中国野生葡萄乙二醛氧化酶基因转化葡萄的研究
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摘要
乙二醛氧化酶是参与植物抗病反应的一类酶,主要参与植物系统抗性的建立、促进植保素的合成。生产上栽培最为广泛的欧洲葡萄几乎均不抗病,而中国野生葡萄有着丰富的抗病基因资源。本研究旨在利用基因工程技术,将携带有中国野生葡萄乙二醛氧化酶基因的植物表达载体pWR306转入感病葡萄欧亚种“佳利酿”、华东葡萄“广西-2”的基因组中,为获得抗病性明显提高的转基因植株提供理论与技术依据。
在以“佳利酿”、“广西-2”的花药为外植体进行体细胞胚诱导与成苗研究的基础上,初步建立了葡萄胚状体再生体系;并对次生胚的诱导、畸形苗的转化进行了初步研究。通过农杆菌介导法,将携带有中国野生葡萄乙二醛氧化酶基因的植物表达载体pWR306转入“广西-2”胚状体及胚性愈伤、合子胚胚性愈伤、“佳利酿”与“广西-2”的带芽茎段。取得的主要结果如下:
1.以“佳利酿”、“广西-2”带芽茎段为材料,研究了不同培养基对外植体生长的影响,获得了适宜“佳利酿”、“广西-2”单芽茎段生长成苗的初代培养基WPM+IBA 0.15mg/L +琼脂6 g/L + AC 0.5 g/L;继代培养基1/2MS + IBA 0.15 mg/L +活性炭(AC)0.5 g/L +蔗糖30 g/L +琼脂6 g/L。
2.培养基ER + 6-BA 1.0 mg/L + 2,4-D 1.0 mg/L + CH 1g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L有利于“佳利酿”花药胚性愈伤组织诱导;所使用的培养基中,B5 + 6-BA 4.0 mg/L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L对“广西-2”花药的胚性愈伤组织诱导较为有利。
3.“广西-2”的胚性愈伤为在B5 + 6-BA 4.0 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L的培养基上能形成胚状体;适宜胚状体萌发的培养基是不含生长调节剂的1/2B5 +蔗糖15 g/L液体培养基;而在液体的B5 + 6-BA 4.0 mg /L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L培养基中,胚状体能有效增殖;在MS + 6-BA 1.5 mg/L + IBA 0.2mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L +琼脂6 g/L的培养基上畸形植株能被诱导形成正常苗;胚状体萌发成苗的适宜的培养基是WPM + IBA 0.15 mg/L + AC 0.5 g/L +蔗糖15 g/L +琼脂6 g/L;在对“广西-2”次生体胚诱导时,B5 + 6-BA 1.0 mg/L + 2,4-D 0.5 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L的培养基较为有效。
4.以“广西-2”单芽茎段和胚状体为试材,潮霉素(Hyg)抗性浓度筛选结果表明,葡萄单芽茎段对Hyg致死浓度为12 mg/L,胚状体对Hyg致死浓度为9 mg/L。
5.通过农杆菌介导法转化,已获得具有潮霉素抗性的“广西-2”胚性细胞团约23块,“佳利酿”抗性植株2个。
Glyoxal Oxidase is one of the enzyme which take part in disease resistance response of plants.It can accelerate the lignin degradation and phytoalexin synthesis. Most of the Vitis vinifera are susceptible to diseases on production, Chinese wild Vitis have abundant resources with resistance gene. This paper is aimed to using genetic engineering techniques, Transformed the novel Glyoxal Oxidase gene which was carried in the pWR306 binary vector was introduced into genomic of susceptible grape“Carignane”and“Guangxi-2”. Results of this study for shorten the breeding cycle, provide a theoretical foundation and technology basis for getting the plant resistance improved obviously.
Based upon this study was to research in regeneration systems after anther culture of“Carignane”and“Guangxi-2”were established by somatic embryogenesis, respectively.And make the basis research on secondary somatic embryos induced,abnormal plants culturaled. The Glyoxal Oxidase gene was introduced into somatic embryo and stem-segment with single bud of“Carignane”and“Guangxi-2”, zygotic embryo callus by Agrobacterium-mediated.The results of the researchs are as follow:
1.The single-bud of grape shoots were investigated of effection of different medium on the growth of“Carignane”and“Guangxi-2”.Selected results indicated that the fitting medium of WPM + IBA 0.15 mg/L + AC 0.5 g/L + Sucrose 30 g/L + Agar 6 g/L.
2.The result of this search indicate that 1/2B5 + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L liquid media is properiated for the somatic embryos formation of“Guangxi-2”; The suitable for proliferation of somatic embryo medium is B5 + 6-BA 4.0 mg/L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L + Agar 6 g/L; And the suitable for germination of somatic embryo medium is liquid medium B5 + 6-BA 4.0 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L;Which media MS + 6-BA 1.5 mg/L + IBA 0.2 mg/L + Sucrose 30 g/L + Agar 6 g/L is beneficial for the abnormal plant conversion into normal plant;The best medium is WPM + IBA 0.15 mg/L + AC 0.5 g/L + Sucrose 15 g/L + Agar 6 g/L for somatic embryogenesis and plantlet formation.
3.The suitable medium for secondary somatic embryos formation is B5 + 6-BA 4.0 mg/L + 2,4-D 0.5 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 30 g/L + Agar 6 g/L.
