荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达
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摘要
为了挖掘荒漠昆虫小胸鳖甲Microdera punctipennis的耐寒性相关基因,从其4℃转录组数据库筛选出差异表达的几丁质酶基因片段394seq2,检测其编码蛋白的几丁质酶活性,研究该基因的表达对低温胁迫的响应情况。采用RACE技术扩增394seq2的5′端和3′端,克隆全长序列,将其ORF构建至原核表达载体pET28a,导入Transetta(DE3)感受态细胞,诱导表达融合蛋白,Western blot法检测表达蛋白的正确性;二硝基水杨酸法测定几丁质酶活性,qRT-PCR技术探究低温表达谱。结果表明,394seq2的5′-UTR为39 bp,3′-UTR为181 bp;ORF为1 140 bp。系统进化树显示该序列与赤拟谷盗几丁质酶8(TcCHT8)聚为一支,命名为MpCht8c。MpCht8c编码379个氨基酸,分子量为41.2 kDa,理论等电点pI为4.67。MpCHT8c含有完整的几丁质酶结构基序,其N端含有几丁质酶催化域,内有一个类18家族几丁质酶的保守基序KXXXXXGGW,中部是一段富PEST的连接区,C端是几丁质酶结合域,属于IV型昆虫几丁质酶。Western blot结果表明His-MpCHT8c在大肠杆菌中正确表达;融合蛋白粗酶液的几丁质酶活性为1.89 U/mL。在4℃冷胁迫0.5 h和5-9 h时Mpcht8c出现两个上调表达峰值,约为对照的2倍。研究表明荒漠昆虫小胸鳖甲的几丁质酶MpCHT8c具有几丁质酶活性,MpCht8c基因的表达可快速响应4℃低温胁迫。研究结果有助于深入研究几丁质酶在小胸鳖甲耐寒性方面的作用机理。
In order to discover more cold related genes from the desert beetle Microdera punctipennis, a gene fragment of a chitinase, 394 seq2, was selected from the transcriptome database of M. punctipennis treated at 4℃. The chitinase activity of its encoding protein was determined, and its expression profiling at low temperature was explored. The 5′-end and 3′-end of 394 seq2 were amplified by RACEs, and the full-length sequence was cloned. The ORF was inserted into plasmid pET28 a, and then transformed into Transetta(DE3) competent cells. The cells were induced by IPTG to over express the fusion protein. The protein was detected by SDS-PAGE and by Western blot. The chitinase activity of the protein was determined by DNS method. qRT-PCR was used to explore its expression profile at 4℃. The results showed that 394 seq2 had 39 bp 5′-UTR, 181 bp 3′-UTR, and 1 140 bp ORF. Phylogenetic tree analysis showed that 394 seq2 and chitinase 8 from Tribolium castaneum(TcCHT8) were clustered together, so 394 seq2 was named as MpCht8c. MpCht8c encoded a protein of 379 amino acids with a molecular mass of 41.2 kDa and a theoretical pI of 4.67. MpCHT8 contained complete motifs of a chitinase, with a chitinase catalytic domain in the N-terminal, a conserved KXXXXXGGW motif of 18 family chitinase in this region. The C-terminal had a chitinase binding domain. A junction region rich in proline(P), glutamic acid(E), serine(S), and threonine(T) connects the chitinase binding domain and the catalytic domain. It belonged to IV insect chitinase. Western blot result showed that His-MpCHT8 c was correctly expressed in E.coli. The chitinase activity of the crude protein was 1.89 U/mL. In conclusion, MpCHT8 c from the desert beetle M. punctipennis has chitinase activity, the expression of its encoding gene rapidly responds to 4℃ low temperature stress. These results will facilitate the study on the mechanism of chitinase in the cold resistance of M. punctipennis.
In order to discover more cold related genes from the desert beetle Microdera punctipennis, a gene fragment of a chitinase, 394 seq2, was selected from the transcriptome database of M. punctipennis treated at 4℃. The chitinase activity of its encoding protein was determined, and its expression profiling at low temperature was explored. The 5′-end and 3′-end of 394 seq2 were amplified by RACEs, and the full-length sequence was cloned. The ORF was inserted into plasmid pET28 a, and then transformed into Transetta(DE3) competent cells. The cells were induced by IPTG to over express the fusion protein. The protein was detected by SDS-PAGE and by Western blot. The chitinase activity of the protein was determined by DNS method. qRT-PCR was used to explore its expression profile at 4℃. The results showed that 394 seq2 had 39 bp 5′-UTR, 181 bp 3′-UTR, and 1 140 bp ORF. Phylogenetic tree analysis showed that 394 seq2 and chitinase 8 from Tribolium castaneum(TcCHT8) were clustered together, so 394 seq2 was named as MpCht8c. MpCht8c encoded a protein of 379 amino acids with a molecular mass of 41.2 kDa and a theoretical pI of 4.67. MpCHT8 contained complete motifs of a chitinase, with a chitinase catalytic domain in the N-terminal, a conserved KXXXXXGGW motif of 18 family chitinase in this region. The C-terminal had a chitinase binding domain. A junction region rich in proline(P), glutamic acid(E), serine(S), and threonine(T) connects the chitinase binding domain and the catalytic domain. It belonged to IV insect chitinase. Western blot result showed that His-MpCHT8 c was correctly expressed in E.coli. The chitinase activity of the crude protein was 1.89 U/mL. In conclusion, MpCHT8 c from the desert beetle M. punctipennis has chitinase activity, the expression of its encoding gene rapidly responds to 4℃ low temperature stress. These results will facilitate the study on the mechanism of chitinase in the cold resistance of M. punctipennis.
