卡拉霉素在转基因黄籽油菜中的应用
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摘要
为筛选出黄籽油菜遗传转化中卡拉霉素或卡拉霉素(kanamycin,Kan or km)最佳质量浓度,首先将油菜子叶接种在不同质量浓度Kan(0~180mg/L)的分化培养基上,结果发现Kan质量浓度小于60 mg/L有利于绿苗分化.然后在不同质量浓度Kan的分化培养基上对农杆菌侵染的下胚轴进行筛选,结果表明初代培养Kan质量浓度以20~40mg/L为宜,同样质量浓度继代培养后可获得抗性愈伤和转基因阳性苗.最后利用Kan抗性对转基因黄籽油菜后代进行研究,苗期到薹花期生长的植株进行叶片涂抹法筛选,Kan最适质量浓度为4g/L,时间为4~10d;T1代抗性分析、GUS染色分析、T1代籽粒的PCR检测说明转基因在T1代、T2代可遗传并出现分离.β-葡萄糖苷酸酶基因(GUS)和聚合酶链式反应(PCR)验证叶片涂抹进行Kan抗性筛选的方法准确性高,可达100%.
Cotyledons of yellow seed rape were inoculated onto differentiation media with different concentrations of kanamycin(Kan)to screen the optimal Kan concentration in genetic transformation,and the results indicated that a Kan concentration of less than 60 mg/L was beneficial to green shoot differentiation.Then,hypocotyls infected by Agrabacterium tumefaciens were transferred to differentiation media containing Kan of different concentrations,and it was found that 20-40 mg/L Kan concentration in the primary culture was appropriate,and resistant calli and regenerated positive plantlets were obtained after successive transfer cultures.Finally,Kan resistance in the transgenic yellow seed rape offspring was studied.The method of leaf daubing was used in transgenic plant growing from the period of young shoot to flowering,and the optimum Kan concentration was found to be 4g/L and the daubing time 4to 10 days.Kan resistance of T1 generation,GUS staining analysis,PCR detection of T1 generation showed that transgenic seeds in T1 and T2generations was inheritable and segregation occurred.GUS and PCR verification in this study demonstrated that the accuracy of the method of leaf daubing to screen resistant plants was as high as 100%.
Cotyledons of yellow seed rape were inoculated onto differentiation media with different concentrations of kanamycin(Kan)to screen the optimal Kan concentration in genetic transformation,and the results indicated that a Kan concentration of less than 60 mg/L was beneficial to green shoot differentiation.Then,hypocotyls infected by Agrabacterium tumefaciens were transferred to differentiation media containing Kan of different concentrations,and it was found that 20-40 mg/L Kan concentration in the primary culture was appropriate,and resistant calli and regenerated positive plantlets were obtained after successive transfer cultures.Finally,Kan resistance in the transgenic yellow seed rape offspring was studied.The method of leaf daubing was used in transgenic plant growing from the period of young shoot to flowering,and the optimum Kan concentration was found to be 4g/L and the daubing time 4to 10 days.Kan resistance of T1 generation,GUS staining analysis,PCR detection of T1 generation showed that transgenic seeds in T1 and T2generations was inheritable and segregation occurred.GUS and PCR verification in this study demonstrated that the accuracy of the method of leaf daubing to screen resistant plants was as high as 100%.
引文
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[3]官春云.油菜的转基因育种[J].中国油料作物学报,2005,27(1):97-103.
[4]谢伶俐,殷家明,李加纳,等.激素对油菜子叶再生及转基因瞬间表达的影响[J].西南师范大学学报:自然科学版,2007,32(5):50-55.
[5]张瑞熙,申敏,殷家明,等.农杆菌介导转化油菜中几种影响玻璃化频率的因素[J].西南大学学报:自然科学版,2012,34(4):9-13.
[6]王关林,方宏筠.植物基因工程[M].2版.北京:科学出版社,2002:527-606.
[7]施荣华,李学宝.外源基因在转基因油菜后代中的表达及遗传学分析[J].华中师范大学学报:自然科学版,2003,34(2):208-212.
[8]林良斌,官春云,周小云,等.转基因抗虫油菜中Bt杀虫蛋白基因稳定遗传和高效表达及抗虫性研究[J].作物学报,2002,28(2):175-178.
[9]WANG Li,ZHAO Chun-mei,WANG Yi-ju,et al.Over Expression of Chloroplast localized Small Molecular Heat-Shock Protein Enhances Chilling Tolerance in Tomato Plant[J].Journal of Plant Physiology and Molecular Biology,2005,31(2):167-174.
[10]刘斌,吴迪,侯文胜.卡那霉素叶片涂抹法筛选转基因大豆植株的有效性[J].西北农林科技大学学报:自然科学版,2006,34(12):80-82.
[11]马丽华,许红霞,胡育昌.转Bt基因棉卡那霉素田间快速检测法[J].中国棉花,2000,27(12):11-12.
[12]韦献雅,牛应泽,张其坤.3种筛选NPT-Ⅱ标记转基因油菜方法研究[J].中国农学通报,2009,25(14):37-41.
[13]周玉,王留明,张学坤.转基因抗虫棉田间快速鉴定方法研究[J].山东农业科学,2000,23(1):48-49.
[14]燕丽萍,夏阳,梁慧敏,等.卡那霉素叶片涂抹法田间筛选转基因苜蓿的研究[J].中国农学通报,2009,25(14):22-26.
[15]赵爽,雷建军,陈国菊,等.卡那霉素在转基因芥菜中的应用[J].遗传,2008,30(4):501-507.