印度南瓜延伸因子基因CmEF1a的克隆与分析
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摘要
真核生物延伸因子(EF1a)是一种重要的内参基因,广泛应用于RT-qPCR中。为获得印度南瓜EF1a基因,开发合适的荧光定量PCR引物,本研究通过转录组测序和RT-PCR方法获得了1条长度为1 701 bp的cDNA,命名为CmEF1a,GeneBank登录号:MH310443。结果表明,CmEF1a包含1个ORF,大小为1 344 bp,编码447个氨基酸,理论分子大小约为49.30 kD,蛋白质等电点为9.14。Wolf Psort分析发现, CmEF1a蛋白亚细胞定位于细胞质基质中;Motif Scan分析显示, CmEF1a蛋白质的氨基酸序列2～432位和322～430位分别为EF1结构域和EF1的C端保守结构域。同源性分析表明,CmEF1a基因编码的蛋白质与同为葫芦科的中国南瓜、甜瓜和黄瓜同源蛋白的相似性达到99%,具有高度的保守性。在此基础上设计了1对RT-qPCR引物,该引物具有较高的特异性和扩增效率,在印度南瓜不同组织和不同胁迫处理下均能稳定表达,适合作为内参基因应用于印度南瓜基因表达研究。本研究结果为印度南瓜重要功能基因的表达分析及相关分子调控机制的研究奠定了一定的理论基础。
Elongation factor 1 alpha(EF1 a) was an important internal reference gene and was always used in RT-qPCR. The study was to obtain the EF1a gene of Pumpkin and design the RT-qPCR primers. A 1 701 bp cDNA was obtained by transcriptome sequencing and RT-PCR. This gene was named CmEF1a and the GenBank accession was MH310443. Bioinformatics analysis results indicated that the sequence contained a size of 1 344 bp ORF encoding 447 amino acids, with a theoretical molecular weight of 49.30 kD and a protein isoelectric point of 9.14. Wolf Psort analysis indicated that CmEF1 a protein was located in the cytoplasmic matrix, and Motif Scan analysis showed that CmEF1 a protein had the EF1 domain and EF1′s c-terminal conserved domain actin in the position of 2-432 and 322-430 sites, respectively. Homology analysis revealed that CmEF1 a shared 99% identity with the homologous proteins from Cucurbita moschata, Cucumis melo and Cucumis sativus which were also belong to cucurbitaceae the same as Cucurbita maxima, proving that it was highly conservative. A pair of RT-qPCR primers was then derived from the CmEF1a gene sequence which had the high specificity and amplification efficiency. The RT-PCR and RT-qPCR indicated that the CmEF1a gene was stable expression in different tissues and under different temperature and drought treatments, so it was suitable as a reference gene for the analysis of gene expression patterns in pumpkin. The result of this study provides an important reference for analysis the expression of critical genes for pumpkin.
Elongation factor 1 alpha(EF1 a) was an important internal reference gene and was always used in RT-qPCR. The study was to obtain the EF1a gene of Pumpkin and design the RT-qPCR primers. A 1 701 bp cDNA was obtained by transcriptome sequencing and RT-PCR. This gene was named CmEF1a and the GenBank accession was MH310443. Bioinformatics analysis results indicated that the sequence contained a size of 1 344 bp ORF encoding 447 amino acids, with a theoretical molecular weight of 49.30 kD and a protein isoelectric point of 9.14. Wolf Psort analysis indicated that CmEF1 a protein was located in the cytoplasmic matrix, and Motif Scan analysis showed that CmEF1 a protein had the EF1 domain and EF1′s c-terminal conserved domain actin in the position of 2-432 and 322-430 sites, respectively. Homology analysis revealed that CmEF1 a shared 99% identity with the homologous proteins from Cucurbita moschata, Cucumis melo and Cucumis sativus which were also belong to cucurbitaceae the same as Cucurbita maxima, proving that it was highly conservative. A pair of RT-qPCR primers was then derived from the CmEF1a gene sequence which had the high specificity and amplification efficiency. The RT-PCR and RT-qPCR indicated that the CmEF1a gene was stable expression in different tissues and under different temperature and drought treatments, so it was suitable as a reference gene for the analysis of gene expression patterns in pumpkin. The result of this study provides an important reference for analysis the expression of critical genes for pumpkin.
