黄瓜（Cucumis sativus L.）遗传转化、植株再生及驯化技术研究
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摘要
黄瓜(Cucumis sativus L.)是世界范围内的重要蔬菜。一方面,建立一套稳定的遗传转化体系可为运用过量表达、报告基因表达和RNAi等技术进行黄瓜功能基因组学研究提供技术支撑；另一方面,由于黄瓜现有栽培品种的遗传基础十分狭窄,与黄瓜属其他野生资源远缘杂交的亲合性很低,采用传统育种方法很难在短时间内获得对生物或非生物胁迫具有抗性的优良品种,而植物基因工程是扩大黄瓜现有资源遗传基础的有效途径。为此,本试验建立了稳定的黄瓜瞬时表达和成株遗传转化体系,主要结果如下：
1构建了由CaMV35S启动子调控的、2种黄瓜a-半乳糖苷酶的增强型绿色荧光蛋白(enhanced green flurescent protein, EGFP)融合表达载体。运用聚合酶链式反应(polymerase chain reaction, PCR)技术,以质粒pCambia1303为模板扩增了CaMV35S启动子序列；以质粒pEGFP-N1为模板扩增了EGFP编码序列；运用反转录聚合酶链式反应(Reverse transcript-PCR, RT-PCR)技术,以黄瓜总RNA为模板扩增了黄瓜酸性α-半乳糖苷酶Ⅰ或碱性α-半乳糖苷酶Ⅰ编码序列。将上述3个片段插入表达载体pCambia1381*c的多克隆位点,分别构建了EGFP位于目的基因C端的2个α-半乳糖苷酶基因的EGFP融合表达载体。
2在NH4NO3浓度为2.0g/L、6-BA浓度为1.0mg/L、NAA浓度为0.1mg/L的MS培养基中添加酵母提取物5g/L有利于产生质地疏松的愈伤组织,适合作为外源基因瞬时表达的受体。基因枪法将上述经测序验证的2个表达载体分别导入洋葱表皮和黄瓜愈伤组织细胞,共聚焦显微镜下观察EGFP的表达情况。结果表明黄瓜酸性α-半乳糖苷酶Ⅰ在包括液泡在内的整个细胞中均有表达,而黄瓜碱性α-半乳糖苷酶Ⅰ主要在细胞核和细胞周边表达。
3通过比较两种野生型黄瓜品种的不同外植体,发现欧洲型黄瓜EP-6适合用子叶为外植体诱导再生植株,华北型黄瓜津春4号适合用茎节作为外植体诱导再生植株。适合EP-6子叶再生芽的最佳芽诱导培养基为(MS±0.5mg/L ABA+2.0mg/L6-BA+2.0mg/L AgNO3);适合津春4号茎节再生芽的最佳诱导培养基为(MS±0.5mg/L6-BA);最佳生根培养基为(1/2MS+15g/L蔗糖+0.6mg/L IBA)。
4EP-6子叶转化体系的建立：将子叶在上述最佳诱芽培养基上预培养2d,以菌液pCambia2201/EHA105侵染20min,在含50μmol/LAS的最佳芽诱导培养基中黑暗条件下共培养2d,转入选择培养基(最佳诱芽培养基+400mg/L Cef)中筛选抗性芽,光照条件下培养25d左右,有抗性芽长出时转入伸长培养基中培养。抗性芽2cm左右时转入生根培养基,获得抗性株系,伸长培养和生根培养时添加60mg/L Kan和400mg/L Cef进行筛选。
5津春4号的茎节转化体系的建立：将茎节在最佳诱芽培养基上预培养2d,以菌液pCambia2201/EHA105侵染20min,在含50μmol/LAS的最佳芽诱导培养基中黑暗条件下共培养2d,转入选择培养基(最佳诱芽培养基+400mg/L Cef)中筛选抗性芽,光照条件下培养,12d左右有抗性芽长出时转入伸长培养基中培养。抗性芽2cm左右时转入生根培养,获得抗性株系,伸长培养和生根培养时添加80mg/L Kan和400mg/L Cef进行筛选。
6统计结果显示,以津春4号的黄瓜茎节为外植体时,从共培养开始到转化苗生根平均需42d；以EP-6黄瓜的子叶作为外植体时,从共培养开始到转化苗生根平均需59d。若以RT-PCR阳性作为判断最终转化成功的标准,津春4号茎节转化体系的转化率为0.83%,EP-6子叶转化体系的转化率为0.36%
7将转基因组培苗于蔗糖浓度为6%的1/2MS培养基中生根4周,随后移栽至蔗糖为3%的培养基中培养1周,在组培苗6叶1心时,移栽于草碳：珍珠岩：河沙=1：1：1的基质中驯化,最有利于组培苗根系的发育,驯化成活率可达98.7%。
Cucumber (Cucumis sativus L.) is grown worldwide as an important vegetable. On the one hand, establishing an efficient cucumber transformation system is important for functional genomics research through techniques such as gene over-expression, reporter gene expression and RNA interference. On the other hand, Improving the biotic or abiotic resistance of cucumber through conventional breeding is limited by its narrow genetic basis and incompatibility barriers to wild Cucumis species. Therefore, it would be desirable to apply genetic engineering to expand the germplasm base of this crop. In this study, a European genotype EP-6and a Northern China genotype Jinchun4were selected to study the callus fomation, transformation, heterogenous gene transient expression and plant regeneration technology of cucumber.
