梅PGIP基因转化盆栽小菊的研究
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摘要
菊花(Dendranthema×grandiflorum Kitamu)为菊科菊属植物,是我国十大传统名花和世界四大切花之一,具有观赏、食用、药用等多种价值。菊花的黑斑病在我国各地普遍发生,对其商品性和观赏性影响很大。菊花黑斑病除危害菊花外,还危害野菊、除虫菊、甘菊等多种菊科植物,极大地限制了菊花的生产发展,因此,提高菊花抗病性是菊花育种的目标之一。
目前防治病害主要还是依赖于栽培措施、人工摘除和化学防治措施,只能在一定程度上控制病害的流行而不能真正解决问题。本研究在建立菊花高频的再生体系和遗传转化体系的基础上,通过农杆菌介导法,将梅PGIP基因转入菊花中。通过基因工程的手段定向改良品种的抗病性,获得具有一定抗性的转基因株系。主要研究内容如下：
1选择花色、株型等各种观赏性状都比较好,但是田间观察黑斑病发病非常严重的三个盆栽小菊品种‘05-44-2’,‘04-1-6’,‘国庆意大利红’进行愈伤组织的诱导和植株再生,通过调整培养基、植物生长调剂浓度及配比、不同的外植体、不同的再生方式等,结果表明,品种‘05-44-2’在MS+6-BA2.0mg·L-1+NAA0.2mg·L-1培养基上诱导叶片再生率最高为85%,最适增殖培养基为0.1mg·L-1最适生根培养基为MS。
2以再生力能力高的盆栽小菊‘05-44-2’为材料,通过对影响转化率的因素：预培养时间、农杆菌OD600值、侵染时间、共培养时间、延迟培养时间等进行了研究探讨,建立了菊花‘05-44-2’的高频遗传转化体系。在菊花转化体系建立中,以Carb 500mg·L-1和Hyg 10mg·L-1作为适宜的抑菌素浓度和选择压。结果显示适宜的预培养时间是2d,侵染时工程农杆菌的浓度以OD600值为0.5,侵染10min转化效果最好。25℃共培养3d,延迟培养5d,有利于菊花遗传转化。
3采用农杆菌介导法将PGIP基因转入盆栽小菊‘05-44-2’中,得232株潮霉素抗性苗。对其中40株进行PCR检测,有8株扩增出目的条带；对其中8株进行RT-PCR检测,发现5个株系均有目的条带出现,初步确定目的基因已经整合到菊花基因组中。
4从菊花病叶上分离、纯化菊花黑斑病的病原菌,并对其致病性和菌落形态和分生孢子的观察,鉴定为细极链格孢(Alternaria tenuissima)。对不同株系的转基因菊花进行抗病性鉴定,分为苗期接种和田间自然发病情况的调查。研究表明,转基因菊花不同株系对黑斑病的抗病存在差异,且重复性好,鉴定结果能明显的反应了转基因菊花抗病性的提高程度。与对照相比,转基因株系对黑斑病有不同程度的抗性,使发病延迟,病情指数降低。田间抗病性调查结果表明,发病情况比苗期接种严重。株系7抗性最强,苗期病情指数最小为33。
Dendranthema grandiflora Kitamu is one of ten traditional famous flowers in China and four cuffed-flowers in the world, and it can be served as ornamental plants, edible vegetables and medicinal materials. The fungi disease of chrysanthemum harms very seriously. Disease control mainly relies on the cultivation measures, artificial pick and chemical substances. These measures can control disease to some extent but not really solve problem. We transformed chrysanthemum with PGIP gene from Prunus mume by agrobacterium-mediated in establishing that highly efficient regeneration system and transformation system. We improve cultivar resistance in definite direction by genetic engineering methods to obtain transformation resistance plants. Main study contents is as follows:
1 Three potted chrysanthemum cultivars'05-44-2','04-1-6','guo qing yi da li hong' induced callus and regenerated by adjusting the factors such as culture medium, concentration of growth hormone, different explants and different regeneration methods and so on. The results showed that the cultivar'05-44-2'induced leaf regeneration efficiency 85% on the MS+6-BA2.0 mg·L-1+NAA0.2 mg·L-1 culture medium, the most suitable proliferation medium was MS+6-BA1.0 mg·L-1+NAA0.1 mg·L-1, the most suitable rooting medium was MS.
2 High efficient and transformation system of potted chrysanthemum'05-44-6'was obtained. By studying the effec t of transformation frequency factors such as preculture duration Agrobacterium concentration (OD600)、infection time、co-cultivated times、prolonged culture time and so on. The regeneration shoots were cultured in selection medium containing Carb 500mg·L-1 and Hyg 10mg·L-1. The results show that preculture duration 2 day, OD600 0.5, infection time10 min, co-cultivated times 3 day at 25℃, prolonged culture time 5 day are suitable for transformation.
3 we transformed potted chrysanthemum'05-44-6'with PGIP gene by agrobacterium-mediated. Through PCR、RT-PCR test, the exogenous gene were introduced into the regeneration plants genome.
4 Lines of pathogens was isolated and purified from pathologic leafs of chrysanthemum. We observed pathogens pathogenicity, colony morphology and conidiophore and identified as Alternaria tenuissima. By seeding inoculation and field disease investigations identified resistance of different transgenic plants. The results show that it had diversity on different transgenic plants and good repetition. Results reflect resistance degree of transgenic plants. Compare with CK, transgenic plants have different degree resistance, postpone disease and reduce disease index. Field diseases harm seriously in comparing with seeding inoculation. No.7 of transgenic plants has the highest resistance and disease index minimum at seeding stage is 33.
