苦瓜α-苦瓜素基因启动子克隆与单倍型分析
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摘要
为探明α-苦瓜素基因的表达调控规律,从苦瓜种质Y5中克隆了α-苦瓜素基因上游包括起始密码子在内的1 620 bp序列,选取转录起始位点上游1 500 bp序列,利用Plant CARE在线启动子预测工具进行分析。结果表明,α-苦瓜素启动子除了含有TATA-box和CAAT-box等核心元件外,还含有光响应元件、赤霉素响应元件、热胁迫响应元件、干旱应答元件、水杨酸应答元件、茉莉酸应答元件等。以28份苦瓜种质为材料,分析了α-苦瓜素启动子区域SNP和In Del分布情况,共发现24个SNP位点和1个In Del,28份苦瓜种质资源共存在4种单倍型。3个SNP位于转录因子结合位点,可能对α-苦瓜素基因的表达调控有重要作用。
In order to explore the expression regulation of α-momorcharin gene,the upstream sequence,1 620 bp in length including start codon of α-momorcharin gene was cloned in bitter gourd( Y5). The 1 500 bp upstream of the transcription start site was selected for analysis based on Plant CARE promoter prediction tools. The results showed that in addition to containing the core elements of TATA-box and CAAT-box,the α-momorcharin promoter also contained light response elements,gibberellin response element,heat stress response element,drought response element,salicylic acid response element,jasmonic acid response element etc. Taking 28 bitter gourd germplasm as materials,the SNP and In Del distribution in promoter region of α-momorcharin was analyzed. Twenty-four SNP loci and1 In Del were found. Four haplotypes were identified in α-momorcharin gene promoter through SNP and In Del grouping. Three SNPs were located in transcription factor binding sites,which may play important roles in regulating the expression of α-momorcharin gene.
In order to explore the expression regulation of α-momorcharin gene,the upstream sequence,1 620 bp in length including start codon of α-momorcharin gene was cloned in bitter gourd( Y5). The 1 500 bp upstream of the transcription start site was selected for analysis based on Plant CARE promoter prediction tools. The results showed that in addition to containing the core elements of TATA-box and CAAT-box,the α-momorcharin promoter also contained light response elements,gibberellin response element,heat stress response element,drought response element,salicylic acid response element,jasmonic acid response element etc. Taking 28 bitter gourd germplasm as materials,the SNP and In Del distribution in promoter region of α-momorcharin was analyzed. Twenty-four SNP loci and1 In Del were found. Four haplotypes were identified in α-momorcharin gene promoter through SNP and In Del grouping. Three SNPs were located in transcription factor binding sites,which may play important roles in regulating the expression of α-momorcharin gene.
引文
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[10]沈富林,曹东亮,邓斐,等.α-苦瓜素体内抗人乳腺癌MCF-7移植瘤实验[J].成都医学院学报2014,9(6):686-689.SHEN F L,CAO D L,DENG F,et al.The anti-tumor experiments of alpha-momorcharin on human breast MCF-7 xenograft in vivo[J].Journal of Chengdu Medical College,2014,9(6):686-689.(in Chinese with English abstract)
[11]WANG P,HUANG S,WANG F,et al.Cyclic AMP-response element regulated cell cycle arrestsin cancer cells[J].PLo S One,2013,8(6):e65661.
[12]LIU F,WANG B,WANG Z,et al.Trichosanthin down-regulates Notch signaling and inhibits proliferation of the nasopharyngeal carcinoma cell line CNE2 in vitro[J].Fitoterapia,2012,83(5):838-842.
[13]蔡秀清.α-苦瓜素在毕赤酵母中表达及生物活性分析[D].儋州:华南热带农业大学,2005.CAI X Q.Expression and bioactivity assay of alpha-momorcharin gene in Pichia pastoris[D].Danzhou:South China University of Tropical Agriculture,2005.(in Chinese with English abstract)
[14]欧阳永长,庄东红,胡忠,等.α-苦瓜素基因的克隆和原核表达[J].武汉植物学研究,2008,26(1):7-11.OUYANG Y C,ZHUANG D H,HU Z,et al.Cloning and prokaryotic expression of alpha-momorcharin gene[J].Journal of Wuhan Botanical Research,2008,26(1):7-11.(in Chinese with English abstract)
[15]王书珍.重组核糖体失活蛋白alpha-苦瓜素的生物学活性分析[D].武汉:武汉大学,2013.WANG S Z.Analysis of the biological activities of a recombinant ribosome-inactivating protein(alpha-momorcharin)[D].Wuhan:Wuhan University,2013.(in Chinese with English abstract)
[16]QIAN Q,HUANG L,YI R,et al.Enhanced resistance to blast fungus in rice(Oryza sativa L.)by expressing the ribosome-inactivating proteinα-momorcharin[J].Plant Science,2014,217/218(1):1-7.
