YANG Minghui,CHEN Meng,LIU Haifeng.Analysis of Two UDP-glycosyl Transferase Genes of Citrullus colocynthis[J].Northern Horticulture,2019,43(23):135-140.[doi:10.11937/bfyy.20191288]
药西瓜（Citrullus colocynthis）UDP-糖基转移酶基因表达分析
- Title:
- Analysis of Two UDP-glycosyl Transferase Genes of Citrullus colocynthis
- 文献标志码:
- A
- 摘要:
- 以药西瓜为试材，采用连接转化、双酶切等方法，在前期克隆得到的2个药西瓜UDP-糖基转移酶基因[STBX]UDP-E1、UDP-E2的基础上，将目的基因UDP-E1、UDP-E2[STBZ]与pET28a连接，构建重组质粒pET28a-[STBX]UDP-E1[STBZ]和pET28a-[STBX]UDP-E2[STBZ]，将重组质粒转化至E.coli BL21中，经过不同浓度的IPTG诱导，期望出现目的蛋白条带，且上清中的目的蛋白条带较沉淀与对照组明显。结果表明：成功构建原核表达重组质粒，IPTG浓度为1.0 mmol?L-1时，UDP-E1的融合蛋白分子量约为49 kDa，IPTG浓度为0.8 mmol?L-1时，UDP-E2的融合蛋白分子量约为32 kDa，目的蛋白在大肠杆菌BL21中得到了高效表达。
- Abstract:
- In this experiment,the experimental material was Citrulus colocynthis,and such methods as ligation conversion and double enzyme digestion were used.In early experiment,two Citrulus colocynthis UDP-glycolyltransferase genes UDP-E1,UDP-E2 were extracted by cloning.On the basis of this,the target genes UDP-E1 and UDP-E2 were connected with pET28a,constructed recombinant plasmids pET28a-UDP-E1 and pET28a-UDP-E2.The recombinant plasmids were transformed into E.coli BL21.Through the different concentration of IPTG induction,the target protein bands were expected to appear,and the target protein bands in the supernatant were more obvious than the precipitation and control group.The results showed that when the concentration of IPTG was 1.0 mmol?L-1,the molecular weight of UDP-E1 fusion protein was about 49 kDa,and when the concentration of IPTG was 0.8 mmol?L-1,the molecular weight of UDP-E2 fusion protein was about 32 kDa.The target protein was highly expressed in E.coli BL21.
参考文献/References:
[1]何江,程波,徐元,等.基于ITS2条形码鉴定维药药西瓜及其误用品[J].中国现代中药,2016,18(10):1261-1264.[2]赵千婧,程瑶,王佳,等.糖基转移酶合成相关糖苷类化合物研究进展[J].北京化工大学学报(自然科学版),2018,45(5):92-99.[3]陈蒙,袁赫海,杨铭慧,等.山葡萄类黄酮-3-O-葡萄糖基转移酶基因的表达分析[J].河南农业科学,2018,47(11):95-98.[4]刘海峰,杨成君,于淼,等.山葡萄UDP-葡萄糖:类黄酮-3-O-葡萄糖基转移酶基因(3GT)cDNA的克隆和分析[J].植物生理学通讯,2009,45(8):748-752.[5]马永硕.黄瓜中苦味素的生物合成、调控及转运机制[D].北京:中国农业科学院,2017.[6]凌冰,张茂新,王玉赞.葫芦素的生态功能及其应用前景[J].生态学报,2010,30(3):780-793.[7]彭朝霞,曹敏,张运杰,等.HPLC测定天花粉中葫芦素B的含量[J].中国现代应用药学,2009,26(11):946-948.[8]周姝婧,王祥瑞.双氢葫芦素B的抗炎和抗肿瘤作用[J].上海医学,2011,34(6):478-482.[9]王力先.中药单体葫芦素E对人外周血活化淋巴细胞的影响及其抗炎机制[C].北京:第十三届亚洲运动医学大会论文汇编,2014.[10]贾庆云.葫芦素E通过PI3K/Akt/NF-KB信号通路抑制TNF-α诱导的MH7A细胞症因子的分泌[D].济南:山东中医药大学,2015.[11]刘颖菊,蒋远明.葫芦素B对大鼠肝细胞凋亡的保护作用[J].四川生理科学杂志,2001,23(2):66-68.[12]曹淑桂,昌友权,曲红光,等.葫芦素B对过氧化氢诱导的大鼠肝细胞凋亡的抑制作用[J].食品科学,2005,26(9):527-530.[13]刘亭彦.葫芦素B治疗喉鳞状细胞癌的实验研究[D].北京:中国医科大学,2008.[14]乔静.葫芦素E对脂多糖诱导的小鼠巨噬细胞RAW 264.7 炎症反应的抑制作用及其机制研究[D].广州:暨南大学,2013.[15]BALLIANO G,CAPUTO O,VIOLA F,et al.Cyclization of squalene-2,3-epoxide to 10α-cucurbita-5,24-dien-3β-ol by microsomes from Cucurbita maxima seedlings[J].Phytochemistry,1983,22:915-921.[16]SHANG Y,GUO X X,LI W W,et al.Cucurbitacin-B inhibits neuroblastoma cell proliferation through up-regulation of PTEN[J].Eur Rev Med Pharmacol Sci,2014,18(21):3297-3303.[17]LIBY K T,YORE M M,SPORN M B.Triterpenoids and rexinoids as multifunctional agents for the prevention and treatment of cancer[J].Nature Reviews Cancer,2007,7(5):357-369.[18]吉宏.葫芦素及其药理学研究[J].国外医学中医中药分册,1996,18(6):13-14.[19]朱靖静,邹坤.葫芦素类四环三萜化合物的研究进展[J].三峡大学学报(自然科学版),2009,31(5):82-87.[20]孙婷婷,孙煦茵,王旭彤,等.桑黄鲨烯环氧酶基因原核表达及过表达载体的构建[J].中草药,2018,49(11):2632-2639.[21]王碧霞,徐德宏,谭朝阳,等.小叶女贞糖基转移酶基因的克隆和原核表达研究[J].中国中药杂志,2018,43(4):704-711.[22]张婷婷,梁会超,巩婷,等.人参糖基转移酶PgUGT74AE2催化生成新型人参三醇皂苷研究[J].药学学报,2018,53(9):1565-1570.[23]李佳兴,莫歌,周良云,等.欧洲花楸糖基转移酶基因的全长克隆与表达分析[J].中国实验方剂学杂志,2019,25(5):167-172.[24]王源杰,郭雪峰,赵蕾,等.毛竹叶黄酮碳苷C-糖基化途径及C-糖基转移酶的分析[J].林业科学,2018,54(12):60-69.
备注/Memo
第一作者简介：杨铭慧（1994-），女，硕士研究生，研究方向为药西瓜基因克隆。E-mail：1918637877@qq.com.责任作者：刘海峰（1973-），男，博士，副教授，硕士生导师，研究方向为山葡萄与药西瓜基因克隆。E-mail：Liufeng_1989@163.com.基金项目：国家自然科学基金资助项目（31511140284）。收稿日期：2019-07-15