1Two kinds of cucumber a-galactosidase-EGFP fusion expression vector controlled by the CaMV35S promoter were constructed. CaMV35S promoter sequence was amplified by polymerase chain reactions (PCR) from vector pCambia1303; The entire coding region of EGFP was amplified by PCR from vector pEGFP-Nl; The entire coding region of cucumber acid a-galactosidase I and alkaline a-galactosidase I were amplified by Reverse transcript PCR using cucumber leaf total RNAs as template. The three above-mentioned fragments were Inserted into the mμltiple cloning sites of expression vector pCambia1381*c. Then two a-galactosidase-EGFP fusion expression vectors were constructed respectively and the EGFP gene was located at the C-terminal of the target genes.
2To obtain the suitalbe material for transient expression of heterogenous genes, friable calli can be produced on the medium with6-BA1.0mg/L,NAA0.1mg/L,NH4NO32.0g/L and Yeast extrat5g/L. Two recombinant plasmids were transformed into onion epidermal cells and cucumber callus cells by particle bombardment. Green fluorescence signals were monitored by a confocal microscope. The results showed that cucumber acid a-galactosidase I is located in the whole cells includeing the vacuole, while cucumber alkaline a-galactosidase I is located at the nucleus and the peripheral region of the cell.
3Regeneration of cucumber plants:Cotyledon is a suitable explant for in vitro shoot induction of EP-6, while for Jinchun4, highest shoot induction rate was obtained if stem node was used as explant. The optimal culture medium of shoot induction of EP-6cotyledons was MS+0.5mg/L ABA+2.0mg/L6-BA+2.0mg/L AgNO3, while the best culture medium for Jinchun4shoot induction was MS+0.5mg/L6-BA. The highest root induction rate can be observed when the regenerated shoots were cultured on the medium1/2MS+15g/L sucrose+0.6mg/L IBA.
4The transfomation system of EP-6cotyledons:After2days of pre-culture on the optimized shoot induction medium, the cotyledons were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about25d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained60mg/L Kan and400mg/L Cef.
5The transfomation system of Jinchun4stem nodes:After2days of pre-culture on the optimized shoot induction medium, the stem nodes were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about12d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained80mg/L Kan and400mg/L Cef.
6From explant co-culture to well rooted putative transgenic plantlets, the transfomation systems of Jinchun4stem nodes and EP-6cotyledons needed42d and59d respectively. The transformation efficiency can be evaluated as0.83%for the Jinchun4stem node system and0.36%for the EP-6cotyledon system by RT-PCR positive plantlets.
