中国水仙花色相关基因的分离和反义Psy基因的遗传转化
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摘要
中国水仙(Narcissus tazetta L.var. chinensis Roem)隶属石蒜科水仙属,多年生草本植物,是多花水仙的一个变种。中国水仙主要产地在福建漳州,上海崇明,浙江舟山,其中以漳州水仙最为有名。目前,种球退化、繁殖速度慢、花色单一已成为制约中国水仙发展的主要障碍,除依靠高效栽培管理措施和脱毒种苗的快繁体系外,非常重要的就是进行品种改良。
本研究以中国水仙品种“金盏银台”为材料,从中国水仙花瓣中分离得到控制水仙花色的相关基因,构建了八氢番茄红素合成酶(Psy)基因的反义表达载体,在建立中国水仙遗传转化体系的基础上,对中国水仙进行了遗传转化。获得了抗性芽,并进行了初步的分子验证。为创建中国水仙新种质提供技术基础,为水仙品种改良提供新的途径,也为阐明中国水仙花色调控的分子机理提供了研究基础。主要研究结果如下:
1.提取中国水仙花瓣的RNA,反转录为cDNA二链,并以此为模板克隆类胡萝卜素合成途径中的关键基因Psy、LycB(番茄红素环化酶)和Pds(八氢番茄红素脱饱和酶)。Psy基因全长1272bp,编码423个氨基酸,LycB基因全长1515bp,编码504个氨基酸,Pds基因全长1713bp,编码570个氨基酸。
2.在已经得到的Psy基因全长的基础上,分离554bp的Psy基因的片段,BamH Ⅰ和Xba Ⅰ双酶切后,连接到同样用BamH Ⅰ和Xba Ⅰ双酶切的pCAMBIA1301双元载体上,构建CaMV35S组成型启动子反义表达载体pCAM35S-PSY。
3.建立了中国水仙的遗传转化体系。中国水仙鳞茎球双鳞片经消毒处理后在MS+10mg·L~(-1)BAP+0.5mg·L~(-1)NAA+3%蔗糖+0.7%琼脂糖预培养7d,在添加200mg·L~(-1)头孢霉素的基础上筛选潮霉素抗性浓度,30mg·L~(-1)的潮霉素即可使未经转化的外植体完全不分化;侵染时间为15-20min,侵染后的外植体在加入AS的共培养基中黑暗培养3d时瞬时表达效率最高,达92.9%。将共培养后的材料转移到筛选培养基(MS+5mg·L~(-1)BAP+0.1mg·L~(-1)NAA+3%蔗糖+0.7%琼脂糖+200mg·L~(-1)Cef+30mg·L~(-1)Hyg)上进行筛选。20d左右转接一次,直至生长出抗性芽,当抗性幼苗长至2-3cm时,从愈伤组织处切下幼苗,在继代培养基上培养到生长成健壮的小苗。
4.在已经建立的中国水仙遗传转化体系的基础上,将Psy基因的反义表达载体pCAM35S-PSY转化入预培养7d的中国水仙外植体,经过共培养和分化培养,得到抗性芽,并对抗性芽进行了初步的分子检测,证明得到了转基因植株。
Narcissus tazetta L. var. chinensis Roem is one of the ten Chinese traditional flowers is a kind of perennial plant with beautiful color and sweet fragrance. And it is a favorite flower of common Chinese and a famed traditional export flower for a long time. Chinese narcissus has widely spread in China, including Zhangzhou, Putuo and Chongming, especially the Zhangzhou narcissus is the most fomous. But for the slowly multiplication severe degeneration of varieties and simplex color, the production of Chinese narcissus can not satisfy the demand of global market and its development has been seriously restricted. So it's imperative to develop new varieties with modern breeding methods for the narcissus industry.In order to modify the Narcissus flower color by genetic engineering, the expressinal plasmid of objective genes and regenerate system from callus after gene transformation are two key steps. In present study, we cloned flower color genes from the petal of Chinese narcissus, and constructed plant expressing plasmid vector(pCAM35S-PSY), after the establishment of an efficient gene transformation system, The anti-PSY(phytoene synthase) gene was introduced into Agrobacterium tumefaciens strain LBA4404 and then transferred to the callus tissue of Chinese narcissus.Some hygromycin-resistant shoots were identified with molecular analysis. The main results are as follow:(1) The RNA extracted from Narcissus flower petal was used for the isolation of the Psy, LycB and Pds genes by RT-PCR. The results of analysis of the coding sequences of the genes demonstrated that the gene of Psy, 1272 bp, encoded a protein of 423 amino acid;ZycB(Lycopene (3-cyclase), 1515bp, encoded a protein of 504 amino acid, and Pds(Phytoene desaturase), 1713 bp, encodes a protein of 570 amino acid(2) In order to expression the Psy in plant, A plant expression vecter was constructed with a binary vector pCAMBIA1301. The antisense gene fragment was amplified from the coding region of Psy gene having the sequence of 554 bp, degisted by BamH I and Xba I, then introduced to binary expressional vecter pCAMBIA1301 under the constitutive CaMV 35S promotor, and the resulting construction with antisense Psy was name d pCAM35S-PSY. Results from PCR amplification and restriction digestion showed that the fragment of antisense gene Psy was correctly introduced into pCAMBIA1301 plasmid and then the expression vector pCAM35S-PSY was successfully transferred into Agrobacterium.