毛果杨半胱氨酸蛋白酶基因PtCP3 的基因克隆和原核表达分析

doi:10.11924/j.issn.1000-6850.casb16010111

中国农学通报 ›› 2016, Vol. 32 ›› Issue (14): 50-55.doi: 10.11924/j.issn.1000-6850.casb16010111

所属专题：生物技术

毛果杨半胱氨酸蛋白酶基因PtCP3 的基因克隆和原核表达分析

要笑云,张强,撖静宜,王莹,曹山,蒋璐瑶,李丽红,李慧,陆海

(北京林业大学生物科学与技术学院,北京 100083）

收稿日期:2016-01-22 修回日期:2016-03-16 接受日期:2016-03-17 出版日期:2016-05-17 发布日期:2016-05-17
通讯作者: 陆海
基金资助:
国家自然科学基金项目“杨树CEP1类半胱氨酸蛋白酶参与木质部细胞分化和程序化死亡的分子机制研究”(31570582)，“杨树4CL基因家族调控木质素生物合成机制研究”(30671697)，“杨树半胱氨酸蛋白酶基因结构与酶学特性研究”(J1103516)。

Cloning of Cysteine Protease Gene PtCP3 and Its Prokaryotic Expression in Populus trichocarpa Torr. & Gray

Yao Xiaoyun, Zhang Qiang, Han Jingyi, Wang Ying, Cao Shan, Jiang Luyao, Li Lihong, Li Hui, Lu Hai

(College of Biological Science and Technology, Beijing Forestry University, Beijing 100083)

Received:2016-01-22 Revised:2016-03-16 Accepted:2016-03-17 Online:2016-05-17 Published:2016-05-17

摘要/Abstract

摘要： 为研究毛果杨半胱氨酸蛋白酶基因PtCP3的酶学特征和生理功能，本实验对PtCP3进行了基因克隆和原核表达分析。从毛果杨(Populus trichocarpa Torr. & Gray)中克隆得到半胱氨酸蛋白酶基因PtCP3，将其克隆至pMD-18T载体。进一步构建原核表达载体pET30a-PtCP3，并在大肠杆菌(Escherichia coli)中成功诱导表达重组蛋白，然后通过包涵体洗涤法对目的蛋白进行纯化。测序结果表明，PtCP3基因CDS序列全长1074 bp，含有木瓜蛋白酶的保守催化位点和组织蛋白酶B的保守结构域。序列分析结果显示，半胱氨酸蛋白酶基因PtCP3编码357个氨基酸，预测N-末端含有长度为27个氨基酸残基的信号肽序列，去除信号肽后蛋白酶大小为36.515 kDa，理论等电点为7.44。SDS-PAGE电泳结果显示，可以检测到大小约为42 kDa的目的条带，并可通过包涵体洗涤法在体外得到了高表达量、单一的重组蛋白PtCP3。

关键词: 水稻, 水稻, 稻瘟病菌, 白叶枯菌, 病原菌分子诱导的抗病性, 效应蛋白诱导的抗病性, 效应蛋白-抗病蛋白互作

Abstract: The objective was to study the enzymatic characteristics and physiological functions of PtCP3 gene of cysteine protease. PtCP3 gene cloning and prokaryotic expression analysis were conducted. The cysteine protease gene PtCP3 was cloned from Populus trichocarpa in this research. PtCP3 was constructed into the expression vector pMD-18T and then constructed the prokaryotic expression vector pET30a-PtCP3. The recombinant protein was induced and expressed in Escherichia coli successfully. The target protein was purified by washing method on inclusion body. The sequencing results showed that the full-length CDS sequence of PtCP3 was 1074 bp, and contained the conservative catalytic sites of papain and the conservative structural domain of cathepsin B. The sequence analysis showed that cysteine protease gene PtCP3 encoded 357 amino acids, and predicted N-terminus hydrophobic region containing a signal peptide with 27 amino acid residues. The protease getting rid of signal peptide was 36.515 kDa, and the isoelectric point was 7.44. The SDS-PAGE showed that the target band with 42 kDa molecular weight was obtained. The high-express and single recombinant protein PtCP3 was obtained by washing method on inclusion body.

