草莓果实特异性启动子的克隆与功能分析
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摘要
组成型启动子是植物基因工程中广泛应用的启动子类型之一,可启动外源基因在受体植物中非特异性的持续、稳定的表达,但不能从时间和空间上对外源基因的表达进行有效的调控,这可能会造成植物在能源利用上的浪费,甚至会对转基因植物造成伤害。组织特异型启动子可以启动外源基因在受体植物的特定组织器官中高效表达,果实特异性启动子作为重要的组织特异性启动子,可以控制外源基因在转基因植物的果实中特异表达。克隆和研究果实特异性启动子,可为改良果实品质奠定基础。
从Genbank中筛选了一个由差减文库筛选得到的草莓基因组EST序列(Accession number:DY633382),将推测对应的基因简称DY基因。对此EST序列进行同源性分析,发现它与葡萄中的一个与果实成熟相关的丝氨酸/苏氨酸激酶基因有很高的相似性(83%)。进而,对草莓(Fragaria ananassa,Duch.,cvChandler)中DY基因的表达模式进行RT-PCR分析,结果显示DY基因在果实中特异表达,且在不同发育时期表达量存在差异。在绿色果实期转录水平最高,在白色果实期转录水平最低,在转变期和红色果实期转录水平较白色果实期有所升高,但比绿色果实期稍低。RT-PCR结果表明DY基因是果实表达特异性的基因。
根据EST:DY633382序列设计特异引物,利用TAIL-PCR克隆得到了DY基因的起始密码子上游的1021bp启动子序列。利用在线生物信息学软件NSITE和PLACE分析该序列,结果表明DY基因启动子区TATA-box位于起始位点上游的-32～-27区段,该序列包含有18个可能的顺式作用元件,如CCGTCG motif、GA-1、AT-rich F等。利用PCR获得了DY基因启动子序列三个缺失片段,分别构建植物表达载体pCAMBIA1305-DⅠ-GUS、pCAMBIA1305-DⅡ-GUS、pCAMBIA1305-DⅢ-GUS。然后利用农杆菌浸染的方法转化烟草,通过PCR和Sourthern杂交分析的方法鉴定单拷贝插入的转基因植株。转基因烟草植株的组织化学染色分析结果表明启动子序列的不同缺失片段都可以启动GUS基因在果实(子房)中特异性表达,在叶片、根、茎和花中没有表达。GUS组织染色结果说明在启动子的-523到转录起始位点的区段存在调控基因果实特异表达的顺式元件。植物表达载体pCAMBIA1305-DⅠ-GUS、pCAMBIA1305-DⅡ-GUS、pCAMBIA1305-DⅢ-GUS在烟草果实(子房)中的表达强度比组成型表达载体pCAMBIA1305在烟草果实(子房)中的GUS基因表达强度要低。在烟草果实发育时期Ⅰ,转pCAMBIA1305-DⅡ-GUS和pCAMBIA1305-DⅡ-GUS表达载体的转基因烟草果实的GUS酶活性比pCAMBIA1305-DⅢ-GUS的表达强度高,且差异较大,推测存在于DY调控序列-699～-523区段的AT-rich F有增强基因表达的作用。
HyPRP基因在草莓果实中特异表达,其产物HyPRP是一类富含脯氨酸的细胞壁结构蛋白,与草莓果实成熟过程中果实内多酚的锚定有关。利用PCR方法得到了HyPRP基因的778bp的启动子序列。利用在线生物信息学软件NSITE和PLACE分析该778bp启动子序列,结果表明在HyPRP启动子序列中,TATA-box位于起始密码子上游-59～-56区段,包含有23个可能的上游元件,如ABRE、ACGTbox、GT motif等。利用PCR扩增了HyPRP基因启动子序列的两个缺失片段,分别构建了植物表达载体pCAMBIA1305-HⅠ-GUS、pCAMBIA1305-HⅡ-GUS,然后利用农杆菌介导的方法转化烟草。对转基因烟草的GUS活性分析显示,pCAMBIA1305-HⅠ-GUS和pCAMBIA1305-HⅡ-GUS中的GUS基因在转基因烟草果实(子房)中特异表达,说明启动子序列-581到起始密码子的序列上存在调控基因果实特表达的顺式作用元件。通过GUS酶活分析发现,转化pCAMBIA1305-HⅠ-GUS的转基因烟草果实(子房)酶活性大大高于转化pCAMBIA1305-HⅡ-GUS的。可能在-778～-581区段存在增强基因表达的顺式作用元件,pCAMBIA1305-HⅡ-GUS由于缺失了该元件而导致GUS基因表达减弱。
Constitutive promoter is one kind of promoters used in plant genetic engineering. This kind of promoter can drive the non-specific,sustained,stable expression of target gene;but not regulate the gene expression with the temporal and spatial control, which may result in the waste of plant nutrition,and even damage to the transgenic plant.Tissue-specific promoters can induce high expression of target gene in one or several specific tissues.As one important kind of tissue-specific promoters, fruit-specific promoter can allow the specific expression of a transgene in fruit tissue(s),which would offer more precise time and spatial control to the target genes. Cloning and studying of fruit-specific promoters can make foundation for improving the quality of fruit.