7The highest survival rate of98.7%of transgenic plants can be obtained when the plantlets were rooted in the1/2MS with6%sucrose for4weeks, then transferred to the1/2MS with3%sucrose for1week, and finally transplanted to the substrate peat:perlite:sand=1:1:1.
1构建了由CaMV35S启动子调控的、2种黄瓜a-半乳糖苷酶的增强型绿色荧光蛋白(enhanced green flurescent protein, EGFP)融合表达载体。运用聚合酶链式反应(polymerase chain reaction, PCR)技术,以质粒pCambia1303为模板扩增了CaMV35S启动子序列；以质粒pEGFP-N1为模板扩增了EGFP编码序列；运用反转录聚合酶链式反应(Reverse transcript-PCR, RT-PCR)技术,以黄瓜总RNA为模板扩增了黄瓜酸性α-半乳糖苷酶Ⅰ或碱性α-半乳糖苷酶Ⅰ编码序列。将上述3个片段插入表达载体pCambia1381*c的多克隆位点,分别构建了EGFP位于目的基因C端的2个α-半乳糖苷酶基因的EGFP融合表达载体。
2在NH4NO3浓度为2.0g/L、6-BA浓度为1.0mg/L、NAA浓度为0.1mg/L的MS培养基中添加酵母提取物5g/L有利于产生质地疏松的愈伤组织,适合作为外源基因瞬时表达的受体。基因枪法将上述经测序验证的2个表达载体分别导入洋葱表皮和黄瓜愈伤组织细胞,共聚焦显微镜下观察EGFP的表达情况。结果表明黄瓜酸性α-半乳糖苷酶Ⅰ在包括液泡在内的整个细胞中均有表达,而黄瓜碱性α-半乳糖苷酶Ⅰ主要在细胞核和细胞周边表达。
3通过比较两种野生型黄瓜品种的不同外植体,发现欧洲型黄瓜EP-6适合用子叶为外植体诱导再生植株,华北型黄瓜津春4号适合用茎节作为外植体诱导再生植株。适合EP-6子叶再生芽的最佳芽诱导培养基为(MS±0.5mg/L ABA+2.0mg/L6-BA+2.0mg/L AgNO3);适合津春4号茎节再生芽的最佳诱导培养基为(MS±0.5mg/L6-BA);最佳生根培养基为(1/2MS+15g/L蔗糖+0.6mg/L IBA)。
4EP-6子叶转化体系的建立：将子叶在上述最佳诱芽培养基上预培养2d,以菌液pCambia2201/EHA105侵染20min,在含50μmol/LAS的最佳芽诱导培养基中黑暗条件下共培养2d,转入选择培养基(最佳诱芽培养基+400mg/L Cef)中筛选抗性芽,光照条件下培养25d左右,有抗性芽长出时转入伸长培养基中培养。抗性芽2cm左右时转入生根培养基,获得抗性株系,伸长培养和生根培养时添加60mg/L Kan和400mg/L Cef进行筛选。
5津春4号的茎节转化体系的建立：将茎节在最佳诱芽培养基上预培养2d,以菌液pCambia2201/EHA105侵染20min,在含50μmol/LAS的最佳芽诱导培养基中黑暗条件下共培养2d,转入选择培养基(最佳诱芽培养基+400mg/L Cef)中筛选抗性芽,光照条件下培养,12d左右有抗性芽长出时转入伸长培养基中培养。抗性芽2cm左右时转入生根培养,获得抗性株系,伸长培养和生根培养时添加80mg/L Kan和400mg/L Cef进行筛选。
6统计结果显示,以津春4号的黄瓜茎节为外植体时,从共培养开始到转化苗生根平均需42d；以EP-6黄瓜的子叶作为外植体时,从共培养开始到转化苗生根平均需59d。若以RT-PCR阳性作为判断最终转化成功的标准,津春4号茎节转化体系的转化率为0.83%,EP-6子叶转化体系的转化率为0.36%
7将转基因组培苗于蔗糖浓度为6%的1/2MS培养基中生根4周,随后移栽至蔗糖为3%的培养基中培养1周,在组培苗6叶1心时,移栽于草碳：珍珠岩：河沙=1：1：1的基质中驯化,最有利于组培苗根系的发育,驯化成活率可达98.7%。
Cucumber (Cucumis sativus L.) is grown worldwide as an important vegetable. On the one hand, establishing an efficient cucumber transformation system is important for functional genomics research through techniques such as gene over-expression, reporter gene expression and RNA interference. On the other hand, Improving the biotic or abiotic resistance of cucumber through conventional breeding is limited by its narrow genetic basis and incompatibility barriers to wild Cucumis species. Therefore, it would be desirable to apply genetic engineering to expand the germplasm base of this crop. In this study, a European genotype EP-6and a Northern China genotype Jinchun4were selected to study the callus fomation, transformation, heterogenous gene transient expression and plant regeneration technology of cucumber.
