中国南瓜内参基因18S rRNA的克隆及其引物开发
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摘要
【目的】克隆获得中国南瓜18SrRNA,并设计合适的荧光定量PCR内参,为开展中国南瓜重要功能基因的表达模式和调控机制研究奠定基础。【方法】以中国南瓜‘密本’品种的基因组DNA为模板,通过PCR方法克隆中国南瓜18SrRNA基因序列,再利用Primer Premier软件设计荧光定量PCR引物,对该内参基因在中国南瓜不同组织、生长阶段及非生物胁迫条件下的表达稳定性进行检测。【结果】首次克隆得到中国南瓜18SrRNA基因序列,其长度为1 857bp,GenBank登录号为KM979454,该基因与西瓜、西葫芦等瓜类蔬菜18SrRNA基因同源性大都在90%以上;以中国南瓜内参基因18SrRNA核苷酸全长序列为基础设计1对荧光定量PCR引物,以中国南瓜果肉总RNA逆转录的cDNA第一链为模板进行PCR扩增,该引物扩增的片段大小为132bp,扩增效率高,特异性强;18SrRNA基因在中国南瓜不同组织、生长发育阶段及非生物胁迫条件下均能稳定表达。【结论】克隆获得中国南瓜18SrRNA基因,该基因适合在中国南瓜基因表达研究中作为内参基因。
【Objective】The study cloned 18 SrRNA gene of Cucurbita moschata and designed suitable qRT-PCR primers for analyzing expression patterns and regulation mechanisms of important critical genes.【Method】Sequence of 18 SrRNA gene was cloned by PCR using the genomic DNA of Cucurbitamoschata cultivar‘Miben'as template.A pair of qRT-PCR primers were then designed by Primer Premier software.Furthermore,the stability of 18 SrRNA gene was determined in Cucurbita moschata in different tissues,growth stages and abiotic stress conditions.【Result】The 18 SrRNA gene of Cucurbita moschata wascloned firstly(GenBank accession number:KM979454).It was 1 857 bp long and shared more than 90%homology with 18 SrRNA genes from melons vegetables such as watermelon and squash.According to the deduced 18 SrRNA gene sequence,apair of qRT-PCR primers were then designed.The fragment of 132 bp was successfully amplified by the primers using the first-strand cDNA as template,which was reverse-transcribed from the total RNA of Cucurbita moschata.The primers had high specificity and amplification efficiency.RT-PCR indicated that the 18 SrRNA gene was stably expressed under different growth stages and abiotic stresses.【Conclusion】The 18 SrRNA gene was suitable as a reference gene for analyzing gene expression patterns in Cucurbita moschata.
【Objective】The study cloned 18 SrRNA gene of Cucurbita moschata and designed suitable qRT-PCR primers for analyzing expression patterns and regulation mechanisms of important critical genes.【Method】Sequence of 18 SrRNA gene was cloned by PCR using the genomic DNA of Cucurbitamoschata cultivar‘Miben'as template.A pair of qRT-PCR primers were then designed by Primer Premier software.Furthermore,the stability of 18 SrRNA gene was determined in Cucurbita moschata in different tissues,growth stages and abiotic stress conditions.【Result】The 18 SrRNA gene of Cucurbita moschata wascloned firstly(GenBank accession number:KM979454).It was 1 857 bp long and shared more than 90%homology with 18 SrRNA genes from melons vegetables such as watermelon and squash.According to the deduced 18 SrRNA gene sequence,apair of qRT-PCR primers were then designed.The fragment of 132 bp was successfully amplified by the primers using the first-strand cDNA as template,which was reverse-transcribed from the total RNA of Cucurbita moschata.The primers had high specificity and amplification efficiency.RT-PCR indicated that the 18 SrRNA gene was stably expressed under different growth stages and abiotic stresses.【Conclusion】The 18 SrRNA gene was suitable as a reference gene for analyzing gene expression patterns in Cucurbita moschata.