By searching in GenBank database,an EST sequence(DY633382),obtained from a subtractive library of strawberry,was selected for further analysis,which has high similarity(83%)with a grape serine/ammonia acid kinase gene related to fruit ripening.The expression pattern of the gene corresponding gene(DY gene)in strawberry(Fragaria ananassa,Duch.,cv Chandler)was also analyzed by RT-PCR, which showed the presence of DY transcripts only in fruit.The result indicated that the level of transcription in strawberry fruit was the highest in green fruit stage,the lowest in the white fruit stage,and then rose in the turning stage and red fruit stage. The RT-PCR analysis revealed the specific-expression of DY gene in strawberry fruit.
According to the sequence of EST DY633382,specific primers were designed for Tail-PCR to obtain upstream sequence of DY gene.A fragment containing 1021 bps of upstream sequence 5' of the DY gene start codon was isolated using the TaiI-PCR.The 1021bps 5' flanking sequence of the DY gene was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE on-line software.The results indicated that TATA box(TATAAT)is located at-32～-27bp of the ATG stat codon.DY gene promoter contains 18 putative cis-elements,such as,CCGTCG motif,GA-1,AT-rich F et al.
To analyze the transcriptional regulation pattern of DY gene promoter,three plant expression vector pCAMBIA1305-DI-GUS,pCAMBIA1305-Ⅱ-GUS, pCAMBIA1305-DⅢ-GUS,which contained 1021bp,699bp and 523bp promoter fragment,respectively,were constructed.These vectors were introduced into tobacco via A.tumefaciens-mediated transformation.Transgenic plants were confirmed by PCR and Southern Blotting analysis.The transgenic plants containing a single copy insertion were selected for the analysis of transcriptional regulation pattern of DY gene promoeter.
Using histochemical staining method,Gus activity was detected in the fruit(ovary) parts for transgenic tobacco plants carrying the three constructs pCAMBIA1305-DⅠ-GUS,pCAMBIA1305-DⅡ-GUS,pCAMBIA 1305-DⅢ-GUS.In contrast,staining of roots,shoots,leaves and flowers structures were not observed in these lines.Thus,the GUS gene can be specific-induced by the DY promoter,which indicated that the region(-523-ATG)contained some cis-element to regulate the gene fruit-specific expression.Temporal and spatial expression of GUS gene was studied at different development stages of fruit(ovary)in transgenic lines.The GUS activity is higher in development stageⅠcompared with in the other development stages.During the fruit stageⅠ,GUS activity in the transgenic plants containing pCAMBIA1305-DⅡ-GUS and pCAMBIA1305-DⅡwere higher than that in the transgenic lines containing pCAMBIA1305-DⅢ.The results indicated that the cis-element AT-rich F located in the-699～-523 of DY promoter might enhance expression of genes.
HyPRR is specific-expressed gene in strawberry fruit,and its product HyPRP is a hybrid proline-rich structure protein in cell wall,which plays a role in the anchoring of polyphenols in the strawberry fruit during the growth and ripening.We isolated a 778bp fragment of the HyPRR promoter using PCR.The sequence of the HyPRR promoter was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE database on line.TATA-box(TATAAT)is located at-59～-56bp of the ATG start codon,and HyPRR gene promoter contains 23 putative cis-elements,such as, ABRE,ACGT box,GT motif et al.Two fusion vectors containing the GUS gene under the control of promoter fragments of different length(581bp and 778bp)was constructed,named pCAMBIA1305-HⅠ-GUS and pCAMBIA1305-HⅡ-GUS,and transformed separately into tobacco.Transgenic plants were used to analyze the expression of GUS gene through Histochemical analysis.The GUS signal was only seen in fruit(ovary)tissue,indicating that the promoter can specifically regulate the gene expression in fruit,and the region(-581-ATG)contained the cis-element to regulate the gene fruit-specific expression.The activity of GUS in transgenic lines transformed with CAMBIA1305-HⅠ-GUS was considerably higher than that in lines with CAMBIA1305-HⅡ-GUS construct.These results showed there might be some cis-elements to increase the gene expression level in the-778—581 region of promoter.