1Two kinds of cucumber a-galactosidase-EGFP fusion expression vector controlled by the CaMV35S promoter were constructed. CaMV35S promoter sequence was amplified by polymerase chain reactions (PCR) from vector pCambia1303; The entire coding region of EGFP was amplified by PCR from vector pEGFP-Nl; The entire coding region of cucumber acid a-galactosidase I and alkaline a-galactosidase I were amplified by Reverse transcript PCR using cucumber leaf total RNAs as template. The three above-mentioned fragments were Inserted into the mμltiple cloning sites of expression vector pCambia1381*c. Then two a-galactosidase-EGFP fusion expression vectors were constructed respectively and the EGFP gene was located at the C-terminal of the target genes.
2To obtain the suitalbe material for transient expression of heterogenous genes, friable calli can be produced on the medium with6-BA1.0mg/L,NAA0.1mg/L,NH4NO32.0g/L and Yeast extrat5g/L. Two recombinant plasmids were transformed into onion epidermal cells and cucumber callus cells by particle bombardment. Green fluorescence signals were monitored by a confocal microscope. The results showed that cucumber acid a-galactosidase I is located in the whole cells includeing the vacuole, while cucumber alkaline a-galactosidase I is located at the nucleus and the peripheral region of the cell.
3Regeneration of cucumber plants:Cotyledon is a suitable explant for in vitro shoot induction of EP-6, while for Jinchun4, highest shoot induction rate was obtained if stem node was used as explant. The optimal culture medium of shoot induction of EP-6cotyledons was MS+0.5mg/L ABA+2.0mg/L6-BA+2.0mg/L AgNO3, while the best culture medium for Jinchun4shoot induction was MS+0.5mg/L6-BA. The highest root induction rate can be observed when the regenerated shoots were cultured on the medium1/2MS+15g/L sucrose+0.6mg/L IBA.
4The transfomation system of EP-6cotyledons:After2days of pre-culture on the optimized shoot induction medium, the cotyledons were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about25d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained60mg/L Kan and400mg/L Cef.
5The transfomation system of Jinchun4stem nodes:After2days of pre-culture on the optimized shoot induction medium, the stem nodes were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about12d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained80mg/L Kan and400mg/L Cef.
6From explant co-culture to well rooted putative transgenic plantlets, the transfomation systems of Jinchun4stem nodes and EP-6cotyledons needed42d and59d respectively. The transformation efficiency can be evaluated as0.83%for the Jinchun4stem node system and0.36%for the EP-6cotyledon system by RT-PCR positive plantlets.
7The highest survival rate of98.7%of transgenic plants can be obtained when the plantlets were rooted in the1/2MS with6%sucrose for4weeks, then transferred to the1/2MS with3%sucrose for1week, and finally transplanted to the substrate peat:perlite:sand=1:1:1.
引文
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