根癌农杆菌介导观赏羽衣甘蓝遗传转化体系优化
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摘要
为建立稳定的羽衣甘蓝遗传转化体系,以羽衣甘蓝自交系'Mark 10','W02-7'和'Curly 6'为试材,通过单因子试验,比较羽衣甘蓝子叶、子叶柄、子叶片、下胚轴4种外植体的再生能力,筛选培养基中的适宜激素浓度,优化了羽衣甘蓝不同外植体的再生体系。将播种后6d的子叶于最适再生培养基上进行黑暗预培养,以根瘤农杆菌为介导侵染子叶后,于黑暗条件下进行农杆菌与植物材料的共培养。随后转入含有头孢霉素的抑菌培养基中进行抑菌培养,再将子叶移入含有卡那霉素浓度的筛选培养基中进行抗性芽选择,并提取植物基因组DNA进行目的基因的PCR验证,从而建立高效的羽衣甘蓝遗传转化体系。结果表明:最佳的外植体是羽衣甘蓝的子叶,在培养基MS+6-BA 1.0mg·L-1+NAA 0.1mg·L-1上,不定芽分化率可达100%;子叶经2～3d预培养,将OD600值为0.3～0.5的携带DFR基因表达载体的农杆菌菌液离心,以50mL MS重悬液重悬,侵染外植体5min,共培养2d;采用头孢霉素300mg·L-1,抑菌培养3d后,采用10mg·L-1卡那霉素进行抗性芽选择。在播种后30～40d共获得了24个抗性芽,以抗性芽的基因组DNA为模板进行PCR扩增,9株呈DFR阳性,显示外源基因已经成功整合到羽衣甘蓝基因组中,平均转化率为37.5%。本研究结果可以为羽衣甘蓝重要性状基因的遗传转化提供依据。
In order to establish a stable kale genetic transformation system, kale inbred lines 'Mark 10', 'W02-7' and 'Curly 6'were employed as test materials using single-factor test to compare the regenerative capacity of explants cotyledons, cotyledonary petiole, cotyledonary blade and hypocotyl, and to select the suitable hormone concentration, and optimize the regeneration system of different explants of ornamental kale. In order to establish an efficient genetic transformation system in kale, we pre-cultured6-day cotyledons on the optimal regeneration medium, then the cotyledons were infected by Agrobacterium tumefaciens. The coculture of plant material and Agrobacterium was carried out under dark conditions. Subsequently, the infected cotyledons were transferred to an antibacterial medium containing cephalosporin. Then the cotyledons were transferred into a screening medium containing kanamycin for selection of resistant buds. Genomic DNA of transgenic plant was extracted for target gene verification by polymerase chain reaction. The results showed that the best explants were cotyledons of kale, and the adventitious bud differentiation rate was 100% in the medium MS+6-BA 1.0 mg·L-1+ NAA 0.1 mg·L-1; cotyledons were pre-cultured for 2-3 days, and bacteriostatic culture containing DFR gene expression vector was cultured to OD600.3-0.5 and resuspended in 50 mL of MS and used to infect the explants for 5 min, culturing for 2 days; after 3 days of bacteriostatic culture with cephalosporin300 mg·L-1, 10 mg·L-1 kanamycin was used for selection of resistant buds. In this experiment, 24 resistant buds were obtained from 30-40 d after sowing, and the genomic DNA of resistant buds was used as a template for PCR amplification, and 9 strains were positive for DFR, indicating that the foreign gene has been successfully integrated into the kale genome. The average transformation rate is 37.5%. These results can provide basic information for the genetic transformation of kale-like genes.
In order to establish a stable kale genetic transformation system, kale inbred lines 'Mark 10', 'W02-7' and 'Curly 6'were employed as test materials using single-factor test to compare the regenerative capacity of explants cotyledons, cotyledonary petiole, cotyledonary blade and hypocotyl, and to select the suitable hormone concentration, and optimize the regeneration system of different explants of ornamental kale. In order to establish an efficient genetic transformation system in kale, we pre-cultured6-day cotyledons on the optimal regeneration medium, then the cotyledons were infected by Agrobacterium tumefaciens. The coculture of plant material and Agrobacterium was carried out under dark conditions. Subsequently, the infected cotyledons were transferred to an antibacterial medium containing cephalosporin. Then the cotyledons were transferred into a screening medium containing kanamycin for selection of resistant buds. Genomic DNA of transgenic plant was extracted for target gene verification by polymerase chain reaction. The results showed that the best explants were cotyledons of kale, and the adventitious bud differentiation rate was 100% in the medium MS+6-BA 1.0 mg·L-1+ NAA 0.1 mg·L-1; cotyledons were pre-cultured for 2-3 days, and bacteriostatic culture containing DFR gene expression vector was cultured to OD600.3-0.5 and resuspended in 50 mL of MS and used to infect the explants for 5 min, culturing for 2 days; after 3 days of bacteriostatic culture with cephalosporin300 mg·L-1, 10 mg·L-1 kanamycin was used for selection of resistant buds. In this experiment, 24 resistant buds were obtained from 30-40 d after sowing, and the genomic DNA of resistant buds was used as a template for PCR amplification, and 9 strains were positive for DFR, indicating that the foreign gene has been successfully integrated into the kale genome. The average transformation rate is 37.5%. These results can provide basic information for the genetic transformation of kale-like genes.
