龙眼胚胎发育相关基因F3H和FRK的克隆与分析
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摘要
本研究以“立冬本”龙眼合子胚为材料,采用RT-PCR和RACE技术,克隆了龙眼胚胎F3H基因cDNA全长和FRK基因cDNA片段,并对F3H基因进行了原核表达的研究和序列分析,对揭示龙眼胚胎发育与分化的分子本质,对进一步利用基因工程技术培育优质果奠定实验基础。主要实验结果如下:
1采用RT-PCR和RACE技术克隆了龙眼胚胎F3H基因cDNA全长
龙眼胚胎F3H基因cDNA全长为1404bp,其中包含119bp的5′非编码区和187bp的3′非编码区,编码365个氨基酸,等电点为5.55,分子量为41.112kDa。对龙眼胚胎F3H基因cDNA编码产物进行分析,推断龙眼胚胎F3H基因为亲水性多肽。将该F3H基因cDNA序列与其它植物F3H基因cDNA进行比较,结果表明,龙眼胚胎F3H基因cDNA序列与许多植物的F3H基因具有很高的同源性,与棉花F3H的同源性达84%,与柑橘F3H的同源性达82%,与草莓F3H的相似系数达83%,对其所推导的氨基酸序列进行分析发现,龙眼F3H的氨基酸序列与棉花F3H同源性最高,达90.76%,与葡萄的同源性达89.04%,与柑橘的同源性达88.49%。该基因在GenBank中登录号为EF468104。
2龙眼胚胎F3H基因cDNA的原核表达
将分离出来的龙眼胚胎F3H基因cDNA的编码区序列插入pET-29a载体上构建原核表达载体,在细菌BL21细胞中进行原核表达。试验结果显示,该编码区片段在原核细胞中不表达。推测是由于表达质粒和表达宿主菌选择不当,不适合龙眼胚胎F3H基因的表达。
3采用RT-PCR和RACE技术克隆了龙眼胚胎FRK基因cDNA片段
试验获得了一个长850bp的FRK基因cDNA片段。经BLAST分析,龙眼胚胎FRK基因cDNA片段序列与许多植物的FRK基因具有很高的同源性。与柑橘FRK的同源性达87%,与番茄FRK的同源性达81%,与拟南芥FRK的同源性达75%。该cDNA片段在GenBank中登录号为EU600093。
In the present study,the full length cDNA of flavanone 3-hydroxylase(F3H)and cDNA fragment of Fructokinase(FRK)from longan(Dimdcarpus longana Lour.) embryo were cloned by RT-PCR and RACE.The recombinant prokaryotic expression vector pET-29a inserted F3H gene was constructed and attempted to express in E.coli BL21.It set up a foundation for discovering the molecular inbeing of development and differentiation in longan embryo as well as adopting gene engineering technology to cultivate high quality fruit.
1 Cloning of the full length cDNA of flavanone 3-hydroxylase(F3H)by RT-PCR and RACE
The full-length cDNA of longan F3H is 1404 nucleotides long,including an ORF encoding 365 amino acids(Mw=41.112kDa,pI=5.55),a 5' untranslated region of 119 bp and a 3' flanking sequence of 118 bp.It was concluded that longan F3His a hydrophilic protein.When the nucleotide sequence compared with Gossypium hirsutum F3H gene and Citrus sinensis F3H gene and Fragaria x ananassa F3H gene, it showed that identity were 84%and 82%and 83%,respectively.The comparison between the putative amino acid sequence with Gossypium hirsutum F3H and Citrus sinensis F3H and Vitis vinifera F3H showed that identity were 90.76%and 89.04% and 88.49%,respectively.This cDNA has been registered in GenBank with accession number EF468104.
2 Expression of longan F3H proteins in E.coli.
The longan F3H gene cDNA was inserted into the vector pET-29a.The expression construct was transformed into BL21 and was induced.The result showed that longan F3H gene has failed to express.It was concluded that the expression vector and host were not suitable for expression of longan F3H.
3 Cloning of cDNA fragment of Fructokinase(FRK)by RT-PCR and RACE
A 850 bp cDNA fragment was obtained.Compared with FRKs of other plants, longan FRK gene cDNA had 87%,81%,75%identity,with that of Citrus sinensis, lycopersicon esculenturn and Arabidopsis thaliana FRK genes,respectively. This cDNA sequence has been registered in GenBank with accession number EU600093.