引文
[1] 段颖, 向成钢, 刘新艳, 马玮, 孙廷珍, 王长林. 印度南瓜果皮结构与色素组成对果皮颜色的影响[J]. 中国蔬菜, 2017(11):33-39
[2] 郭言言, 郭卫丽, 孙丽, 范文秀, 李新峥. 印度南瓜果实营养成分分析[J]. 北方园艺, 2017(15):42-47
[3] 王洋洋, 单文琪, 徐文龙, 崔崇士, 屈淑平. 印度南瓜转录组SSR信息分析及其多态性研究[J]. 园艺学报, 2016, 43(3):578-586
[4] 刘小俊, 李跃建, 梁根云, 房超, 刘独臣, 杨宏. 印度南瓜“甜栗”果实主要营养成份的动态研究[J]. 西南农业学报, 2010, 23(5):1507-1509
[5] 朱白婢, 蔡兴来, 周曼, 云天海, 胡艳平, 李雪峤, 王小娟, 王敏. 基于ISSR标记的印度南瓜种质资源遗传多样性分析[J]. 分子植物育种, 2016, 14(12):3596-3600
[6] 郑扬, 张国裕, 张帆, 田佳星, 张沙沙, 王建书, 李海真. 南瓜育种相关基础研究进展[J]. 中国蔬菜, 2018(12):16-23
[7] 周晓慧, 刘军, 庄勇. 喀西茄内参基因实时荧光定量PCR表达稳定性评价[J]. 园艺学报, 2014, 41(86):1731-1738
[8] 蒋晓梅, 张新全, 严海东, 张瑜, 杨盛婷, 黄琳凯. 柳枝稷根组织实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2014, 22(1):55-63
[9] Wan H J, Zhao Z G, Qian C T, Sui Y H, Malik A A, Chen J F. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399(2): 257-261
[10] Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros L M G, Romano E, Grossi-de-Sa M F, Vaslin M, Alves-Ferreira M. Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR[J]. Molecular Breeding, 2009, 23(4): 607-616
[11] Andersen C L, Jensen J L, Orntoft T F. Normalization of Real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research, 2004, 64(15): 5245-5250
[12] 朱海生, 陈敏氡, 温庆放, 蓝新隆, 李永平, 王彬, 张前荣, 吴卫东. 丝瓜18S rRNA基因克隆及其作为内参基因的应用[J]. 核农学报, 2016, 30(1): 35-41
[13] 魏秀清, 章希娟, 许玲, 陈长忠, 许家辉. 莲雾实时荧光定量PCR内参基因的筛选和验证[J]. 果树学报, 2018, 35(4): 402-411
[14] Chen J H, Sun Y, Sun F, Xia X L, Yin W L. Tobacco plants ectopically expressing the Ammopiptanthus mongolicus AmCBL1 gene display enhanced tolerance to multiple abiotic stresses[J]. Plant Growth Regulation, 2011, 63(3): 259-269
[15] Zhou L, Wang Y, Peng Z H. Molecular characterization and expression analysis of chalcone synthase gene during flower development in tree peony (Paeonia suffruticosa)[J]. African Journal of Biotechnology, 2011, 10(8): 1275-1284
[16] 王彦杰, 董丽, 张超, 王晓庆. 牡丹实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2012, 20(5): 521-528
[17] 刘晓婷, 王顺利, 薛璟祺, 薛玉前, 吕英民, 张秀新. 朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选[J]. 园艺学报, 2018, 45(5): 919-930
[18] 周峰, 刘燕, 马永贵, 黄原. 真核翻译延伸因子1A 蛋白家族功能位点的进化踪迹分析[J]. 中国生物化学与分子生物学报, 2013, 29(8): 773-782
[19] Gross S R, Kinzy T G. Translation elongation factor IA is essential for regulation of the actin cytoskeleton and cell morphology[J]. Nature Structural & Molecular Biology, 2005, 12(9):772-778
[20] Ruggiero I, Cantiello P, Lamberti A, SorrentinoB A, Martucci N M. Biochemical characterisation of the D60A mutant of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus[J]. Biochimie, 2009, 91(7): 835-842
[21] Zhu H S, Liu J T, Wen Q F, Chen M D, Wang B, Zhang Q R, Xue Z Z. De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica ‘Fusi-3’ fruits[J]. PLoS One, 2017, 12(11): e0187117