从Genbank中筛选了一个由差减文库筛选得到的草莓基因组EST序列(Accession number:DY633382),将推测对应的基因简称DY基因。对此EST序列进行同源性分析,发现它与葡萄中的一个与果实成熟相关的丝氨酸/苏氨酸激酶基因有很高的相似性(83%)。进而,对草莓(Fragaria ananassa,Duch.,cvChandler)中DY基因的表达模式进行RT-PCR分析,结果显示DY基因在果实中特异表达,且在不同发育时期表达量存在差异。在绿色果实期转录水平最高,在白色果实期转录水平最低,在转变期和红色果实期转录水平较白色果实期有所升高,但比绿色果实期稍低。RT-PCR结果表明DY基因是果实表达特异性的基因。
根据EST:DY633382序列设计特异引物,利用TAIL-PCR克隆得到了DY基因的起始密码子上游的1021bp启动子序列。利用在线生物信息学软件NSITE和PLACE分析该序列,结果表明DY基因启动子区TATA-box位于起始位点上游的-32～-27区段,该序列包含有18个可能的顺式作用元件,如CCGTCG motif、GA-1、AT-rich F等。利用PCR获得了DY基因启动子序列三个缺失片段,分别构建植物表达载体pCAMBIA1305-DⅠ-GUS、pCAMBIA1305-DⅡ-GUS、pCAMBIA1305-DⅢ-GUS。然后利用农杆菌浸染的方法转化烟草,通过PCR和Sourthern杂交分析的方法鉴定单拷贝插入的转基因植株。转基因烟草植株的组织化学染色分析结果表明启动子序列的不同缺失片段都可以启动GUS基因在果实(子房)中特异性表达,在叶片、根、茎和花中没有表达。GUS组织染色结果说明在启动子的-523到转录起始位点的区段存在调控基因果实特异表达的顺式元件。植物表达载体pCAMBIA1305-DⅠ-GUS、pCAMBIA1305-DⅡ-GUS、pCAMBIA1305-DⅢ-GUS在烟草果实(子房)中的表达强度比组成型表达载体pCAMBIA1305在烟草果实(子房)中的GUS基因表达强度要低。在烟草果实发育时期Ⅰ,转pCAMBIA1305-DⅡ-GUS和pCAMBIA1305-DⅡ-GUS表达载体的转基因烟草果实的GUS酶活性比pCAMBIA1305-DⅢ-GUS的表达强度高,且差异较大,推测存在于DY调控序列-699～-523区段的AT-rich F有增强基因表达的作用。
HyPRP基因在草莓果实中特异表达,其产物HyPRP是一类富含脯氨酸的细胞壁结构蛋白,与草莓果实成熟过程中果实内多酚的锚定有关。利用PCR方法得到了HyPRP基因的778bp的启动子序列。利用在线生物信息学软件NSITE和PLACE分析该778bp启动子序列,结果表明在HyPRP启动子序列中,TATA-box位于起始密码子上游-59～-56区段,包含有23个可能的上游元件,如ABRE、ACGTbox、GT motif等。利用PCR扩增了HyPRP基因启动子序列的两个缺失片段,分别构建了植物表达载体pCAMBIA1305-HⅠ-GUS、pCAMBIA1305-HⅡ-GUS,然后利用农杆菌介导的方法转化烟草。对转基因烟草的GUS活性分析显示,pCAMBIA1305-HⅠ-GUS和pCAMBIA1305-HⅡ-GUS中的GUS基因在转基因烟草果实(子房)中特异表达,说明启动子序列-581到起始密码子的序列上存在调控基因果实特表达的顺式作用元件。通过GUS酶活分析发现,转化pCAMBIA1305-HⅠ-GUS的转基因烟草果实(子房)酶活性大大高于转化pCAMBIA1305-HⅡ-GUS的。可能在-778～-581区段存在增强基因表达的顺式作用元件,pCAMBIA1305-HⅡ-GUS由于缺失了该元件而导致GUS基因表达减弱。
Constitutive promoter is one kind of promoters used in plant genetic engineering. This kind of promoter can drive the non-specific,sustained,stable expression of target gene;but not regulate the gene expression with the temporal and spatial control, which may result in the waste of plant nutrition,and even damage to the transgenic plant.Tissue-specific promoters can induce high expression of target gene in one or several specific tissues.As one important kind of tissue-specific promoters, fruit-specific promoter can allow the specific expression of a transgene in fruit tissue(s),which would offer more precise time and spatial control to the target genes. Cloning and studying of fruit-specific promoters can make foundation for improving the quality of fruit.