1采用RT-PCR和RACE技术克隆了龙眼胚胎F3H基因cDNA全长
龙眼胚胎F3H基因cDNA全长为1404bp,其中包含119bp的5′非编码区和187bp的3′非编码区,编码365个氨基酸,等电点为5.55,分子量为41.112kDa。对龙眼胚胎F3H基因cDNA编码产物进行分析,推断龙眼胚胎F3H基因为亲水性多肽。将该F3H基因cDNA序列与其它植物F3H基因cDNA进行比较,结果表明,龙眼胚胎F3H基因cDNA序列与许多植物的F3H基因具有很高的同源性,与棉花F3H的同源性达84%,与柑橘F3H的同源性达82%,与草莓F3H的相似系数达83%,对其所推导的氨基酸序列进行分析发现,龙眼F3H的氨基酸序列与棉花F3H同源性最高,达90.76%,与葡萄的同源性达89.04%,与柑橘的同源性达88.49%。该基因在GenBank中登录号为EF468104。
2龙眼胚胎F3H基因cDNA的原核表达
将分离出来的龙眼胚胎F3H基因cDNA的编码区序列插入pET-29a载体上构建原核表达载体,在细菌BL21细胞中进行原核表达。试验结果显示,该编码区片段在原核细胞中不表达。推测是由于表达质粒和表达宿主菌选择不当,不适合龙眼胚胎F3H基因的表达。
3采用RT-PCR和RACE技术克隆了龙眼胚胎FRK基因cDNA片段
试验获得了一个长850bp的FRK基因cDNA片段。经BLAST分析,龙眼胚胎FRK基因cDNA片段序列与许多植物的FRK基因具有很高的同源性。与柑橘FRK的同源性达87%,与番茄FRK的同源性达81%,与拟南芥FRK的同源性达75%。该cDNA片段在GenBank中登录号为EU600093。
In the present study,the full length cDNA of flavanone 3-hydroxylase(F3H)and cDNA fragment of Fructokinase(FRK)from longan(Dimdcarpus longana Lour.) embryo were cloned by RT-PCR and RACE.The recombinant prokaryotic expression vector pET-29a inserted F3H gene was constructed and attempted to express in E.coli BL21.It set up a foundation for discovering the molecular inbeing of development and differentiation in longan embryo as well as adopting gene engineering technology to cultivate high quality fruit.
1 Cloning of the full length cDNA of flavanone 3-hydroxylase(F3H)by RT-PCR and RACE
The full-length cDNA of longan F3H is 1404 nucleotides long,including an ORF encoding 365 amino acids(Mw=41.112kDa,pI=5.55),a 5' untranslated region of 119 bp and a 3' flanking sequence of 118 bp.It was concluded that longan F3His a hydrophilic protein.When the nucleotide sequence compared with Gossypium hirsutum F3H gene and Citrus sinensis F3H gene and Fragaria x ananassa F3H gene, it showed that identity were 84%and 82%and 83%,respectively.The comparison between the putative amino acid sequence with Gossypium hirsutum F3H and Citrus sinensis F3H and Vitis vinifera F3H showed that identity were 90.76%and 89.04% and 88.49%,respectively.This cDNA has been registered in GenBank with accession number EF468104.
2 Expression of longan F3H proteins in E.coli.
The longan F3H gene cDNA was inserted into the vector pET-29a.The expression construct was transformed into BL21 and was induced.The result showed that longan F3H gene has failed to express.It was concluded that the expression vector and host were not suitable for expression of longan F3H.
3 Cloning of cDNA fragment of Fructokinase(FRK)by RT-PCR and RACE
A 850 bp cDNA fragment was obtained.Compared with FRKs of other plants, longan FRK gene cDNA had 87%,81%,75%identity,with that of Citrus sinensis, lycopersicon esculenturn and Arabidopsis thaliana FRK genes,respectively. This cDNA sequence has been registered in GenBank with accession number EU600093.
引文
[1]吕柳新,陈荣木,陈景渌.荔枝胚胎发育过程的观察[J].亚热带植物通讯,1985(1):1-5.
[2]李燕.龙眼胚胎发育不同时期的蛋白质组分析[D].福州:福建农林大学生命科学学院,2006.
[31]Lotan T,Ohto M.,Matsudaira Yee,et al.Arabidopsis LEAFYCOTYLEDON1 is sufficient to induce embryo development in vegetative cells[J].Cell,19.98,93:1195-1205.
[4]Stone S L,Kwong L W,Yee K M,et a.l.LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[J].Proc Natl Acad Sci USA,2001,98:11806-11811.
[5]Kwong R W,BuiA Q,Lee H,et al.LEAFYCOTYLEDON1-LIKE defines a class of regulators essential for embryo development[J].Plant Cell,2003,15:5-18.