[17]刘子记,牛玉,朱婕,等.苦瓜核心种质资源构建方法的比较[J].华南农业大学学报,2017,38(1):31-37.LIU Z J,NIU Y,ZHU J,et al.Comparison of different methods to construct core collections of Momordica charantia[J].Journal of South China Agricultural University,2017,38(1):31-37.(in Chinese with English abstract)
[18]刘子记,牛玉,杨衍.热研一号油绿苦瓜种子纯度的SSR鉴定[J].热带作物学报,2013,34(11):2179-2182.LIU Z J,NIU Y,YANG Y.Identification of bitter gourd‘Reyan No.1’seed purity by SSR markers[J].Chinese Journal of Tropical Crops,2013,34(11):2179-2182.(in Chinese with English abstract)
[19]URASAKI N,TAKAGI H,NATSUME S,et al.Draft genome sequence of bitter gourd(Momordica charantia),a vegetable and medicinal plant in tropical and subtropical regions[J].DNA Research:An International Journal for Rapid Publication of Reports on Genes and Genomes,2017,24(1):51-58.
[20]LESCOT M,DEHAIS P,MOREAU Y,et al.Plant CARE:a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.
[21]STIRPE F,BARBIERI L,BATTELLI M G,et al.Ribosome-inactivating proteins from plants:present status and future prospects[J].Nature Biotechnology,1992,10(4):405-412.
[22]VALERIE R,MICHAEL K,PETER O.Transcriptional regulation of gene expression in C.elegans[J].Worm Book,2013,4(6):1-34.
[23]LEE T I,YOUNG R A.Transcriptional regulation and its misregulation in disease[J].Cell,2013,152(6):1237-1251.
[24]黄玉,杨波,迟小华,等.真核生物启动子的研究及应用[J].生物技术通讯,2010,21(2):275-279.HUANG Y,YANG B,CHI X H,et al.Investigation and application of promoters of eukaryotic organism[J].Letters in Biotechnology,2010,21(2):275-279.(in Chinese with English abstract)
[25]ZHU F,ZHANG P,MENG Y F,et al.Alpha-momorcharin,a RIP produced by bitter melon,enhances defense response in tobacco plants against diverse plant viruses and shows antifungal activity in vitro[J].Planta,2013,237(1):77-88.
[26]凌冰,向亚林,王国才,等.苦瓜叶提取物对美洲斑潜蝇取食和产卵行为的抑制作用[J].应用生态学报,2009,20(4):836-842.LING B,XIANG Y L,WANG G C,et al.Antifeedant and antioviposition activities of Momordica charantia leaf ethanol extract against Liriomyza sativae[J].Chinese Journal of Applied Ecology,2009,20(4):836-842.(in Chinese with English abstract)
[27]魏周玲,彭浩然,潘琪,等.核糖体失活蛋白(α-MC)亚细胞定位及对TMV的抑制作用[J].中国农业科学,2017,50(5):840-848.WEI Z L,PENG H R,PAN Q,et al.Subcellular localization of the ribosome-inactivating proteinα-MC and its antiviral effect on TMV[J].Scientia Agricultura Sinica,2017,50(5):840-848.(in Chinese with English abstract)
[28]FANG E F,NG T B,SHAW P C,et al.Recent progress in medicinal investigations on trichosanthin and other ribosome inactivating proteins from the plant genus trichosanthes[J].Current Medical Chemistry,2011,18(1):4410-4417.
[29]MOCK J W,NG T B,WONG R N,et al.Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha-and beta-momorcharins[J].Life Science,1996,59(22):1853-1859.