[21] Ara N, Nakkanong K, Lv W, Yang J, Hu Z, Zhang M. Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species (“Cucurbita maxima” and “Cucurbita moschata”) and their interspecific inbred line “Maxchata”[J]. International Journal of Molecular Sciences, 2013, 14(12): 24008-24028
[23] 温庆放, 刘建汀, 朱海生, 陈敏氡, 王彬, 张前荣. 丝瓜过氧化氢酶基因CAT1 的克隆及表达分析[J]. 园艺学报, 2016, 43(10): 2039-2048
[24] Mascia T, Santovitto E, Gallitelli D, Collo F. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants[J]. Molecular Plant Pathology, 2010, 11(6): 805-816
[25] 张力维, 李勇鹏, 姚瑶, 梁优, 杜丽. 香樟延伸因子EF1a 基因片段的克隆及表达分析[J]. 中南林业科技大学学报, 2015, 35 (5): 122-128
[26] Nicot N, Hausman J F, Hoffmann L, Evers D. Housek eep ing gene select ion for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005, 56(421): 2907-2914
[27] 李钱峰, 蒋美艳, 于恒秀, 辛世文, 顾铭洪, 刘巧泉. 水稻胚乳RNA定量RT-PCR分析中参照基因选择[J]. 扬州大学学报(农业与生命科学版), 2008, 29(2):61-66
[28] 周峰, 刘燕, 马永贵, 黄原. 真核翻译延伸因子1A 蛋白家族功能位点的进化踪迹分析[J]. 中国生物化学与分子生物学报, 2013, 29(8): 773-782
[29] Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737
[30] Sang J, Wang Z N, Li M, Cao J B, Niu G Y, Xia L, Zou D, Wang F, Xu X J, Han X J, Fan J Q, Yang Y, Zuo W Z, Zhang Y, Zhao W M, Bao Y M, Xiao J F, Hu S N, Hao L L, Zhang Z. ICG:A wiki-driven knowledgebase of internal control genes for RT-qPCR normalization[J]. Nucleic Acid Research, 2017, 47(5):121-126
[31] Chen L, Zhong H Y, Kuang J F, Li J G, Lu W J, Chen J Y. Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions[J]. Planta, 2011, 234(2):377-390
[32] Wei L B, Miao H G, Zhao R H, Han X H, Zhang T D, Zhang H Y. Identi?cation and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR[J]. Planta, 2013, 237(3):873-889
[33] Xu X Y, Yang Z P, Sun X L, Zhang L G, Fang Z Y. Selection of reference genes for quantitative real-time PCR during ?ower bud development in CMS7311 of heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. Acta Physiol Plant, 2014, 36(3):809-814
[34] Chen Y, TanZ, Hu B, Yang Z, Xu B, Zhuang L, Huang B. Selection and validation of reference genes for target gene analysis with quantitative RT-PCR in leaves and roots of bermudagrass under four different abiotic stresses[J]. Physiologia Plantarum, 2015, 155(2):138-148
[35] 蒋素华, 王默霏, 宋彩霞, 梁芳, 李艳辉, 崔波. 萼脊兰EF1a基因片段的克隆及序列分析[J]. 贵州农业科学, 2016, 44(5):5-8
[36] Mallona Ⅰ, Lischewski S, Weiss J, Hause B, Egeacortines M. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J]. BMC Plant Biology, 2010, 10: 4-14
[37] 张岗, 赵明明, 张大为, 郭顺星. 铁皮石斛实时定量PCR内参基因的筛选[J]. 中国药学杂志, 2013, 48(19):1664-1668
[38] Wan H, Zhao Z, Qian C, Sui Y, Malik A. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399(2): 257-261
[39] Qi J, Yu S, Zhang F, Shen X, Zhao X, Yu Y, Zhang D. Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage (Brassica rapa ssp. pekinensis) [J]. Plant Molecular Biology Reporter, 2010, 28(4): 597-604
[40] 王彦杰, 陈叶清, 薛泽云, 周华, 金奇江, 徐迎春. 荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证[J]. 南京农业大学学报, 2017, 40(3):408-415