[6]Shevell D E,leu W M,Gilimor C S,et al.EMB30 is essential for normal cell division,cell expansion,and cell adhesion in Arabidopsis and encodes a protein that has similarity to Sec7[J].Cell,1994,77:1015-1063.
[7]Torres Ruiz R A,Lohner A,Jurgens G.The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo[J].Plant J,1996,10:1005-1016.
[8]Smith L G,Greene B,Veit B,et al.A dominant mutation in the maize homeobox gene,Knotted-1,causes its ectopic expression in leaf cells with altered fates[J].Development,1992,116:21-30.
[9]Hall Q.,and Cannon,M.C.The cell wall hydroxyproline-rich glycoprotein RSH is essential for normal embryo development in Arabidopsis[J].Plant Cell,2002,14:1161-1172.
[10]Schrick K,Mayer U,Horrichs A,et al.FACKEL is a sterol C-14reductase required for organized cell division and expansion in Arabidopsis embryogenesis[J].Genes Dev,2000,14:1471-1484.
[11]Boutilier K,Offringa R.,Sharma V K,et al.Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth[J].Plant Cell,2002,14:1737-1749.
[12]Hardtke C S,Berleth T.The Arabidopsis gene MONOPTEROUS encodes a transcription factor mediating embryo axis formation and vascular development[J].EMBO J,1998,17:1405-1411.
[13]Long J A,Moan E I,Medford J I,et al.A member of the KNOTTED class ofhomeodomain proteins encoded by STM gene in Arabidopsis[J].Nature,1996,379:66-69.
[14]Kaydamony C,Tewes A,Adler K,et al.Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv[J].Biochim Biophys Acta,2000,1491(1-3):143-160.
[15]ZuoJ,Niu QW,Frugis G,et al.The WUSCHEL gene promotes vegetative-to-embryonic in Arabidopsis[J].Plant J,2002,30(3):349-359.
[16]Dong J Z,Dunstan D I.Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce[Picea glauca(Moench)Voss][J].Planta.1996,200(1):85-91.
[17]王风华.龙眼体细胞胚胎发生过程中的基因差别表达[D].福州:福建农林大学园艺学院,2003.
[18]郭志雄.龙眼胚胎乙醇脱氢酶的分离纯化、鉴定及其cDNA克隆[D].福州:福建农林大学园艺学院,2006.
[19]孟丽,戴思兰.F3'5'H基因与蓝色花的形成[J].分子植物育种,2004,2(3):413-420.
[20]张学英,张上隆,骆军,等.果实花色素苷合成研究进展[J].果树学报,2004,21(5):456-460.
[21]Forkmann G,Heller W.Grisebach H.Anthocyanin biosynthesis in flowers of Matthiola incana.Flavanone 3- and Flavonoid 3'-hydroxylase[J].Zeitschrift Fur Naturforschung C-A Journal of Biosciences C,1980,35:691-695.
[22]Martin C,Prescott A,Mackay S.Control of anthocyanin biosynthesis in flowers of Antirrhinum majus[J].Plant J,1991(1):37-49.
[23]Britsch L,Ruhnau-Brich B,Forkmann G.Molecular cloning,sequence analysis,and invitro expression of flavanone 3-β-hydroxylase from Petunia hybrida[J].Biol Chem,1992,267:5380-5387.
[24]张大生,崔丽洁,王景明,等.红巴梨F3H基因的克隆及植物表达载体的构建[J].南京林业大学学报,2005,29(2):65-68.
[25]Deboo G B,Albertsen M C,Taylor L P.Flavanone 3-β-hydroxylase transcripts and flavonol accumulation are temporally coordinate in Maize anthers[J].Plant J,1995,7(5):703-713.
[26]Charrier B,Coronado C,Kondorosi A,et al.Molecular characterization and expression of alfafa flavanone 3-hydroxylase and dihydroflavono1-4-reductase encoding genes[J].Plant Mol Biol,1995,29(4):773-786.
[27]Pelletier M K,Shirley B W.Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings.Coordinate regulation with chalcone synthase and chalcone isomerase[J].Plant Physiol,1996,111(1):339-345.
[28]Bartlett J.The genetic control of Anthocyanin biosynethesis in Antirrhinum majus[D].Norwich:England,University of East Anglia,1991.
[29]Renz A,and Stitt M.Substrate specificity and product inhibition of different forms of fructokinases and hexokinases developing potato tubers[J].Planta,1993,190:165-175.
[30]Filip Rolland,Brandon Moore,Jen Sheen.Sugar Sensing and Signaling in Plants[J].Plant Cell,2002,185-205.