(3) Effect of hygromycin was studied on the regeneration of Narcissus Tazetta bulb-scales in vitro culture. An efficient gene transformation system is established by co-culturing the bulds pretreated for 7 days with Agrobacterium tumefaciens introducing the plasmid vector pCAMBIA1301. Explants from plantlets are the most sensitive to hygromucin, and 30 mg·L~(-1) hygromycin would inhibit the regeneration. The effect of acetosyringone(AS) on transformation of Chinese narcissus by Agrobacterium tumefaciens was tested in this papers. The results showed that added 100 mg·L~(-1) AS, then co-cultured 3
days, the instant expression activity of gus gene reached 92.9%.(4) An efficient gene transformation system was established, and the optimum conditions for the transformation of antisense gene Psy fragment were as follow: After preculture of 7 days with the bulb-scales as the primary explants, the explants was infected with Agrobacterium tumefaciens strain LBA4404 contained with pCAM35S-PSY lasting 15-20 min. The coculture time was 3 days. Then the cocultured explants were inoculated into the regeneration MS medium containing 5 mg-L"1 BAP, 0.1 mg-L'1 NAA, 200 mg-L"1 Cef, 30 mg-L'1 Hyg, 3% sucrose and 0.7% agar to obtain the HygR shoot, and the shoots were transfer to fresh medium for growth and exchanged per 3 weeks. The results with PCR analysis showed that some tested lines carried the transgenes. the antisense gene fragment was susucesfully transferred into Narcissus tazetta L. var. chinensis Roem. The in-depth biochemical and molecular biological analysis was undertaken. The study on flower color and stability of this traits will be carried out in the field test in future.
本研究以中国水仙品种“金盏银台”为材料,从中国水仙花瓣中分离得到控制水仙花色的相关基因,构建了八氢番茄红素合成酶(Psy)基因的反义表达载体,在建立中国水仙遗传转化体系的基础上,对中国水仙进行了遗传转化。获得了抗性芽,并进行了初步的分子验证。为创建中国水仙新种质提供技术基础,为水仙品种改良提供新的途径,也为阐明中国水仙花色调控的分子机理提供了研究基础。主要研究结果如下:
1.提取中国水仙花瓣的RNA,反转录为cDNA二链,并以此为模板克隆类胡萝卜素合成途径中的关键基因Psy、LycB(番茄红素环化酶)和Pds(八氢番茄红素脱饱和酶)。Psy基因全长1272bp,编码423个氨基酸,LycB基因全长1515bp,编码504个氨基酸,Pds基因全长1713bp,编码570个氨基酸。
2.在已经得到的Psy基因全长的基础上,分离554bp的Psy基因的片段,BamH Ⅰ和Xba Ⅰ双酶切后,连接到同样用BamH Ⅰ和Xba Ⅰ双酶切的pCAMBIA1301双元载体上,构建CaMV35S组成型启动子反义表达载体pCAM35S-PSY。
3.建立了中国水仙的遗传转化体系。中国水仙鳞茎球双鳞片经消毒处理后在MS+10mg·L~(-1)BAP+0.5mg·L~(-1)NAA+3%蔗糖+0.7%琼脂糖预培养7d,在添加200mg·L~(-1)头孢霉素的基础上筛选潮霉素抗性浓度,30mg·L~(-1)的潮霉素即可使未经转化的外植体完全不分化;侵染时间为15-20min,侵染后的外植体在加入AS的共培养基中黑暗培养3d时瞬时表达效率最高,达92.9%。将共培养后的材料转移到筛选培养基(MS+5mg·L~(-1)BAP+0.1mg·L~(-1)NAA+3%蔗糖+0.7%琼脂糖+200mg·L~(-1)Cef+30mg·L~(-1)Hyg)上进行筛选。20d左右转接一次,直至生长出抗性芽,当抗性幼苗长至2-3cm时,从愈伤组织处切下幼苗,在继代培养基上培养到生长成健壮的小苗。
4.在已经建立的中国水仙遗传转化体系的基础上,将Psy基因的反义表达载体pCAM35S-PSY转化入预培养7d的中国水仙外植体,经过共培养和分化培养,得到抗性芽,并对抗性芽进行了初步的分子检测,证明得到了转基因植株。
Narcissus tazetta L. var. chinensis Roem is one of the ten Chinese traditional flowers is a kind of perennial plant with beautiful color and sweet fragrance. And it is a favorite flower of common Chinese and a famed traditional export flower for a long time. Chinese narcissus has widely spread in China, including Zhangzhou, Putuo and Chongming, especially the Zhangzhou narcissus is the most fomous. But for the slowly multiplication severe degeneration of varieties and simplex color, the production of Chinese narcissus can not satisfy the demand of global market and its development has been seriously restricted. So it's imperative to develop new varieties with modern breeding methods for the narcissus