[2] 郭言言, 郭卫丽, 孙丽, 范文秀, 李新峥. 印度南瓜果实营养成分分析[J]. 北方园艺, 2017(15):42-47
[3] 王洋洋, 单文琪, 徐文龙, 崔崇士, 屈淑平. 印度南瓜转录组SSR信息分析及其多态性研究[J]. 园艺学报, 2016, 43(3):578-586
[4] 刘小俊, 李跃建, 梁根云, 房超, 刘独臣, 杨宏. 印度南瓜“甜栗”果实主要营养成份的动态研究[J]. 西南农业学报, 2010, 23(5):1507-1509
[5] 朱白婢, 蔡兴来, 周曼, 云天海, 胡艳平, 李雪峤, 王小娟, 王敏. 基于ISSR标记的印度南瓜种质资源遗传多样性分析[J]. 分子植物育种, 2016, 14(12):3596-3600
[6] 郑扬, 张国裕, 张帆, 田佳星, 张沙沙, 王建书, 李海真. 南瓜育种相关基础研究进展[J]. 中国蔬菜, 2018(12):16-23
[7] 周晓慧, 刘军, 庄勇. 喀西茄内参基因实时荧光定量PCR表达稳定性评价[J]. 园艺学报, 2014, 41(86):1731-1738
[8] 蒋晓梅, 张新全, 严海东, 张瑜, 杨盛婷, 黄琳凯. 柳枝稷根组织实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2014, 22(1):55-63
[9] Wan H J, Zhao Z G, Qian C T, Sui Y H, Malik A A, Chen J F. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399(2): 257-261
[10] Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros L M G, Romano E, Grossi-de-Sa M F, Vaslin M, Alves-Ferreira M. Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR[J]. Molecular Breeding, 2009, 23(4): 607-616
[11] Andersen C L, Jensen J L, Orntoft T F. Normalization of Real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research, 2004, 64(15): 5245-5250
[12] 朱海生, 陈敏氡, 温庆放, 蓝新隆, 李永平, 王彬, 张前荣, 吴卫东. 丝瓜18S rRNA基因克隆及其作为内参基因的应用[J]. 核农学报, 2016, 30(1): 35-41
[13] 魏秀清, 章希娟, 许玲, 陈长忠, 许家辉. 莲雾实时荧光定量PCR内参基因的筛选和验证[J]. 果树学报, 2018, 35(4): 402-411
[14] Chen J H, Sun Y, Sun F, Xia X L, Yin W L. Tobacco plants ectopically expressing the Ammopiptanthus mongolicus AmCBL1 gene display enhanced tolerance to multiple abiotic stresses[J]. Plant Growth Regulation, 2011, 63(3): 259-269
[15] Zhou L, Wang Y, Peng Z H. Molecular characterization and expression analysis of chalcone synthase gene during flower development in tree peony (Paeonia suffruticosa)[J]. African Journal of Biotechnology, 2011, 10(8): 1275-1284
[16] 王彦杰, 董丽, 张超, 王晓庆. 牡丹实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2012, 20(5): 521-528
[17] 刘晓婷, 王顺利, 薛璟祺, 薛玉前, 吕英民, 张秀新. 朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选[J]. 园艺学报, 2018, 45(5): 919-930
[18] 周峰, 刘燕, 马永贵, 黄原. 真核翻译延伸因子1A 蛋白家族功能位点的进化踪迹分析[J]. 中国生物化学与分子生物学报, 2013, 29(8): 773-782
[19] Gross S R, Kinzy T G. Translation elongation factor IA is essential for regulation of the actin cytoskeleton and cell morphology[J]. Nature Structural & Molecular Biology, 2005, 12(9):772-778
[20] Ruggiero I, Cantiello P, Lamberti A, SorrentinoB A, Martucci N M. Biochemical characterisation of the D60A mutant of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus[J]. Biochimie, 2009, 91(7): 835-842
[21] Zhu H S, Liu J T, Wen Q F, Chen M D, Wang B, Zhang Q R, Xue Z Z. De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica ‘Fusi-3’ fruits[J]. PLoS One, 2017, 12(11): e0187117
[21] Ara N, Nakkanong K, Lv W, Yang J, Hu Z, Zhang M. Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species (“Cucurbita maxima” and “Cucurbita moschata”) and their interspecific inbred line “Maxchata”[J]. International Journal of Molecular Sciences, 2013, 14(12): 24008-24028
