葡萄SVP-like MADS-box、MADS12基因的克隆与植物表达载体的构建
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摘要
葡萄是世界上最重要的果树之一,其种植面积和产量都在世界前列。研究花发育的机理对果树栽培、育种工作的发展以及促进果品生产都具有重要的意义。
本试验以藤稔葡萄的花序为试材,应用电子克隆技术从葡萄EST数据库中拼接到两个MADS-box基因,并用RT-PCR方法扩增得到了该基因的编码区全长序列,分别命名为VvSVP和VvMADS12。结果如下：
VvSVP基因序列编码区全长684 bp,编码227个氨基酸残基,与PtMADS1、MdJOINTLESS、PkMADS、SVP分别有84.1%、81.7%、78.9%和74.9%的同源性。该序列具有MADS-box基因典型的MADS-box和K-box结构域。系统发育分析同样将VvSVP基因归入MADS-box基因家族的STMADS11进化系。电子表达谱分析显示,该基因在葡萄的芽、叶片、花序及果实中表达,在花、花梗、茎、根中不表达,推测该基因与葡萄花序及果实发育有关。将VvSVP mRNA与基因组序列比对发现,VvSVP基因由8个外显子,7个内含子组成,与SVP基因的结构不完全相同。
VvMADS12基因序列编码区全长630 bp,编码209个氨基酸残基,与PdMADS-box、AmDEFH7、MODEFH7、SlTDR8、CsMADS-box、PrMADS-box、ZmMADS15分别有70%、63%、62%、60%、66%、45%和50%的同源性。该序列具有MADS-box基因典型的MADS-box和K-box结构域。系统发育分析将VvMADS12基因归入MADS-box基因家族的TM3进化系。分析VvMADS12 mRNA的表达谱,结果表明：VvMADS12基因在葡萄的花序、花梗、茎中表达,在花、果实、叶、根中没有检测到,说明VvMADS12与葡萄花序发育有关。将VvSVP mRNA与基因组序列比对发现,VvSVP基因由8个外显子,7个内含子组成。成功构建了VvSVP和VvMADS12基因的植物双元表达载体。
Grape is one of the most important fruit trees in the world, its culture area and total yields are advanced in the world. it is a great significance to study the mechanism of flower development on fruit trees cultivation, breeding and promoting the development of fruit production.
A SHORT VEGETATIVE PHASE (SVP) homologue was isolated from Vitis vinifera x V. labrusca'Fujiminori'inflorescence by bioformatic analysis and RT-PCR. It is 684 bp, coding a polypeptide of 227 amino acids, named as VvSVP. Homology analysis showed that the deduced VvSVP protein was highly homologous to the MADS-box genes, PMADS1, MdJOINTLESS、PkMADS、SVP, the identity are 84.1%、81.7%、78.9% and74.9%, respectively. Phylogenetic analysis also indicated that VvSVP belongs to the STMADS11 lineage in MADS-box gene family. Expression pattern analysis showed that VvSVP expressed in buds, leaves, inflorescenes, fruits, but not expressed in flowers, pedicels, stems and roots. Blast of VvSVP with a shotgun genome sequence indicated that VvSVP genome is consist of 8 exons and 7 introns, the number and length of exon is not the same as SVP.
Electronic VvMADS12 homologue was isolated from Vitis vinifera×V. labrusca 'Fujiminori'inflorescence by bioformatic analysis and RT-PCR. It is 630 bp, coding a polypeptide of 209 amino acids, named as VvMADS12. Homology analysis showed that the deduced VvMADS12 protein was highly homologous to the MADS-box genes, Pd MADS-box、AmDEFH7、MODEFH7、SlTDR8、CsMADS-box、PrMADS-box、ZmMADS15the identity are 70%、63%、62%、60%、66%、45%、and50%, respectively. Phylogenetic analysis also indicated that VvMADS12 belongs to the TM3 lineage in MADS-box gene family. Expression pattern analysis showed that VvMADS12 expressed in buds, leaves, inflorescenes, fruits, but not expressed in flowers, pedicels, stems and roots. Blast of VvMADS12 with a shotgun genome sequence indicated that VvMADS12 genome is consist of 8 exons and 7 introns.
