rolC基因转化‘美人指’葡萄的研究
摘要
本研究以‘美人指'葡萄(Vitis vinifera L‘Manicure Finger')为材料,建立培养方法简单的不定芽再生体系,并在此基础上采用农杆菌介导法和花粉管通道法,将rolC基因导入‘美人指'葡萄,以达到减缓‘美人指'葡萄的生长势、促进其花芽的形成的目的。研究结果如下:
1.以‘美人指'葡萄叶片、叶柄和茎段为外植体,用不同浓度6-BA、TDZ与IBA组合在不同的基本培养基上诱导再生植株。结果表明:在MS、B_5和NN_(69)3种基本培养基中,外植体均可直接再生不定芽,且再生率和出芽数差异不显著;6-BA与IBA组合中,MS+6-BA3.0 mg/L+IBA0.1 mg/L再生率最高,为6%;TDZ与IBA不同组合中,MS+TDZ 1.0 mg/L+IBA 0.2mg/L再生率最高,为4%。
2.以‘美人指'葡萄离体叶片、叶柄和茎段为农杆菌介导转化rolC基因的受体,对农杆菌侵染时间、共培养时间、卡那霉素选择压及头孢霉素浓度等因素进行了研究。结果表明:农杆菌侵染4min,共培养3d,卡那霉素选择压3mg/L,头孢霉素浓度250mg/L为最佳的农杆菌介导法‘美人指'葡萄遗传转化体系。
3.采用优化的农杆菌介导转化体系,对‘美人指'进行转化,共获得了12株抗性苗。利用GUS组织化学染色、PCR扩增及RT-PCR检测,结果表明,有2株抗性苗GUS染色呈阳性,其中有1株通过PCR和RT-PCR检测均扩增到540bp的条带,与rolC基因完全一致,证实rolC基因已经整合进入‘美人指'葡萄基因组中,并得到了表达。
4.以‘美人指'葡萄花序为农杆菌介导转化rolC基因的受体,利用花粉管通道法对‘美人指'葡萄进行转化,共获得了40株后代单株。利用GUS组织化学染色、PCR及RT-PCR方法进行检测,结果表明,5株GUS染色呈阳性,GUS阳性率为12.5%;对其中的22株抗性株系进行PCR和RT-PCR检测,证实rolC基因已经整合进入2个株系的基因组中,并得到了表达。
An effective system regeneration system of 'Manicure Finger' grapevinevine was developed in this study.Agrobacterium-mediated transformation and pollen-tube pathway were adopted to introduce the role gene into 'Manicure Finger' grapevine to improve the ability of abloom.The detailed results were Listed as follows:
1.Plant regeneration were induced from leaf,petiole and stem explants of 'Manicure Finger' grapevine with difference media supplemented with different combinations of 6-BA,TDZ and IBA through organogenesis.The results indicated that the basic media MS,B_5 and NN_(69) all induce adventitious buds from leaf,petiole and stem explants.The highest regeneration rate of adventitious buds was 6.00%,when explants were incubated on MS medium with 6-BA 3.0 mg/L and IBA 0.1 mg/L.The regeneration rate of adventitious buds was 4.00%when explants were incubated on MS+TDZ1.0mg/L+IBA0.2 mg/L.
2.Transgenic plants were generated from leaves,petiole and stem explants of 'Manicure Finger' grapevine by Agrobacterium-mediated transformation with rolC gene,and the following factors were investigated:Agrobacterium dipping time,co=culture time,concentration of Kanamycin,and concentration of Cefotaxine.The optimal conditions included that infected 4 min by Agrobacterium,co-cultured 3d,Km3.0mg/L,and Cef 250 mg/L.
3.Twelve Km-resistant shoots of 'Manicure Finger' grapevine were obtained by Agrobacterium-mediated transformation.Histochemical GUS assay on these resistant shoots showed that two shoots were GUS positive plants;one of them was confirmed that rolC gene was integrated into the host genome of 'Manicure Finger' grapevine by both PCR and RT-PCR analysis.
4.Agrobacterium-mediated transformation was adopted to introduce the rolC gene into the inflorescence of 'Manicure Finger' grapevine via the pollen-tube pathway.40 planflets were obtained from 'Manicure Finger' seeds.By GUS histochemistry assay、PCR amplifiation and RT-PCR analysis,five plants showed GUS positive with a 12.5%positive rate,and two shoots showed PCR and RT-PCR positive,which initially demonstrated that the rolC gene was integrated into the genome of 'Manicure Finger' grapevine.