[30]FANG E F,ZHANG C Z,FONG W P,et al.RNase MC2:a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKS and induction of caspase pathways[J].Apoptosis,2011,17(4):377-387.
[2]毛娟,陆璐,陈佰鸿,等.甜瓜Cm ACOI启动子组织特异性表达研究[J].园艺学报,2014,40(6):1101-1109.MAO J,LU L,CHEN B H,et al.Studies on Tissue specific regulation of Cm ACOⅠpromoter in melon[J].Acta Horticulturae Sinica,2014,40(6):1101-1109.(in Chinese with English abstract)
[3]BUTLER J E,KADONAGA J T.The RNA polymerase II core promoter:a key component in the regulation of gene expression[J].Genes&Development,2002,16(20):2583-2592.
[4]屈玮,陈彦光,吴祖强,等.苦瓜提取物抑制3T3-L1脂肪细胞脂肪沉淀研究[J].食品科学,2014,35(5):188-192.QU W,CHEN Y G,WU Z Q,et al.Anti-adipogenic effects of bitter melon extracts in 3T3-L1 adipocytes[J].Food Science,2014,35(5):188-192.(in Chinese with English abstract)
[5]董英,钱希文,白娟,等.苦瓜改善胰岛素抵抗功能与作用机制研究进展[J].食品科学,2013,34(21):369-374.DONG Y,QIAN X W,BAI J,et al.Research progress in functions and mechanisms of Momordica charantia L.for ameliorating insulin resistance[J].Food Science,2013,34(21):369-374.(in Chinese with English abstract)
[6]LICASTRO F,FRANCESCHI C,BARBIERI L,et al.Toxicity of Momordica charantia lectin and inhibitor for human normal and leukaemic lymphocytes[J].Virchows Archiv B,1980,33(2):257-265.
[7]NG T B,WONG C M,LI W W,et al.Isolation and characterization of a galactose binding lectin with insulinomimetic activities[J].International Journal of Peptide&Protein Research,1986,28(2):163-172.
[8]TSAO S W,NG T B,YEUNG H W.Toxicities of trichosanthin and alpha-momorcharin,abortifacient proteins from Chinese medicinal plants,on cultured tumor cell lines[J].Toxicon,1990,28(1):1183-1192.
[9]NG T B,LIU W K,SZE S F,et al.Action ofα-momorcharin,a ribosome inactivating protein,on cultured tumor cell lines[J].General Pharmacology the Vascular System,1994,25(1):75-77.
[10]沈富林,曹东亮,邓斐,等.α-苦瓜素体内抗人乳腺癌MCF-7移植瘤实验[J].成都医学院学报2014,9(6):686-689.SHEN F L,CAO D L,DENG F,et al.The anti-tumor experiments of alpha-momorcharin on human breast MCF-7 xenograft in vivo[J].Journal of Chengdu Medical College,2014,9(6):686-689.(in Chinese with English abstract)
[11]WANG P,HUANG S,WANG F,et al.Cyclic AMP-response element regulated cell cycle arrestsin cancer cells[J].PLo S One,2013,8(6):e65661.
[12]LIU F,WANG B,WANG Z,et al.Trichosanthin down-regulates Notch signaling and inhibits proliferation of the nasopharyngeal carcinoma cell line CNE2 in vitro[J].Fitoterapia,2012,83(5):838-842.
[13]蔡秀清.α-苦瓜素在毕赤酵母中表达及生物活性分析[D].儋州:华南热带农业大学,2005.CAI X Q.Expression and bioactivity assay of alpha-momorcharin gene in Pichia pastoris[D].Danzhou:South China University of Tropical Agriculture,2005.(in Chinese with English abstract)
[14]欧阳永长,庄东红,胡忠,等.α-苦瓜素基因的克隆和原核表达[J].武汉植物学研究,2008,26(1):7-11.OUYANG Y C,ZHUANG D H,HU Z,et al.Cloning and prokaryotic expression of alpha-momorcharin gene[J].Journal of Wuhan Botanical Research,2008,26(1):7-11.(in Chinese with English abstract)
[15]王书珍.重组核糖体失活蛋白alpha-苦瓜素的生物学活性分析[D].武汉:武汉大学,2013.WANG S Z.Analysis of the biological activities of a recombinant ribosome-inactivating protein(alpha-momorcharin)[D].Wuhan:Wuhan University,2013.(in Chinese with English abstract)
[16]QIAN Q,HUANG L,YI R,et al.Enhanced resistance to blast fungus in rice(Oryza sativa L.)by expressing the ribosome-inactivating proteinα-momorcharin[J].Plant Science,2014,217/218(1):1-7.