中图分类号:

要笑云,张强,撖静宜,王莹,曹山,蒋璐瑶,李丽红,李慧,陆海. 毛果杨半胱氨酸蛋白酶基因PtCP3 的基因克隆和原核表达分析[J]. 中国农学通报, 2016, 32(14): 50-55.

Yao Xiaoyun,Zhang Qiang,Han Jingyi,Wang Ying,Cao Shan,Jiang Luyao,Li Lihong,Li Hui and Lu Hai. Cloning of Cysteine Protease Gene PtCP3 and Its Prokaryotic Expression in Populus trichocarpa Torr. & Gray[J]. Chinese Agricultural Science Bulletin, 2016, 32(14): 50-55.

参考文献

[1] Zhang D, Liu D, Lv X, et al. The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis[J]. Plant Cell, 2014,26(7):2939-2961.
[2] Khanna-Chopra R, Srivalli B, Ahlawat Y S. Drought induces many forms of cysteine proteases not observed during natural senescence[J]. Biochemical and Biophysical Research Communications,1999,255(2):324-327.
[3] Bar-Ziv A, Levy Y, Citovsky V, et al. The Tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1.[J]. Biochemical & Biophysical Research Communications, 2015, 460(3):525-529.
[4] Trobacher C P, Senatore A, Greenwood J S. Masterminds or minions Cysteine proteinases in plant programmed cell death[J]. Canadian Journal of Botany, 2006,84: 651-667.
[5] McLellan H, Gilroy E M, Yun B W, et al. Functional redundancy in the Arabidopsis Cathepsin B gene family contributes to basal defence, the hypersensitive response and senescence[J]. New Phytologist, 2009,183(2):408-418.
[6] Wan L, Xia Q, Qiu X, et al. Early stages of seed development in Brassica napus: a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument[J]. Plant Journal, 2002,30(1):1-10.
[7] Ueda T, Seo S, Ohashi Y, et al. Circadian and senescence-enhanced expression of a tobacco cysteine protease gene[J]. Plant Molecular Biology, 2000, 44(5):649-657.
[8] Wagstaff C, Leverentz M K, Griffiths G, et al. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals[J]. Journal of Experimental Botany, 2002,53(367):233-240.
[9] Grudkowska M, Zagdańska B. Multifunctional role of plant cysteine proteinases[J]. Acta Biochimica Polonica, 2004,51(3):609-624.
[10] Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, et al. Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[J]. Gene, 1993,129(2):175-182.
[11] Mackey D, Holt B F 3rd, Wiig A, et al. RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis[J]. Cell, 2002,108(6):743-754.
[12] Barrett A J, Rawlings N D. Evolutionary lines of cysteine peptidases[J]. Biological Chemistry, 2001,382(5):727-733.
[13] Bar-Ziv A, Levy Y, Citovsky V, et al. The Tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteineprotease CYP1[J]. Biochemical and biophysical research communications, 2015,460(3):525-529.
[14] Wiederanders B. Structure-function relationships in class CA1 cysteine peptidase propeptides[J]. Acta Biochimica Polonica, 2003,50(3):691-713.
[15] 闫龙凤,杨青川,韩建国,等.植物半胱氨酸蛋白酶研究进展[J].草业报, 2005,14(5):11-18.
[16] Richau K H, Kaschani F, Verdoes M, et al. Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics[J]. Plant Physiology, 2012,158(4):1583-1599.
[17] Karrer K M, Peiffer S L, DiTomas M E. Two distinct gene subfamilies within the family of cysteine protease genes[J]. Proceedings of the National Academy of Sciences of the United States of America,1993,90(7):3063-3067.
[18] Wiederanders B. Structure-function relationship in class CA1 cysteine peptidase propeptides[J]. Acta Biochemica PolonIca, 2003,50(3):691-713.
[19] Riese R J, Chapman H A. Cathepsins and compartmentalization in antigen presentation[J]. Current Opinion in Immunology, 2000,12(1):107-13.
[20] Martinez M, Diaz I. The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship[J].BMC Evolutionary Biology, 2008,8:198.

毛果杨半胱氨酸蛋白酶基因PtCP3 的基因克隆和原核表达分析

Cloning of Cysteine Protease Gene PtCP3 and Its Prokaryotic Expression in Populus trichocarpa Torr. & Gray

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