引文
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Dotsenko AS, Alexander VG, Pavel VV. N-Linked glycosylation of recombinant cellobiohydrolase I (Cel7A) From Penicillium verruculosum and its effect on the enzyme activity [J]. Biotechnology and Bioengineering, 2016, 113 (2): 283-291.
Funkhouser JD, Aronson-Jr NN. Chitinase family GH18:Evolutionary insights from the genomic history of a diverse protein family [J]. BMC Evolutionary Biology, 2007, 96 (7): 1-16.
Genta FA, Blanes L, Cristofoletti PT. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut [J]. Insect Biochemistry and Molecular Biology, 2006, 36: 789-800.
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Lu XY, Li JQ, Ma J,et al. Characterization and expression analysis of six chitinase genes from the desert beetle Microdera punctipennis in response to low temperature [J]. CryoLetters, 2014, 35 (5): 438-448.
Mazila-Ramli AN, Mahadi NM, Rabu A. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12 [J]. Microbial Cell Factories, 2011, 10 (94): 2-13.
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Su YC, Xu LP, Fu ZW, et al. ScChi, encoding an acidic class III chitinase of sugarcane, confers positive responses to biotic and abiotic stresses in sugarcane [J]. International Journal of Molecular Sciences, 2014, 15: 2738-2760.
Takao A, Noriko K, Shoko S,et al. Crystal structures of the catalytic domain of a novel glycohydrolase family 23 chitinase from Ralstonia sp.A-471 reveals a unique arrangement of the catalytic residues for inverting chitin hydrolysis [J]. Journal of Biological Chemistry, 2013, 288 (26): 18696-18706.
Tomcala A, Tollarová M, Overgaard J,et al. Seasonal acquisition of chill tolerance and restructuring of membrane glycerophospholipids in an overwintering insect: Triggering by low temperature, desiccation and diapause progression [J]. The Journal of Experimental Biology, 2006, 209: 4102-4114.
Wang XH, Chi NY, Fengwu B,et al. Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6 [J]. Extremophiles, 2016, 20: 167-176.
Worland MR, Convey P. Rapid cold hardening in Antarctic microarthropods [J]. Functional Ecology, 2001, 15: 515-524.
Yang JK, Yan Y, Li J,et al. Characterization and functional analyses of the chitinase-encoding genes in the nematodetrapping fungus Arthrobotrys [J]. Archives of Microbiology, 2013, 195: 453-462.
Zhang DW, Qian ZM, Zhang ZL,et al. The new research progress in function of insectchitinase gene family [J]. Journal of Environmental Entomology, 2016, 38 (1): 193-199. [张道伟, 钱正敏, 张正玲, 等. 昆虫几丁质酶基因家族功能研究进展 [J]. 环境昆虫学报, 2016, 38 (1): 193-199]
Zhang JZ, Zhang X, Yasuyuki A,et al. Comparative genomic analysis of chitinase and chitinase-Like genes in the African malaria mosquito (Anopheles gambiae) [J]. PLoS ONE, 2011, 6: 1-14.
Zheng MX, Chen Z, Liu B. The characteristics of chitinase and secondary mtobolites in Paenibacillus citinolyticus [J]. Fujian Journol of Agricultural Sciences, 2014, 29 (5): 492-487. [郑梅霞, 陈峥, 刘波. 解几丁质类芽胞杆菌几丁质酶活性及其次生代谢物的测定 [J]. 福建农业学报, 2014, 29 (5): 492-487]
Babiker MA, Abdel B, Daizo K. Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase [J]. The Journal of Biological Chemistry, 2002, 277: 30524-30534.
Cai QL, Lu XY, Ma J. Molecular cloning and expression profiling of a cold stress-related chitinase gene Mpcht19 in the desert beetle Microdera punctipennis (Coleoptera:Tenebrionidae) [J]. Acta Entomologica Sinica, 2017, 60 (3): 286-296. [蔡卿龄, 陆雪莹, 马纪. 荒漠甲虫小胸鳖甲低温响应几丁质酶基因Mpcht19的克隆及表达模式分析 [J]. 昆虫学报, 2017, 60 (3): 286-296]
Clark MS, Worland MR. How insects survive the cold: Molecular mechanisms [J]. Journal of Comparative Physiology, 2008, 178: 917-933.