引文
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[4]张箭.南瓜发展传播史初探[J].烟台大学学报(哲学社会科学版),2010,23(1):100-108.Zhang J.An exploration of development of the history of pumpkin[J].Journal of Yantai University(Philosophy and Social Science),2010,23(1):100-108.
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[7]贺小琼,陈彦红,肖建春,等.南瓜粉开发及营养成份分析[J].昆明医学院学报,1990,20(3):46-48.He X Q,Chen Y H,Xiao J C,et al.The exploitation and composition of pumpkin powder[J].Academic Journal of Kunming Medical College,1990,20(3):46-48.
[8]范文秀,李新峥.南瓜营养成分分析及功能特性的研究[J].广东微量元素科学,2005,12(2):38-41.Fan W X,Li X Z.Study on determination of nutritive composition and functional properties of pumpkin[J].Guangdong Trace Elements Science,2005,12(2):38-41.
[9]张拥军,姚惠源.南瓜活性多糖的降糖作用及其组分分析[J].无锡轻工大学学报,2002,21(2):173-175.Zhang Y J,Yao H Y.Composition analysis of pumpkin polysaccharide and its glucatonic effect[J].Journal of Wuxi University of Light Industry,2002,21(2):173-175.
[10]Wu J X,Chang Z J,Wu Q S,et al.Molecular diversity of Chinese Cucurbita moschata germplasm collections detected by AFLP markers[J].Scientia Horticulturae,2011,128(8):7-13.
[11]Heid C A,Stevens J,Livak K J,et al.Real time quantitative PCR[J].Genome Research,1996,6(10):986-994.
[12]Huggett J,Dheda K,Bustin S,et al.Real-time RT-PCR normalization strategies and considerations[J].Genes Immunity,2005,6(4):279-284.
[13]Derveaux S,Vandesompele J,Hellemans J.How to do successful gene expression analysis using Real-time PCR[J].Methods,2010,50(4):227-230.
[14]刘金泊,欧静,姚富姣,等.磷化氢诱导下赤拟谷盗实时定量PCR内参基因的筛选[J].农业生物技术学报,2014,22(2):257-264.Liu J B,Ou J,Yao F J,et al.Studies by quantitative real-time PCR in Tribolium castaneum after exposure to phosphine[J].Journal of Agricultural Biotechnology,2014,22(2):257-264.
[15]黄春红,高燕会,朱玉球,等.石蒜黄烷酮3-羟化酶基因LrF3 H的克隆及表达分析[J].园艺学报,2013,40(5):960-970.Huang C H,Gao Y H,Zhu Y Q,et al.Cloning and expression analysis of flavanone 3-hydroxylase gene LrF3 HfromLycoris radiata[J].Acta Horticulturae Sinica,2013,40(5):960-970.
[16]王彦杰,董丽,张超,等牡丹实时定量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.Wang Y J,Dong L,Zhang C,et al.Reference gene selection forreal-time quantitative PCR nonllalization in tree peony(Paenoia suffruticosa Andr.)[J].Journal of AgricuItural Biotechnology,2012,20(5):521-528.
[17]魏永赞,赖彪,胡福初,等.用于荔枝qPCR分析的内参基因克隆及稳定性分析[J].华南农业大学学报,2012,33(3):301-303.Wei Y Z,Lai B,Hu F C,et al.Cloning and stability analysis of reference genes for expression studies by quantitativer Realtime PCR in litchi[J].Joumal of South China Agricultural University,2012,33(3):301-303.
[18]郝姗.茶树不同逆境条件下QRT-PCR适宜内参基因的筛选[D].南京:南京农业大学,2012.Hao S.Selection of appropriate reference genes for expression studies in camellia sinensis by rReal-time polymerase chain reaction[D].Nanjing:Nanjing Agricultural University,2012.
[19]王慧,赵升,魏珉,等.砧木南瓜硅转运蛋白基因CmLsi3的克隆与表达分析[J].园艺学报,2015,42(10):2075-2082.Wang H,Zhao S,Wei M,et al.Cloning and expression analysis of silicon transporter gene CmLsi3in roots of pumpkin[J].Acta Horticulturae Sinica,2015,42(10):2075-2082.