We constructed plant overexpression vector of VvSVP and VvMADS12.
本试验以藤稔葡萄的花序为试材,应用电子克隆技术从葡萄EST数据库中拼接到两个MADS-box基因,并用RT-PCR方法扩增得到了该基因的编码区全长序列,分别命名为VvSVP和VvMADS12。结果如下：
VvSVP基因序列编码区全长684 bp,编码227个氨基酸残基,与PtMADS1、MdJOINTLESS、PkMADS、SVP分别有84.1%、81.7%、78.9%和74.9%的同源性。该序列具有MADS-box基因典型的MADS-box和K-box结构域。系统发育分析同样将VvSVP基因归入MADS-box基因家族的STMADS11进化系。电子表达谱分析显示,该基因在葡萄的芽、叶片、花序及果实中表达,在花、花梗、茎、根中不表达,推测该基因与葡萄花序及果实发育有关。将VvSVP mRNA与基因组序列比对发现,VvSVP基因由8个外显子,7个内含子组成,与SVP基因的结构不完全相同。
VvMADS12基因序列编码区全长630 bp,编码209个氨基酸残基,与PdMADS-box、AmDEFH7、MODEFH7、SlTDR8、CsMADS-box、PrMADS-box、ZmMADS15分别有70%、63%、62%、60%、66%、45%和50%的同源性。该序列具有MADS-box基因典型的MADS-box和K-box结构域。系统发育分析将VvMADS12基因归入MADS-box基因家族的TM3进化系。分析VvMADS12 mRNA的表达谱,结果表明：VvMADS12基因在葡萄的花序、花梗、茎中表达,在花、果实、叶、根中没有检测到,说明VvMADS12与葡萄花序发育有关。将VvSVP mRNA与基因组序列比对发现,VvSVP基因由8个外显子,7个内含子组成。成功构建了VvSVP和VvMADS12基因的植物双元表达载体。
Grape is one of the most important fruit trees in the world, its culture area and total yields are advanced in the world. it is a great significance to study the mechanism of flower development on fruit trees cultivation, breeding and promoting the development of fruit production.
A SHORT VEGETATIVE PHASE (SVP) homologue was isolated from Vitis vinifera x V. labrusca'Fujiminori'inflorescence by bioformatic analysis and RT-PCR. It is 684 bp, coding a polypeptide of 227 amino acids, named as VvSVP. Homology analysis showed that the deduced VvSVP protein was highly homologous to the MADS-box genes, PMADS1, MdJOINTLESS、PkMADS、SVP, the identity are 84.1%、81.7%、78.9% and74.9%, respectively. Phylogenetic analysis also indicated that VvSVP belongs to the STMADS11 lineage in MADS-box gene family. Expression pattern analysis showed that VvSVP expressed in buds, leaves, inflorescenes, fruits, but not expressed in flowers, pedicels, stems and roots. Blast of VvSVP with a shotgun genome sequence indicated that VvSVP genome is consist of 8 exons and 7 introns, the number and length of exon is not the same as SVP.
Electronic VvMADS12 homologue was isolated from Vitis vinifera×V. labrusca 'Fujiminori'inflorescence by bioformatic analysis and RT-PCR. It is 630 bp, coding a polypeptide of 209 amino acids, named as VvMADS12. Homology analysis showed that the deduced VvMADS12 protein was highly homologous to the MADS-box genes, Pd MADS-box、AmDEFH7、MODEFH7、SlTDR8、CsMADS-box、PrMADS-box、ZmMADS15the identity are 70%、63%、62%、60%、66%、45%、and50%, respectively. Phylogenetic analysis also indicated that VvMADS12 belongs to the TM3 lineage in MADS-box gene family. Expression pattern analysis showed that VvMADS12 expressed in buds, leaves, inflorescenes, fruits, but not expressed in flowers, pedicels, stems and roots. Blast of VvMADS12 with a shotgun genome sequence indicated that VvMADS12 genome is consist of 8 exons and 7 introns.
We constructed plant overexpression vector of VvSVP and VvMADS12.
引文
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