Dotsenko AS, Alexander VG, Pavel VV. N-Linked glycosylation of recombinant cellobiohydrolase I (Cel7A) From Penicillium verruculosum and its effect on the enzyme activity [J]. Biotechnology and Bioengineering, 2016, 113 (2): 283-291.
Funkhouser JD, Aronson-Jr NN. Chitinase family GH18:Evolutionary insights from the genomic history of a diverse protein family [J]. BMC Evolutionary Biology, 2007, 96 (7): 1-16.
Genta FA, Blanes L, Cristofoletti PT. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut [J]. Insect Biochemistry and Molecular Biology, 2006, 36: 789-800.
Huang RX, Hu HY, Wu W,et al. Formation and evolution of desert insect in Xinjiang and its adjacent areas [J]. Arid Land Geography, 2005, 28 (1): 38-44. [黄人鑫, 胡红英, 吴卫, 等. 新疆及其毗邻地区荒漠昆虫区系的形成与演变 [J]. 干旱区地理, 2005, 28 (1): 38-44]
Li Y, Fan XJ. Insect chitinase and its application in insect pest control [J]. Chinese Journal of Applied Entomology, 2011, 48 (5): 1489-1494. [李瑶,范晓军. 昆虫几丁质酶及其在害虫防治中的应用 [J]. 应用昆虫学报, 2011, 48 (5): 1489-1494]
Lu XY, Li JQ, Ma J,et al. Characterization and expression analysis of six chitinase genes from the desert beetle Microdera punctipennis in response to low temperature [J]. CryoLetters, 2014, 35 (5): 438-448.
Mazila-Ramli AN, Mahadi NM, Rabu A. Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12 [J]. Microbial Cell Factories, 2011, 10 (94): 2-13.
Ouyang SW, Liu JL, Feng LX, et al. Research on insect chitinases and their application [J]. Journal of Mountain Agriculture and Biology, 2001, 20 (2): 147-153. [欧阳石文, 刘江良, 冯兰香, 等. 昆虫几丁质酶的研究与应用山地农业生物学报, 2001, 20 (2): 147-153]
Shi CH, Hu JR, Li CR,et al. Research progress in the cold tolerance mechanism of insects under environmental stress [J]. Plant Protection, 2016, 42 (6): 21-28. [史彩华, 胡静荣, 李传仁, 等. 环境胁迫下昆虫的耐寒适应机制研究进展 [J]. 植物保护, 2016, 42 (6): 21-28]
Su YC, Xu LP, Fu ZW, et al. ScChi, encoding an acidic class III chitinase of sugarcane, confers positive responses to biotic and abiotic stresses in sugarcane [J]. International Journal of Molecular Sciences, 2014, 15: 2738-2760.
Takao A, Noriko K, Shoko S,et al. Crystal structures of the catalytic domain of a novel glycohydrolase family 23 chitinase from Ralstonia sp.A-471 reveals a unique arrangement of the catalytic residues for inverting chitin hydrolysis [J]. Journal of Biological Chemistry, 2013, 288 (26): 18696-18706.
Tomcala A, Tollarová M, Overgaard J,et al. Seasonal acquisition of chill tolerance and restructuring of membrane glycerophospholipids in an overwintering insect: Triggering by low temperature, desiccation and diapause progression [J]. The Journal of Experimental Biology, 2006, 209: 4102-4114.
Wang XH, Chi NY, Fengwu B,et al. Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6 [J]. Extremophiles, 2016, 20: 167-176.
Worland MR, Convey P. Rapid cold hardening in Antarctic microarthropods [J]. Functional Ecology, 2001, 15: 515-524.
Yang JK, Yan Y, Li J,et al. Characterization and functional analyses of the chitinase-encoding genes in the nematodetrapping fungus Arthrobotrys [J]. Archives of Microbiology, 2013, 195: 453-462.
Zhang DW, Qian ZM, Zhang ZL,et al. The new research progress in function of insectchitinase gene family [J]. Journal of Environmental Entomology, 2016, 38 (1): 193-199. [张道伟, 钱正敏, 张正玲, 等. 昆虫几丁质酶基因家族功能研究进展 [J]. 环境昆虫学报, 2016, 38 (1): 193-199]
Zhang JZ, Zhang X, Yasuyuki A,et al. Comparative genomic analysis of chitinase and chitinase-Like genes in the African malaria mosquito (Anopheles gambiae) [J]. PLoS ONE, 2011, 6: 1-14.
Zheng MX, Chen Z, Liu B. The characteristics of chitinase and secondary mtobolites in Paenibacillus citinolyticus [J]. Fujian Journol of Agricultural Sciences, 2014, 29 (5): 492-487. [郑梅霞, 陈峥, 刘波. 解几丁质类芽胞杆菌几丁质酶活性及其次生代谢物的测定 [J]. 福建农业学报, 2014, 29 (5): 492-487]