农杆菌介导茄子叶转基因体系的建立
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摘要
本文选择3份茄核心种质,研究Hyg B浓度、农杆菌浓度(OD600)、侵染时间、共培养时间对茄遗传转化效果的影响。同时,选择7份核心种质进行转化研究,以确定茄遗传转化的最佳受体。结果表明,筛选阳性植株的最适Hyg B浓度为50mg/L;菌液浓度(OD_(600))0.6,侵染外植体15min和共培养4d时,抗性愈伤百分率最高;获得的Hyg B抗性植株,经PCR检测及GUS组织化学鉴定,确认外源基因GUS已成功整合到茄基因组中;不同茄遗传型转化效果差异较大,最高转化率可达到10%。
Three eggplant core germplasm resources were selected to study the effects of hygromycin B concentration,Agrobacterium concentration(OD_(600)),infection time and co-culture time on the genetic transformation.At the same time,seven core germplasm resources were used to confirm the best receptor for eggplant genetic transformation.The results showed that the most suitable hygromycin B concentration for screening positive plants was 50 mg/L.The percentage of resistant calluses was the highest with the combination of 0.6 OD_(600) for Agrobacterium,15 minutes of explant infection duration and 4 days of coculture duration.PCR and GUS analysis of hygromycin B-resistant plants showed that the GUSgene was successfully integrated into the eggplant genome,and there was great variation in transformation rate among different genotypes,the highest transformation rate being 10%.
Three eggplant core germplasm resources were selected to study the effects of hygromycin B concentration,Agrobacterium concentration(OD_(600)),infection time and co-culture time on the genetic transformation.At the same time,seven core germplasm resources were used to confirm the best receptor for eggplant genetic transformation.The results showed that the most suitable hygromycin B concentration for screening positive plants was 50 mg/L.The percentage of resistant calluses was the highest with the combination of 0.6 OD_(600) for Agrobacterium,15 minutes of explant infection duration and 4 days of coculture duration.PCR and GUS analysis of hygromycin B-resistant plants showed that the GUSgene was successfully integrated into the eggplant genome,and there was great variation in transformation rate among different genotypes,the highest transformation rate being 10%.
引文
[1]中国人民共和国农业部官方网站[EB/OL].http://www.moa.gov.cn.
[2]金丹丹,梁美霞,谢立波,等.茄子组织培养与基因工程研究进展[J].分子植物育种,2004,2(6):861-866.
[3]Rotino G L,Perri E,Acciarri N,et al.Development of eggplant varietal resistance to insects and diseases via plant breeding[J].Advances in Horticultural Science,1997,11(4):193-201.
[4]Prabhavathi V,Yadav J S,Kumar P A,et al.Abiotic stress tolerance in transgenic eggplant(Solanum melongena L.)by introduction of bacterial mannitol phosphodehydrogenase gene[J].Molecular Breeding,2002,9(2):137-147.
[5]Papolu P K,Dutta T K,Tyagi N,et al.Expression of a Cystatin transgene in eggplant provides resistance to root-knot nematode,Meloidogyne incognita[J].Frontiers in Plant Science,2016,7,1122.
[6]胡晓琴,贾士荣.水稻巯基蛋白酶抑制剂基因导入马铃薯和茄子[J].园艺学报,1998,25(1):65-69.
[7]王凤华,李光远,陈双臣,等.农杆菌介导的茄子愈伤组织遗传转化研究[J].河南农业大学学报,2009,43(4):389-392.
[8]孟平红,万发香,王永清,等.冷诱导转录因子CBF3转化茄子的初步研究[J].中国蔬菜,2013,10:36-43.
[9]张明华,陈钰辉,刘富中,等.农杆菌介导抗根结线虫Bt cry6A基因转化茄子的研究[J].核农学报,2015,29(4):686-691.
[10]包崇来,杜黎明,胡天华,等.紫红线茄转基因体系优化及SmARF8基因RNA干涉表达载体的遗传转化[J].园艺学报,2014,41(6):1105-1114.
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[12]李洪清,李美茹.影响农杆菌介导植物基因转化的因素问题[J].植物生理学通讯,1994,35(2):145-151.