By searching in GenBank database,an EST sequence(DY633382),obtained from a subtractive library of strawberry,was selected for further analysis,which has high similarity(83%)with a grape serine/ammonia acid kinase gene related to fruit ripening.The expression pattern of the gene corresponding gene(DY gene)in strawberry(Fragaria ananassa,Duch.,cv Chandler)was also analyzed by RT-PCR, which showed the presence of DY transcripts only in fruit.The result indicated that the level of transcription in strawberry fruit was the highest in green fruit stage,the lowest in the white fruit stage,and then rose in the turning stage and red fruit stage. The RT-PCR analysis revealed the specific-expression of DY gene in strawberry fruit.
According to the sequence of EST DY633382,specific primers were designed for Tail-PCR to obtain upstream sequence of DY gene.A fragment containing 1021 bps of upstream sequence 5' of the DY gene start codon was isolated using the TaiI-PCR.The 1021bps 5' flanking sequence of the DY gene was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE on-line software.The results indicated that TATA box(TATAAT)is located at-32～-27bp of the ATG stat codon.DY gene promoter contains 18 putative cis-elements,such as,CCGTCG motif,GA-1,AT-rich F et al.
To analyze the transcriptional regulation pattern of DY gene promoter,three plant expression vector pCAMBIA1305-DI-GUS,pCAMBIA1305-Ⅱ-GUS, pCAMBIA1305-DⅢ-GUS,which contained 1021bp,699bp and 523bp promoter fragment,respectively,were constructed.These vectors were introduced into tobacco via A.tumefaciens-mediated transformation.Transgenic plants were confirmed by PCR and Southern Blotting analysis.The transgenic plants containing a single copy insertion were selected for the analysis of transcriptional regulation pattern of DY gene promoeter.
Using histochemical staining method,Gus activity was detected in the fruit(ovary) parts for transgenic tobacco plants carrying the three constructs pCAMBIA1305-DⅠ-GUS,pCAMBIA1305-DⅡ-GUS,pCAMBIA 1305-DⅢ-GUS.In contrast,staining of roots,shoots,leaves and flowers structures were not observed in these lines.Thus,the GUS gene can be specific-induced by the DY promoter,which indicated that the region(-523-ATG)contained some cis-element to regulate the gene fruit-specific expression.Temporal and spatial expression of GUS gene was studied at different development stages of fruit(ovary)in transgenic lines.The GUS activity is higher in development stageⅠcompared with in the other development stages.During the fruit stageⅠ,GUS activity in the transgenic plants containing pCAMBIA1305-DⅡ-GUS and pCAMBIA1305-DⅡwere higher than that in the transgenic lines containing pCAMBIA1305-DⅢ.The results indicated that the cis-element AT-rich F located in the-699～-523 of DY promoter might enhance expression of genes.
HyPRR is specific-expressed gene in strawberry fruit,and its product HyPRP is a hybrid proline-rich structure protein in cell wall,which plays a role in the anchoring of polyphenols in the strawberry fruit during the growth and ripening.We isolated a 778bp fragment of the HyPRR promoter using PCR.The sequence of the HyPRR promoter was used to analyze plant cis-regulatory DNA elements using NSITE and PLACE database on line.TATA-box(TATAAT)is located at-59～-56bp of the ATG start codon,and HyPRR gene promoter contains 23 putative cis-elements,such as, ABRE,ACGT box,GT motif et al.Two fusion vectors containing the GUS gene under the control of promoter fragments of different length(581bp and 778bp)was constructed,named pCAMBIA1305-HⅠ-GUS and pCAMBIA1305-HⅡ-GUS,and transformed separately into tobacco.Transgenic plants were used to analyze the expression of GUS gene through Histochemical analysis.The GUS signal was only seen in fruit(ovary)tissue,indicating that the promoter can specifically regulate the gene expression in fruit,and the region(-581-ATG)contained the cis-element to regulate the gene fruit-specific expression.The activity of GUS in transgenic lines transformed with CAMBIA1305-HⅠ-GUS was considerably higher than that in lines with CAMBIA1305-HⅡ-GUS construct.These results showed there might be some cis-elements to increase the gene expression level in the-778—581 region of promoter.
引文
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