[31]Sheen J,Zhou L,and Jang JC.Sugars as signaling molecules[J].Curr Opin Plant Biol,1999,2:410-418.
[32]程建徽,谢鸣,蒋桂华,等.植物己糖激酶的信号转导作用[J].细胞生物学杂志,2004,26:594-598.
[33]Renz A,Merlo L,Stitt M.Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity[J].Planta,1993,190:156-165.
[34]Martinez-Barajas E,and Randall DD.Purification and characterization of fructokinase from developing tomato(Lycopersicon esculentum Mill.)fruits[J].Planta,1996,199:451-458.
[35]Kanayama Y,Granot D,Dai N,et al.Tomato fructokinase exhibit differential expression and substrate regulation[J].Plant Physiol,1998,117:85-90.
[36]Hoepfner S W,Botha F C.Expression of fructokinase isoforms in sugarcane culm[J].Plant Physiol and Biochem,2003,41:741-747.
[37]王少丽,盛承发,乔传令.eDNA末端快速扩增技术及其应用.遗传[J],2004,26(3):419-423.
[38]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.黄培堂.第3版[M].北京:科学出版社,2002:644-651,1217-1270.
[39]唐克轩,开国银,张磊,等.RACE的研究及其在植物基因克隆上的应用[J].复旦学报(自然科学版),2002,41(6):704-709.
[41]Matthew K,Pelletier,Brenda W,Shirley.Analysis of flavanone 3-hydroxylase in arabidopsis seedlings[J].Plant Physiol,1996,111:339-345.
[40]李明,姚玉新,刘志,等.苹果果实中细胞质型苹果酸酶基因(NADP-ME)的克隆与表达分析[J].中国农学通报,2007,23(7):95-100.
[42]邓晓军,陈晓亚,杜家纬.植物挥发性物质及其代谢工程.植物生理与分子生物学学报[J],2004,30(1):11-18.
[2]李燕.龙眼胚胎发育不同时期的蛋白质组分析[D].福州:福建农林大学生命科学学院,2006.
[31]Lotan T,Ohto M.,Matsudaira Yee,et al.Arabidopsis LEAFYCOTYLEDON1 is sufficient to induce embryo development in vegetative cells[J].Cell,19.98,93:1195-1205.
[4]Stone S L,Kwong L W,Yee K M,et a.l.LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development[J].Proc Natl Acad Sci USA,2001,98:11806-11811.
[5]Kwong R W,BuiA Q,Lee H,et al.LEAFYCOTYLEDON1-LIKE defines a class of regulators essential for embryo development[J].Plant Cell,2003,15:5-18.
[6]Shevell D E,leu W M,Gilimor C S,et al.EMB30 is essential for normal cell division,cell expansion,and cell adhesion in Arabidopsis and encodes a protein that has similarity to Sec7[J].Cell,1994,77:1015-1063.
[7]Torres Ruiz R A,Lohner A,Jurgens G.The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo[J].Plant J,1996,10:1005-1016.
[8]Smith L G,Greene B,Veit B,et al.A dominant mutation in the maize homeobox gene,Knotted-1,causes its ectopic expression in leaf cells with altered fates[J].Development,1992,116:21-30.
[9]Hall Q.,and Cannon,M.C.The cell wall hydroxyproline-rich glycoprotein RSH is essential for normal embryo development in Arabidopsis[J].Plant Cell,2002,14:1161-1172.
[10]Schrick K,Mayer U,Horrichs A,et al.FACKEL is a sterol C-14reductase required for organized cell division and expansion in Arabidopsis embryogenesis[J].Genes Dev,2000,14:1471-1484.
[11]Boutilier K,Offringa R.,Sharma V K,et al.Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth[J].Plant Cell,2002,14:1737-1749.
[12]Hardtke C S,Berleth T.The Arabidopsis gene MONOPTEROUS encodes a transcription factor mediating embryo axis formation and vascular development[J].EMBO J,1998,17:1405-1411.
[13]Long J A,Moan E I,Medford J I,et al.A member of the KNOTTED class ofhomeodomain proteins encoded by STM gene in Arabidopsis[J].Nature,1996,379:66-69.
[14]Kaydamony C,Tewes A,Adler K,et al.Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv[J].Biochim Biophys Acta,2000,1491(1-3):143-160.
[15]ZuoJ,Niu QW,Frugis G,et al.The WUSCHEL gene promotes vegetative-to-embryonic in Arabidopsis[J].Plant J,2002,30(3):349-359.