1.以‘美人指'葡萄叶片、叶柄和茎段为外植体,用不同浓度6-BA、TDZ与IBA组合在不同的基本培养基上诱导再生植株。结果表明:在MS、B_5和NN_(69)3种基本培养基中,外植体均可直接再生不定芽,且再生率和出芽数差异不显著;6-BA与IBA组合中,MS+6-BA3.0 mg/L+IBA0.1 mg/L再生率最高,为6%;TDZ与IBA不同组合中,MS+TDZ 1.0 mg/L+IBA 0.2mg/L再生率最高,为4%。
2.以‘美人指'葡萄离体叶片、叶柄和茎段为农杆菌介导转化rolC基因的受体,对农杆菌侵染时间、共培养时间、卡那霉素选择压及头孢霉素浓度等因素进行了研究。结果表明:农杆菌侵染4min,共培养3d,卡那霉素选择压3mg/L,头孢霉素浓度250mg/L为最佳的农杆菌介导法‘美人指'葡萄遗传转化体系。
3.采用优化的农杆菌介导转化体系,对‘美人指'进行转化,共获得了12株抗性苗。利用GUS组织化学染色、PCR扩增及RT-PCR检测,结果表明,有2株抗性苗GUS染色呈阳性,其中有1株通过PCR和RT-PCR检测均扩增到540bp的条带,与rolC基因完全一致,证实rolC基因已经整合进入‘美人指'葡萄基因组中,并得到了表达。
4.以‘美人指'葡萄花序为农杆菌介导转化rolC基因的受体,利用花粉管通道法对‘美人指'葡萄进行转化,共获得了40株后代单株。利用GUS组织化学染色、PCR及RT-PCR方法进行检测,结果表明,5株GUS染色呈阳性,GUS阳性率为12.5%;对其中的22株抗性株系进行PCR和RT-PCR检测,证实rolC基因已经整合进入2个株系的基因组中,并得到了表达。
An effective system regeneration system of 'Manicure Finger' grapevinevine was developed in this study.Agrobacterium-mediated transformation and pollen-tube pathway were adopted to introduce the role gene into 'Manicure Finger' grapevine to improve the ability of abloom.The detailed results were Listed as follows:
1.Plant regeneration were induced from leaf,petiole and stem explants of 'Manicure Finger' grapevine with difference media supplemented with different combinations of 6-BA,TDZ and IBA through organogenesis.The results indicated that the basic media MS,B_5 and NN_(69) all induce adventitious buds from leaf,petiole and stem explants.The highest regeneration rate of adventitious buds was 6.00%,when explants were incubated on MS medium with 6-BA 3.0 mg/L and IBA 0.1 mg/L.The regeneration rate of adventitious buds was 4.00%when explants were incubated on MS+TDZ1.0mg/L+IBA0.2 mg/L.
2.Transgenic plants were generated from leaves,petiole and stem explants of 'Manicure Finger' grapevine by Agrobacterium-mediated transformation with rolC gene,and the following factors were investigated:Agrobacterium dipping time,co=culture time,concentration of Kanamycin,and concentration of Cefotaxine.The optimal conditions included that infected 4 min by Agrobacterium,co-cultured 3d,Km3.0mg/L,and Cef 250 mg/L.
3.Twelve Km-resistant shoots of 'Manicure Finger' grapevine were obtained by Agrobacterium-mediated transformation.Histochemical GUS assay on these resistant shoots showed that two shoots were GUS positive plants;one of them was confirmed that rolC gene was integrated into the host genome of 'Manicure Finger' grapevine by both PCR and RT-PCR analysis.
4.Agrobacterium-mediated transformation was adopted to introduce the rolC gene into the inflorescence of 'Manicure Finger' grapevine via the pollen-tube pathway.40 planflets were obtained from 'Manicure Finger' seeds.By GUS histochemistry assay、PCR amplifiation and RT-PCR analysis,five plants showed GUS positive with a 12.5%positive rate,and two shoots showed PCR and RT-PCR positive,which initially demonstrated that the rolC gene was integrated into the genome of 'Manicure Finger' grapevine.
引文
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