠, 杨慧, 李莉, 王锡锋. 水稻咖啡酰辅酶A-O-甲基转移酶基因的原核表达、抗体制备和应用[J]. 生物技术通报, 2022, 38(8): 135-141.

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.

图/表 9

图1 OsCCoAOMT1基因的PCR扩增结果 M：DL 2000 DNA marker;1：OsCCoAOMT1基因扩增产物

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

图2 重组质粒pET30a（+）-OsCCoAOMT1的酶切鉴定 M：DL 5000 DNA marker;1：Nde I和Hind III酶切pET30a（+）-OsCCoAOMT1;2：pET30a（+）空载体

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

图3 重组质粒pET30a（+）-OsCCoAOMT1不同诱导条件下的SDS-PAGE分析 M：蛋白Marker;PC1：BSA（1 μg）;PC2：BSA（2 μg）;NC：未经诱导的细胞裂解液;1：在15℃下诱导16 h的细胞裂解液;2：在37℃下诱导4 h的细胞裂解液;NC1：未经诱导的细胞裂解液上清;3：经15℃下16 h诱导的细胞裂解液上清;4：37℃下诱导4 h的细胞裂解液上清;NC2：未诱导的细胞裂解液包涵体;5：15℃下诱导16 h的细胞裂解液包涵体;6：37℃下诱导4 h的细胞裂解液包涵体

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℃

图4 重组质粒pET30a（+）-OsCCoAOMT1不同诱导条件下的Western blot分析 M：Western blot marker;1：经15℃下16 h诱导的细胞裂解液上清;2：37℃下诱导4 h的细胞裂解液上清

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

图5 OsCCoAOMT1蛋白的纯化 M：Western blot marker;1：BSA;2：纯化的OsCCoAOMT1蛋白

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

图6 水稻茎、叶中OsCCoAOMT1的Western blot 检测 M：Western blot marker;A：水稻内参抗体（1：水稻茎总蛋白;2：水稻叶总蛋白）;B：OsCCoAOMT1抗体（1：水稻茎总蛋白;2：水稻叶总蛋白）

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）

图7 水稻不同蛋白浓度Western blot检测 M：Western blot marker;A：水稻内参抗体（1：100 μg;2：50 μg;3：25 μg;4：12.5 μg）;B：OsCCoAOMT1抗体（1：100 μg;2：50 μg;3：25 μg;4：12.5 μg）

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）

图8 OsCCoAOMT1蛋白在野生型（A）和转基因（B）水稻茎原生质体中的定位分析

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

图9 OsCCoAOMT1蛋白在水稻细胞质和细胞核中的表达量分析 M：Western blot marker;1：水稻茎细胞质蛋白;2：水稻茎细胞核蛋白

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

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