[16]Dong J Z,Dunstan D I.Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce[Picea glauca(Moench)Voss][J].Planta.1996,200(1):85-91.
[17]王风华.龙眼体细胞胚胎发生过程中的基因差别表达[D].福州:福建农林大学园艺学院,2003.
[18]郭志雄.龙眼胚胎乙醇脱氢酶的分离纯化、鉴定及其cDNA克隆[D].福州:福建农林大学园艺学院,2006.
[19]孟丽,戴思兰.F3'5'H基因与蓝色花的形成[J].分子植物育种,2004,2(3):413-420.
[20]张学英,张上隆,骆军,等.果实花色素苷合成研究进展[J].果树学报,2004,21(5):456-460.
[21]Forkmann G,Heller W.Grisebach H.Anthocyanin biosynthesis in flowers of Matthiola incana.Flavanone 3- and Flavonoid 3'-hydroxylase[J].Zeitschrift Fur Naturforschung C-A Journal of Biosciences C,1980,35:691-695.
[22]Martin C,Prescott A,Mackay S.Control of anthocyanin biosynthesis in flowers of Antirrhinum majus[J].Plant J,1991(1):37-49.
[23]Britsch L,Ruhnau-Brich B,Forkmann G.Molecular cloning,sequence analysis,and invitro expression of flavanone 3-β-hydroxylase from Petunia hybrida[J].Biol Chem,1992,267:5380-5387.
[24]张大生,崔丽洁,王景明,等.红巴梨F3H基因的克隆及植物表达载体的构建[J].南京林业大学学报,2005,29(2):65-68.
[25]Deboo G B,Albertsen M C,Taylor L P.Flavanone 3-β-hydroxylase transcripts and flavonol accumulation are temporally coordinate in Maize anthers[J].Plant J,1995,7(5):703-713.
[26]Charrier B,Coronado C,Kondorosi A,et al.Molecular characterization and expression of alfafa flavanone 3-hydroxylase and dihydroflavono1-4-reductase encoding genes[J].Plant Mol Biol,1995,29(4):773-786.
[27]Pelletier M K,Shirley B W.Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings.Coordinate regulation with chalcone synthase and chalcone isomerase[J].Plant Physiol,1996,111(1):339-345.
[28]Bartlett J.The genetic control of Anthocyanin biosynethesis in Antirrhinum majus[D].Norwich:England,University of East Anglia,1991.
[29]Renz A,and Stitt M.Substrate specificity and product inhibition of different forms of fructokinases and hexokinases developing potato tubers[J].Planta,1993,190:165-175.
[30]Filip Rolland,Brandon Moore,Jen Sheen.Sugar Sensing and Signaling in Plants[J].Plant Cell,2002,185-205.
[31]Sheen J,Zhou L,and Jang JC.Sugars as signaling molecules[J].Curr Opin Plant Biol,1999,2:410-418.
[32]程建徽,谢鸣,蒋桂华,等.植物己糖激酶的信号转导作用[J].细胞生物学杂志,2004,26:594-598.
[33]Renz A,Merlo L,Stitt M.Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity[J].Planta,1993,190:156-165.
[34]Martinez-Barajas E,and Randall DD.Purification and characterization of fructokinase from developing tomato(Lycopersicon esculentum Mill.)fruits[J].Planta,1996,199:451-458.
[35]Kanayama Y,Granot D,Dai N,et al.Tomato fructokinase exhibit differential expression and substrate regulation[J].Plant Physiol,1998,117:85-90.
[36]Hoepfner S W,Botha F C.Expression of fructokinase isoforms in sugarcane culm[J].Plant Physiol and Biochem,2003,41:741-747.
[37]王少丽,盛承发,乔传令.eDNA末端快速扩增技术及其应用.遗传[J],2004,26(3):419-423.
[38]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.黄培堂.第3版[M].北京:科学出版社,2002:644-651,1217-1270.
[39]唐克轩,开国银,张磊,等.RACE的研究及其在植物基因克隆上的应用[J].复旦学报(自然科学版),2002,41(6):704-709.
[41]Matthew K,Pelletier,Brenda W,Shirley.Analysis of flavanone 3-hydroxylase in arabidopsis seedlings[J].Plant Physiol,1996,111:339-345.
[40]李明,姚玉新,刘志,等.苹果果实中细胞质型苹果酸酶基因(NADP-ME)的克隆与表达分析[J].中国农学通报,2007,23(7):95-100.
[42]邓晓军,陈晓亚,杜家纬.植物挥发性物质及其代谢工程.植物生理与分子生物学学报[J],2004,30(1):11-18.