[20]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析[J].中国农业科学,2014,47(6):1216-1226.Liu J,Xiu B L,Xue Y Y,et al.Cloning and expression analysis of PALgene in seed coat of Cucurbita pepo[J].Scientia Agricultura Sinica,2014,47(6):1216-1226.
[21]Athanasiadou R,Polidoros A N,Mermigka G,et al.Differential expression of CmPP16homologues in pumpkin(Curcurbita maxima),winter squash(C.moschata)and their interspecific hybrid[J].Journal of Horticultural Science&Biotechnology,2005,80(5):643-649.
[22]Wu T,Cao J S,Qin Z W,et al.Identification of a novel ga-related bush mutant in pumpkin(Cucurbita moschata duchesne)[J].Pakistan Journal of Botany,2015,47(4):1359-1366.
[23]毕燕会,邹丹燕,周志刚.海带配子体18SrRNA基因的克隆、序列分析及其作为内参基因的应用[J].华北农学报,2009,24(1):49-54.Bi Y H,Zou D Y,Zhou Z G,et al.Cloning and sequence analysis of 18SrRNA gene from Laminaria japonica gametophytes and its application as an internal standard[J].Acta Agriculturae Boreali-Sinica,2009,24(1):49-54.
[24]朱海生,李永平,花秀凤,等.草莓9-顺式-环氧类胡萝卜素双加氧酶基因(FaNCED)的克隆及表达分析[J].园艺学报,2012,39(1):40-48.Zhu H S,Li Y P,Hua X F,et al.Cloning and expression analysis of 9-cis-epoxycarotenoid dioxygenase gene FaNCED in strawberry[J].Acta Horticulturae Sinica,2012,39(1):40-48.
[25]韩振云,宋婷婷,田佶,等.苹果属观赏海棠McUFGT的克隆及其在不同叶色品种间的表达差异分析[J].园艺学报,2014,41(2):301-310.Han Z Y,Song T T,Tian J,et al.Cloning and expression analysis of McUFGTin different cultivars of Crabapple[J].Acta Horticulturae Sinica,2014,41(2):301-310.
[26]邹智,刘建汀,庄美露,等.橡胶树AtSAG12同源基因的克隆与表达特性分析[J].西南农业学报,2014,27(5):2168-2172.Zou Z,Liu J T,Zhuang M L,et al.Molecular cloning and expression analysis of Arabidopsis SAG12homologue from rubber tree[J].Southwest China Journal of Agricultural Sciences,2014,27(5):2168-2172.
[27]朱海生,卢丽芳,温庆放,等.林义章.南瓜ISSR反应体系的建立与优化[J].分子植物育种,2014,12(4):802-809.Zhu H S,Lu L F,Wen Q F,et al.Establishment and optimization of ISSR-PCR amplification system in squash[J].Molecular Plant Breeding,2014,12(4):802-809.
[28]胡瑞波,范成明,傅永福.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报,2009,11(6):30-36.Hu R B,Fan C M,Fu Y F.Reference gene selection in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Journa l of Agricultural Science and Technology,2009,11(6):30-36.
[29]Williams M E,Torabinejad J,Cohick E,et al.Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9lead to over accumulation of PtdIns(4,5)P2and constitutive expression of the stress-response pathway[J].Plant Physiology,2005,138(2):686-700.
[2]李新峥,杨鹏鸣,刘振威,等.中国南瓜主要性状遗传特性的研究[J].华南农业大学学报,2011,32(1):7-10.Li X Z,Yang P M,Liu Z W,et al.Study on genetic character of Cucurbita moschata main traits[J].Journal of South China Agricultural University,2011,32(1):7-10.
[3]闫世江,张继宁,刘洁.南瓜育种研究进展[J].种子,2010,29(7):60-62.Yan S J,Zhang J N,Liu J.Advances in research on breeding of pumpkin[J].Seed,2010,29(7):60-62.