[13]葛海燕.基于SSR标记的茄子遗传多样性及主要农艺性状的关联分析[D].上海:上海交通大学,2013.
[14]张兴国,刘元清,杨正安,等.茄子遗传转化体系的建立[J].西南农业大学学报,2001,23(3):233-234.
[15]Chen C F,Chan K G,Tan B C,et al.Enhancement of Agrobacterium-mediated transformation efficiency of model plant using quorum sensing molecule,N-3-oxo-octanoyl-L-homoserine-lactone[J].Plant Cell Tissue and Organ Culture,2015,121:481-487.
[16]Ge J,Wang L J,Yang C,et al.Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis[J].Plant Cell Reports,2016,35:2045-2053.
[17]胡凤,杨万年.大豆组培苗水培生根与培养基生根比较研究[J].大豆科学,2013,32(30):333-335.
[18]赵锦慧,赖颖,郭婕,等.不同草甘膦筛选方式对玉米抗性愈伤组织继代与分化的影响[J].江苏农业科学,2011,39(4):87-89.
[2]金丹丹,梁美霞,谢立波,等.茄子组织培养与基因工程研究进展[J].分子植物育种,2004,2(6):861-866.
[3]Rotino G L,Perri E,Acciarri N,et al.Development of eggplant varietal resistance to insects and diseases via plant breeding[J].Advances in Horticultural Science,1997,11(4):193-201.
[4]Prabhavathi V,Yadav J S,Kumar P A,et al.Abiotic stress tolerance in transgenic eggplant(Solanum melongena L.)by introduction of bacterial mannitol phosphodehydrogenase gene[J].Molecular Breeding,2002,9(2):137-147.
[5]Papolu P K,Dutta T K,Tyagi N,et al.Expression of a Cystatin transgene in eggplant provides resistance to root-knot nematode,Meloidogyne incognita[J].Frontiers in Plant Science,2016,7,1122.
[6]胡晓琴,贾士荣.水稻巯基蛋白酶抑制剂基因导入马铃薯和茄子[J].园艺学报,1998,25(1):65-69.
[7]王凤华,李光远,陈双臣,等.农杆菌介导的茄子愈伤组织遗传转化研究[J].河南农业大学学报,2009,43(4):389-392.
[8]孟平红,万发香,王永清,等.冷诱导转录因子CBF3转化茄子的初步研究[J].中国蔬菜,2013,10:36-43.
[9]张明华,陈钰辉,刘富中,等.农杆菌介导抗根结线虫Bt cry6A基因转化茄子的研究[J].核农学报,2015,29(4):686-691.
[10]包崇来,杜黎明,胡天华,等.紫红线茄转基因体系优化及SmARF8基因RNA干涉表达载体的遗传转化[J].园艺学报,2014,41(6):1105-1114.
[11]张兴国,刘元清,杨正安,等.茄子遗传转化体系的建立[J].西南农业大学学报,2001,23(3):233-234.
[12]李洪清,李美茹.影响农杆菌介导植物基因转化的因素问题[J].植物生理学通讯,1994,35(2):145-151.
[13]葛海燕.基于SSR标记的茄子遗传多样性及主要农艺性状的关联分析[D].上海:上海交通大学,2013.
[14]张兴国,刘元清,杨正安,等.茄子遗传转化体系的建立[J].西南农业大学学报,2001,23(3):233-234.
[15]Chen C F,Chan K G,Tan B C,et al.Enhancement of Agrobacterium-mediated transformation efficiency of model plant using quorum sensing molecule,N-3-oxo-octanoyl-L-homoserine-lactone[J].Plant Cell Tissue and Organ Culture,2015,121:481-487.
[16]Ge J,Wang L J,Yang C,et al.Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis[J].Plant Cell Reports,2016,35:2045-2053.
[17]胡凤,杨万年.大豆组培苗水培生根与培养基生根比较研究[J].大豆科学,2013,32(30):333-335.
[18]赵锦慧,赖颖,郭婕,等.不同草甘膦筛选方式对玉米抗性愈伤组织继代与分化的影响[J].江苏农业科学,2011,39(4):87-89.