[23] 温庆放, 刘建汀, 朱海生, 陈敏氡, 王彬, 张前荣. 丝瓜过氧化氢酶基因CAT1 的克隆及表达分析[J]. 园艺学报, 2016, 43(10): 2039-2048
[24] Mascia T, Santovitto E, Gallitelli D, Collo F. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants[J]. Molecular Plant Pathology, 2010, 11(6): 805-816
[25] 张力维, 李勇鹏, 姚瑶, 梁优, 杜丽. 香樟延伸因子EF1a 基因片段的克隆及表达分析[J]. 中南林业科技大学学报, 2015, 35 (5): 122-128
[26] Nicot N, Hausman J F, Hoffmann L, Evers D. Housek eep ing gene select ion for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005, 56(421): 2907-2914
[27] 李钱峰, 蒋美艳, 于恒秀, 辛世文, 顾铭洪, 刘巧泉. 水稻胚乳RNA定量RT-PCR分析中参照基因选择[J]. 扬州大学学报(农业与生命科学版), 2008, 29(2):61-66
[28] 周峰, 刘燕, 马永贵, 黄原. 真核翻译延伸因子1A 蛋白家族功能位点的进化踪迹分析[J]. 中国生物化学与分子生物学报, 2013, 29(8): 773-782
[29] Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737
[30] Sang J, Wang Z N, Li M, Cao J B, Niu G Y, Xia L, Zou D, Wang F, Xu X J, Han X J, Fan J Q, Yang Y, Zuo W Z, Zhang Y, Zhao W M, Bao Y M, Xiao J F, Hu S N, Hao L L, Zhang Z. ICG:A wiki-driven knowledgebase of internal control genes for RT-qPCR normalization[J]. Nucleic Acid Research, 2017, 47(5):121-126
[31] Chen L, Zhong H Y, Kuang J F, Li J G, Lu W J, Chen J Y. Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions[J]. Planta, 2011, 234(2):377-390
[32] Wei L B, Miao H G, Zhao R H, Han X H, Zhang T D, Zhang H Y. Identi?cation and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR[J]. Planta, 2013, 237(3):873-889
[33] Xu X Y, Yang Z P, Sun X L, Zhang L G, Fang Z Y. Selection of reference genes for quantitative real-time PCR during ?ower bud development in CMS7311 of heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. Acta Physiol Plant, 2014, 36(3):809-814
[34] Chen Y, TanZ, Hu B, Yang Z, Xu B, Zhuang L, Huang B. Selection and validation of reference genes for target gene analysis with quantitative RT-PCR in leaves and roots of bermudagrass under four different abiotic stresses[J]. Physiologia Plantarum, 2015, 155(2):138-148
[35] 蒋素华, 王默霏, 宋彩霞, 梁芳, 李艳辉, 崔波. 萼脊兰EF1a基因片段的克隆及序列分析[J]. 贵州农业科学, 2016, 44(5):5-8
[36] Mallona Ⅰ, Lischewski S, Weiss J, Hause B, Egeacortines M. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida[J]. BMC Plant Biology, 2010, 10: 4-14
[37] 张岗, 赵明明, 张大为, 郭顺星. 铁皮石斛实时定量PCR内参基因的筛选[J]. 中国药学杂志, 2013, 48(19):1664-1668
[38] Wan H, Zhao Z, Qian C, Sui Y, Malik A. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399(2): 257-261
[39] Qi J, Yu S, Zhang F, Shen X, Zhao X, Yu Y, Zhang D. Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage (Brassica rapa ssp. pekinensis) [J]. Plant Molecular Biology Reporter, 2010, 28(4): 597-604
[40] 王彦杰, 陈叶清, 薛泽云, 周华, 金奇江, 徐迎春. 荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证[J]. 南京农业大学学报, 2017, 40(3):408-415
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