[4]张箭.南瓜发展传播史初探[J].烟台大学学报(哲学社会科学版),2010,23(1):100-108.Zhang J.An exploration of development of the history of pumpkin[J].Journal of Yantai University(Philosophy and Social Science),2010,23(1):100-108.
[5]Akintayo E T.Chemical composition and physico-chemical properties of fluted pumpkin seed and seed oils[J].Rivista Italiana Delle Sostanze Grasse,1997,74(1):13-15.
[6]王萍,赵清岩.南瓜的营养成分药用价值及开发利用[J].长江蔬菜,1998,13(7):1-3.Wang P,Zhao Q Y.Nutrient ingredient,medicinal value and exploitative prospects of pumpkin[J].Journal of Changjiang Vegetables,1998,13(7):1-3.
[7]贺小琼,陈彦红,肖建春,等.南瓜粉开发及营养成份分析[J].昆明医学院学报,1990,20(3):46-48.He X Q,Chen Y H,Xiao J C,et al.The exploitation and composition of pumpkin powder[J].Academic Journal of Kunming Medical College,1990,20(3):46-48.
[8]范文秀,李新峥.南瓜营养成分分析及功能特性的研究[J].广东微量元素科学,2005,12(2):38-41.Fan W X,Li X Z.Study on determination of nutritive composition and functional properties of pumpkin[J].Guangdong Trace Elements Science,2005,12(2):38-41.
[9]张拥军,姚惠源.南瓜活性多糖的降糖作用及其组分分析[J].无锡轻工大学学报,2002,21(2):173-175.Zhang Y J,Yao H Y.Composition analysis of pumpkin polysaccharide and its glucatonic effect[J].Journal of Wuxi University of Light Industry,2002,21(2):173-175.
[10]Wu J X,Chang Z J,Wu Q S,et al.Molecular diversity of Chinese Cucurbita moschata germplasm collections detected by AFLP markers[J].Scientia Horticulturae,2011,128(8):7-13.
[11]Heid C A,Stevens J,Livak K J,et al.Real time quantitative PCR[J].Genome Research,1996,6(10):986-994.
[12]Huggett J,Dheda K,Bustin S,et al.Real-time RT-PCR normalization strategies and considerations[J].Genes Immunity,2005,6(4):279-284.
[13]Derveaux S,Vandesompele J,Hellemans J.How to do successful gene expression analysis using Real-time PCR[J].Methods,2010,50(4):227-230.
[14]刘金泊,欧静,姚富姣,等.磷化氢诱导下赤拟谷盗实时定量PCR内参基因的筛选[J].农业生物技术学报,2014,22(2):257-264.Liu J B,Ou J,Yao F J,et al.Studies by quantitative real-time PCR in Tribolium castaneum after exposure to phosphine[J].Journal of Agricultural Biotechnology,2014,22(2):257-264.
[15]黄春红,高燕会,朱玉球,等.石蒜黄烷酮3-羟化酶基因LrF3 H的克隆及表达分析[J].园艺学报,2013,40(5):960-970.Huang C H,Gao Y H,Zhu Y Q,et al.Cloning and expression analysis of flavanone 3-hydroxylase gene LrF3 HfromLycoris radiata[J].Acta Horticulturae Sinica,2013,40(5):960-970.
[16]王彦杰,董丽,张超,等牡丹实时定量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.Wang Y J,Dong L,Zhang C,et al.Reference gene selection forreal-time quantitative PCR nonllalization in tree peony(Paenoia suffruticosa Andr.)[J].Journal of AgricuItural Biotechnology,2012,20(5):521-528.
[17]魏永赞,赖彪,胡福初,等.用于荔枝qPCR分析的内参基因克隆及稳定性分析[J].华南农业大学学报,2012,33(3):301-303.Wei Y Z,Lai B,Hu F C,et al.Cloning and stability analysis of reference genes for expression studies by quantitativer Realtime PCR in litchi[J].Joumal of South China Agricultural University,2012,33(3):301-303.