[17]刘子记,牛玉,朱婕,等.苦瓜核心种质资源构建方法的比较[J].华南农业大学学报,2017,38(1):31-37.LIU Z J,NIU Y,ZHU J,et al.Comparison of different methods to construct core collections of Momordica charantia[J].Journal of South China Agricultural University,2017,38(1):31-37.(in Chinese with English abstract)
[18]刘子记,牛玉,杨衍.热研一号油绿苦瓜种子纯度的SSR鉴定[J].热带作物学报,2013,34(11):2179-2182.LIU Z J,NIU Y,YANG Y.Identification of bitter gourd‘Reyan No.1’seed purity by SSR markers[J].Chinese Journal of Tropical Crops,2013,34(11):2179-2182.(in Chinese with English abstract)
[19]URASAKI N,TAKAGI H,NATSUME S,et al.Draft genome sequence of bitter gourd(Momordica charantia),a vegetable and medicinal plant in tropical and subtropical regions[J].DNA Research:An International Journal for Rapid Publication of Reports on Genes and Genomes,2017,24(1):51-58.
[20]LESCOT M,DEHAIS P,MOREAU Y,et al.Plant CARE:a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.
[21]STIRPE F,BARBIERI L,BATTELLI M G,et al.Ribosome-inactivating proteins from plants:present status and future prospects[J].Nature Biotechnology,1992,10(4):405-412.
[22]VALERIE R,MICHAEL K,PETER O.Transcriptional regulation of gene expression in C.elegans[J].Worm Book,2013,4(6):1-34.
[23]LEE T I,YOUNG R A.Transcriptional regulation and its misregulation in disease[J].Cell,2013,152(6):1237-1251.
[24]黄玉,杨波,迟小华,等.真核生物启动子的研究及应用[J].生物技术通讯,2010,21(2):275-279.HUANG Y,YANG B,CHI X H,et al.Investigation and application of promoters of eukaryotic organism[J].Letters in Biotechnology,2010,21(2):275-279.(in Chinese with English abstract)
[25]ZHU F,ZHANG P,MENG Y F,et al.Alpha-momorcharin,a RIP produced by bitter melon,enhances defense response in tobacco plants against diverse plant viruses and shows antifungal activity in vitro[J].Planta,2013,237(1):77-88.
[26]凌冰,向亚林,王国才,等.苦瓜叶提取物对美洲斑潜蝇取食和产卵行为的抑制作用[J].应用生态学报,2009,20(4):836-842.LING B,XIANG Y L,WANG G C,et al.Antifeedant and antioviposition activities of Momordica charantia leaf ethanol extract against Liriomyza sativae[J].Chinese Journal of Applied Ecology,2009,20(4):836-842.(in Chinese with English abstract)
[27]魏周玲,彭浩然,潘琪,等.核糖体失活蛋白(α-MC)亚细胞定位及对TMV的抑制作用[J].中国农业科学,2017,50(5):840-848.WEI Z L,PENG H R,PAN Q,et al.Subcellular localization of the ribosome-inactivating proteinα-MC and its antiviral effect on TMV[J].Scientia Agricultura Sinica,2017,50(5):840-848.(in Chinese with English abstract)
[28]FANG E F,NG T B,SHAW P C,et al.Recent progress in medicinal investigations on trichosanthin and other ribosome inactivating proteins from the plant genus trichosanthes[J].Current Medical Chemistry,2011,18(1):4410-4417.
[29]MOCK J W,NG T B,WONG R N,et al.Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha-and beta-momorcharins[J].Life Science,1996,59(22):1853-1859.
[30]FANG E F,ZHANG C Z,FONG W P,et al.RNase MC2:a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKS and induction of caspase pathways[J].Apoptosis,2011,17(4):377-387.