目前防治病害主要还是依赖于栽培措施、人工摘除和化学防治措施,只能在一定程度上控制病害的流行而不能真正解决问题。本研究在建立菊花高频的再生体系和遗传转化体系的基础上,通过农杆菌介导法,将梅PGIP基因转入菊花中。通过基因工程的手段定向改良品种的抗病性,获得具有一定抗性的转基因株系。主要研究内容如下：
1选择花色、株型等各种观赏性状都比较好,但是田间观察黑斑病发病非常严重的三个盆栽小菊品种‘05-44-2’,‘04-1-6’,‘国庆意大利红’进行愈伤组织的诱导和植株再生,通过调整培养基、植物生长调剂浓度及配比、不同的外植体、不同的再生方式等,结果表明,品种‘05-44-2’在MS+6-BA2.0mg·L-1+NAA0.2mg·L-1培养基上诱导叶片再生率最高为85%,最适增殖培养基为0.1mg·L-1最适生根培养基为MS。
2以再生力能力高的盆栽小菊‘05-44-2’为材料,通过对影响转化率的因素：预培养时间、农杆菌OD600值、侵染时间、共培养时间、延迟培养时间等进行了研究探讨,建立了菊花‘05-44-2’的高频遗传转化体系。在菊花转化体系建立中,以Carb 500mg·L-1和Hyg 10mg·L-1作为适宜的抑菌素浓度和选择压。结果显示适宜的预培养时间是2d,侵染时工程农杆菌的浓度以OD600值为0.5,侵染10min转化效果最好。25℃共培养3d,延迟培养5d,有利于菊花遗传转化。
3采用农杆菌介导法将PGIP基因转入盆栽小菊‘05-44-2’中,得232株潮霉素抗性苗。对其中40株进行PCR检测,有8株扩增出目的条带；对其中8株进行RT-PCR检测,发现5个株系均有目的条带出现,初步确定目的基因已经整合到菊花基因组中。
4从菊花病叶上分离、纯化菊花黑斑病的病原菌,并对其致病性和菌落形态和分生孢子的观察,鉴定为细极链格孢(Alternaria tenuissima)。对不同株系的转基因菊花进行抗病性鉴定,分为苗期接种和田间自然发病情况的调查。研究表明,转基因菊花不同株系对黑斑病的抗病存在差异,且重复性好,鉴定结果能明显的反应了转基因菊花抗病性的提高程度。与对照相比,转基因株系对黑斑病有不同程度的抗性,使发病延迟,病情指数降低。田间抗病性调查结果表明,发病情况比苗期接种严重。株系7抗性最强,苗期病情指数最小为33。
Dendranthema grandiflora Kitamu is one of ten traditional famous flowers in China and four cuffed-flowers in the world, and it can be served as ornamental plants, edible vegetables and medicinal materials. The fungi disease of chrysanthemum harms very seriously. Disease control mainly relies on the cultivation measures, artificial pick and chemical substances. These measures can control disease to some extent but not really solve problem. We transformed chrysanthemum with PGIP gene from Prunus mume by agrobacterium-mediated in establishing that highly efficient regeneration system and transformation system. We improve cultivar resistance in definite direction by genetic engineering methods to obtain transformation resistance plants. Main study contents is as follows:
1 Three potted chrysanthemum cultivars'05-44-2','04-1-6','guo qing yi da li hong' induced callus and regenerated by adjusting the factors such as culture medium, concentration of growth hormone, different explants and different regeneration methods and so on. The results showed that the cultivar'05-44-2'induced leaf regeneration efficiency 85% on the MS+6-BA2.0 mg·L-1+NAA0.2 mg·L-1 culture medium, the most suitable proliferation medium was MS+6-BA1.0 mg·L-1+NAA0.1 mg·L-1, the most suitable rooting medium was MS.
2 High efficient and transformation system of potted chrysanthemum'05-44-6'was obtained. By studying the effec t of transformation frequency factors such as preculture duration Agrobacterium concentration (OD600)、infection time、co-cultivated times、prolonged culture time and so on. The regeneration shoots were cultured in selection medium containing Carb 500mg·L-1 and Hyg 10mg·L-1. The results show that preculture duration 2 day, OD600 0.5, infection time10 min, co-cultivated times 3 day at 25℃, prolonged culture time 5 day are suitable for transformation.
3 we transformed potted chrysanthemum'05-44-6'with PGIP gene by agrobacterium-mediated. Through PCR、RT-PCR test, the exogenous gene were introduced into the regeneration plants genome.
4 Lines of pathogens was isolated and purified from pathologic leafs of chrysanthemum. We observed pathogens pathogenicity, colony morphology and conidiophore and identified as Alternaria tenuissima. By seeding inoculation and field disease investigations identified resistance of different transgenic plants. The results show that it had diversity on different transgenic plants and good repetition. Results reflect resistance degree of transgenic plants. Compare with CK, transgenic plants have different degree resistance, postpone disease and reduce disease index. Field diseases harm seriously in comparing with seeding inoculation. No.7 of transgenic plants has the highest resistance and disease index minimum at seeding stage is 33.
引文
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