[18]郝姗.茶树不同逆境条件下QRT-PCR适宜内参基因的筛选[D].南京:南京农业大学,2012.Hao S.Selection of appropriate reference genes for expression studies in camellia sinensis by rReal-time polymerase chain reaction[D].Nanjing:Nanjing Agricultural University,2012.
[19]王慧,赵升,魏珉,等.砧木南瓜硅转运蛋白基因CmLsi3的克隆与表达分析[J].园艺学报,2015,42(10):2075-2082.Wang H,Zhao S,Wei M,et al.Cloning and expression analysis of silicon transporter gene CmLsi3in roots of pumpkin[J].Acta Horticulturae Sinica,2015,42(10):2075-2082.
[20]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析[J].中国农业科学,2014,47(6):1216-1226.Liu J,Xiu B L,Xue Y Y,et al.Cloning and expression analysis of PALgene in seed coat of Cucurbita pepo[J].Scientia Agricultura Sinica,2014,47(6):1216-1226.
[21]Athanasiadou R,Polidoros A N,Mermigka G,et al.Differential expression of CmPP16homologues in pumpkin(Curcurbita maxima),winter squash(C.moschata)and their interspecific hybrid[J].Journal of Horticultural Science&Biotechnology,2005,80(5):643-649.
[22]Wu T,Cao J S,Qin Z W,et al.Identification of a novel ga-related bush mutant in pumpkin(Cucurbita moschata duchesne)[J].Pakistan Journal of Botany,2015,47(4):1359-1366.
[23]毕燕会,邹丹燕,周志刚.海带配子体18SrRNA基因的克隆、序列分析及其作为内参基因的应用[J].华北农学报,2009,24(1):49-54.Bi Y H,Zou D Y,Zhou Z G,et al.Cloning and sequence analysis of 18SrRNA gene from Laminaria japonica gametophytes and its application as an internal standard[J].Acta Agriculturae Boreali-Sinica,2009,24(1):49-54.
[24]朱海生,李永平,花秀凤,等.草莓9-顺式-环氧类胡萝卜素双加氧酶基因(FaNCED)的克隆及表达分析[J].园艺学报,2012,39(1):40-48.Zhu H S,Li Y P,Hua X F,et al.Cloning and expression analysis of 9-cis-epoxycarotenoid dioxygenase gene FaNCED in strawberry[J].Acta Horticulturae Sinica,2012,39(1):40-48.
[25]韩振云,宋婷婷,田佶,等.苹果属观赏海棠McUFGT的克隆及其在不同叶色品种间的表达差异分析[J].园艺学报,2014,41(2):301-310.Han Z Y,Song T T,Tian J,et al.Cloning and expression analysis of McUFGTin different cultivars of Crabapple[J].Acta Horticulturae Sinica,2014,41(2):301-310.
[26]邹智,刘建汀,庄美露,等.橡胶树AtSAG12同源基因的克隆与表达特性分析[J].西南农业学报,2014,27(5):2168-2172.Zou Z,Liu J T,Zhuang M L,et al.Molecular cloning and expression analysis of Arabidopsis SAG12homologue from rubber tree[J].Southwest China Journal of Agricultural Sciences,2014,27(5):2168-2172.
[27]朱海生,卢丽芳,温庆放,等.林义章.南瓜ISSR反应体系的建立与优化[J].分子植物育种,2014,12(4):802-809.Zhu H S,Lu L F,Wen Q F,et al.Establishment and optimization of ISSR-PCR amplification system in squash[J].Molecular Plant Breeding,2014,12(4):802-809.
[28]胡瑞波,范成明,傅永福.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报,2009,11(6):30-36.Hu R B,Fan C M,Fu Y F.Reference gene selection in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Journa l of Agricultural Science and Technology,2009,11(6):30-36.
[29]Williams M E,Torabinejad J,Cohick E,et al.Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9lead to over accumulation of PtdIns(4,5)P2and constitutive expression of the stress-response pathway[J].Plant Physiology,2005,138(2):686-700.