industry.In order to modify the Narcissus flower color by genetic engineering, the expressinal plasmid of objective genes and regenerate system from callus after gene transformation are two key steps. In present study, we cloned flower color genes from the petal of Chinese narcissus, and constructed plant expressing plasmid vector(pCAM35S-PSY), after the establishment of an efficient gene transformation system, The anti-PSY(phytoene synthase) gene was introduced into Agrobacterium tumefaciens strain LBA4404 and then transferred to the callus tissue of Chinese narcissus.Some hygromycin-resistant shoots were identified with molecular analysis. The main results are as follow:(1) The RNA extracted from Narcissus flower petal was used for the isolation of the Psy, LycB and Pds genes by RT-PCR. The results of analysis of the coding sequences of the genes demonstrated that the gene of Psy, 1272 bp, encoded a protein of 423 amino acid;ZycB(Lycopene (3-cyclase), 1515bp, encoded a protein of 504 amino acid, and Pds(Phytoene desaturase), 1713 bp, encodes a protein of 570 amino acid(2) In order to expression the Psy in plant, A plant expression vecter was constructed with a binary vector pCAMBIA1301. The antisense gene fragment was amplified from the coding region of Psy gene having the sequence of 554 bp, degisted by BamH I and Xba I, then introduced to binary expressional vecter pCAMBIA1301 under the constitutive CaMV 35S promotor, and the resulting construction with antisense Psy was name d pCAM35S-PSY. Results from PCR amplification and restriction digestion showed that the fragment of antisense gene Psy was correctly introduced into pCAMBIA1301 plasmid and then the expression vector pCAM35S-PSY was successfully transferred into Agrobacterium.(3) Effect of hygromycin was studied on the regeneration of Narcissus Tazetta bulb-scales in vitro culture. An efficient gene transformation system is established by co-culturing the bulds pretreated for 7 days with Agrobacterium tumefaciens introducing the plasmid vector pCAMBIA1301. Explants from plantlets are the most sensitive to hygromucin, and 30 mg·L~(-1) hygromycin would inhibit the regeneration. The effect of acetosyringone(AS) on transformation of Chinese narcissus by Agrobacterium tumefaciens was tested in this papers. The results showed that added 100 mg·L~(-1) AS, then co-cultured 3
days, the instant expression activity of gus gene reached 92.9%.(4) An efficient gene transformation system was established, and the optimum conditions for the transformation of antisense gene Psy fragment were as follow: After preculture of 7 days with the bulb-scales as the primary explants, the explants was infected with Agrobacterium tumefaciens strain LBA4404 contained with pCAM35S-PSY lasting 15-20 min. The coculture time was 3 days. Then the cocultured explants were inoculated into the regeneration MS medium containing 5 mg-L"1 BAP, 0.1 mg-L'1 NAA, 200 mg-L"1 Cef, 30 mg-L'1 Hyg, 3% sucrose and 0.7% agar to obtain the HygR shoot, and the shoots were transfer to fresh medium for growth and exchanged per 3 weeks. The results with PCR analysis showed that some tested lines carried the transgenes. the antisense gene fragment was susucesfully transferred into Narcissus tazetta L. var. chinensis Roem. The in-depth biochemical and molecular biological analysis was undertaken. The study on flower color and stability of this traits will be carried out in the field test in future.
引文
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