4.Concentration of hygromycin for selection were studied. The number of 23 somatic embryo callus from“Guangxi-2”and two resistant-hygromycin plants from single-bud grape shoots of“Carignane”were obtained.
在以“佳利酿”、“广西-2”的花药为外植体进行体细胞胚诱导与成苗研究的基础上,初步建立了葡萄胚状体再生体系;并对次生胚的诱导、畸形苗的转化进行了初步研究。通过农杆菌介导法,将携带有中国野生葡萄乙二醛氧化酶基因的植物表达载体pWR306转入“广西-2”胚状体及胚性愈伤、合子胚胚性愈伤、“佳利酿”与“广西-2”的带芽茎段。取得的主要结果如下:
1.以“佳利酿”、“广西-2”带芽茎段为材料,研究了不同培养基对外植体生长的影响,获得了适宜“佳利酿”、“广西-2”单芽茎段生长成苗的初代培养基WPM+IBA 0.15mg/L +琼脂6 g/L + AC 0.5 g/L;继代培养基1/2MS + IBA 0.15 mg/L +活性炭(AC)0.5 g/L +蔗糖30 g/L +琼脂6 g/L。
2.培养基ER + 6-BA 1.0 mg/L + 2,4-D 1.0 mg/L + CH 1g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L有利于“佳利酿”花药胚性愈伤组织诱导;所使用的培养基中,B5 + 6-BA 4.0 mg/L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L对“广西-2”花药的胚性愈伤组织诱导较为有利。
3.“广西-2”的胚性愈伤为在B5 + 6-BA 4.0 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L的培养基上能形成胚状体;适宜胚状体萌发的培养基是不含生长调节剂的1/2B5 +蔗糖15 g/L液体培养基;而在液体的B5 + 6-BA 4.0 mg /L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L培养基中,胚状体能有效增殖;在MS + 6-BA 1.5 mg/L + IBA 0.2mg/L + CH 1 g/L + PVP 1 g/L +蔗糖15 g/L +琼脂6 g/L的培养基上畸形植株能被诱导形成正常苗;胚状体萌发成苗的适宜的培养基是WPM + IBA 0.15 mg/L + AC 0.5 g/L +蔗糖15 g/L +琼脂6 g/L;在对“广西-2”次生体胚诱导时,B5 + 6-BA 1.0 mg/L + 2,4-D 0.5 mg/L + CH 1 g/L + PVP 1 g/L +蔗糖30 g/L +琼脂6 g/L的培养基较为有效。
4.以“广西-2”单芽茎段和胚状体为试材,潮霉素(Hyg)抗性浓度筛选结果表明,葡萄单芽茎段对Hyg致死浓度为12 mg/L,胚状体对Hyg致死浓度为9 mg/L。
5.通过农杆菌介导法转化,已获得具有潮霉素抗性的“广西-2”胚性细胞团约23块,“佳利酿”抗性植株2个。
Glyoxal Oxidase is one of the enzyme which take part in disease resistance response of plants.It can accelerate the lignin degradation and phytoalexin synthesis. Most of the Vitis vinifera are susceptible to diseases on production, Chinese wild Vitis have abundant resources with resistance gene. This paper is aimed to using genetic engineering techniques, Transformed the novel Glyoxal Oxidase gene which was carried in the pWR306 binary vector was introduced into genomic of susceptible grape“Carignane”and“Guangxi-2”. Results of this study for shorten the breeding cycle, provide a theoretical foundation and technology basis for getting the plant resistance improved obviously.
Based upon this study was to research in regeneration systems after anther culture of“Carignane”and“Guangxi-2”were established by somatic embryogenesis, respectively.And make the basis research on secondary somatic embryos induced,abnormal plants culturaled. The Glyoxal Oxidase gene was introduced into somatic embryo and stem-segment with single bud of“Carignane”and“Guangxi-2”, zygotic embryo callus by Agrobacterium-mediated.The results of the researchs are as follow:
1.The single-bud of grape shoots were investigated of effection of different medium on the growth of“Carignane”and“Guangxi-2”.Selected results indicated that the fitting medium of WPM + IBA 0.15 mg/L + AC 0.5 g/L + Sucrose 30 g/L + Agar 6 g/L.
2.The result of this search indicate that 1/2B5 + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L liquid media is properiated for the somatic embryos formation of“Guangxi-2”; The suitable for proliferation of somatic embryo medium is B5 + 6-BA 4.0 mg/L + 2,4-D 0.7 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L + Agar 6 g/L; And the suitable for germination of somatic embryo medium is liquid medium B5 + 6-BA 4.0 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 15 g/L;Which media MS + 6-BA 1.5 mg/L + IBA 0.2 mg/L + Sucrose 30 g/L + Agar 6 g/L is beneficial for the abnormal plant conversion into normal plant;The best medium is WPM + IBA 0.15 mg/L + AC 0.5 g/L + Sucrose 15 g/L + Agar 6 g/L for somatic embryogenesis and plantlet formation.
3.The suitable medium for secondary somatic embryos formation is B5 + 6-BA 4.0 mg/L + 2,4-D 0.5 mg/L + CH 1 g/L + PVP 1 g/L + Sucrose 30 g/L + Agar 6 g/L.
4.Concentration of hygromycin for selection were studied. The number of 23 somatic embryo callus from“Guangxi-2”and two resistant-hygromycin plants from single-bud grape shoots of“Carignane”were obtained.
引文
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