引文
[1]祝朋芳,刘丽,周广柱,等.羽衣甘蓝的离体培养研究[J].沈阳农业大学学报,2003,34(4):249-251.
[2]赵秀枢,李名扬,张文玲.观赏羽衣甘蓝高频再生体系的建立[J].基因组学与应用生物学,2009,28(1):141-148.
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[8]段岩娇,张鲁刚,何琼.紫心大白菜花青素积累特性及相关基因表达分析[J].园艺学报,2012,39(11):2159-2167.
[9]焦淑珍,张正言,王林.植物二氢黄酮醇-4-还原酶基因的研究进展[J].南方农业,2016,10(21):186-187,190.
[10]段岩娇,张鲁刚,何琼．紫心大白菜花青素积累特性及相关基因表达分析[J]．园艺学报，2012,39(11):2159-2167.
[11]熊勇,冯馨,程明明,等.几种改良CTAB法提取羽衣甘蓝基因组DNA质量的比较研究[J].中国园艺文摘,2015,31(5):47;74.
[12]廖志强,王小武,孙亮,等.甘蓝型油菜带节子叶再生体系的建立及遗传转化应用[J].中国农学通报,2017,33(34):40-45.
[13]茹磊,张鲁刚,刘学成,等.大白菜离体再生体系的建立及其不定芽对抗生素的敏感性[J].西北农业学报,2011(10):74-78.
[14]刘宏波.甘蓝型油菜抗病虫基因遗传转化及转基因植株抗性鉴定的研究[D].杭州:浙江大学,2010.
[15]许念芳,张恩慧,李殿荣,等.甘蓝CMS下胚轴和子叶的离体培养与植株再生研究[J].西北农林科技大学学报(自然科学版),2010,38(4):115-120.
[16]范爱丽,张鲁刚,惠麦侠,等.大白菜orf224基因植物表达载体的构建及遗传转化[J].西北植物学报,2008(7):1296-1302.
[17]王肖红,曾爱松,高兵,等.结球甘蓝不定芽诱导及遗传转化的影响因子[J].江苏农业学报,2013,29(4):918-920.
[18]张晓东,李利斌,宋宜现,等.大白菜高效稳定遗传转化体系的建立[J].山东农业科学,2010(2):1-7.
[19]朱小彬,于一帆,葛会敏,等.农杆菌介导的甘蓝型油菜下胚轴遗传再生体系研究[J].安徽农业科学,2013,41(31):12257-12261.
[20]杨广东,朱祯,李燕娥,等.大白菜高效遗传转化体系的建立[J].农业生物技术学报,2002(2):127-132.
[21]刘洋,徐漫,赵德刚.甘蓝型油菜花柄遗传转化体系的创建[J].贵州农业科学,2013,41(2):1-5.
[22]孙满芝,尹成涛.植物组培过程中玻璃化现象的发生与解决措施[J].山东林业科技,2001(6):19-20.
[23]何芳兰,李毅,赵明,等.影响高山杜鹃试管苗玻璃化的几个因素研究[J].西北林学院学报,2008(1):104-107.
[24]张鹏,傅爱根,王爱国.AgNO3在植物离体培养中的作用及可能的机制[J].植物生理学通讯,1997,33(5):376-379.
[25]曹家树,余小林,黄爱军,等.提高白菜离体培养株再生频率的研究[J].园艺学报,2000,27(6):452-454.
[26]王火旭,王关林,王晓岩,等.大白菜AB-81高频再生系统的建立及gus A基因瞬时表达的研究[J].园艺学报,2001,28(1):74-76.
[27]景巧丽.大白菜半胱氨酸脱巯基酶突变植株的构建[D].太原:山西大学,2017.
[2]赵秀枢,李名扬,张文玲.观赏羽衣甘蓝高频再生体系的建立[J].基因组学与应用生物学,2009,28(1):141-148.
[3] HOSOKI T,SHIRASHI K,KIGO T,et al.Transformation and regeneration of ornamental kale(Brassica oleracea var.acephala DC)mediated by Agrobacterium rhizogenes[J].Plant Genetics and Breeding,1989,40(3):259-266.
[4] CAO J,SHELTON AM,EARLE ED.Development of transgenic collards(Brassica oleracea var.acephala)expressing a cry1Ac or cry1C Bt gene for control of the diamond back moth[J].Crop Protection,2005,24:804-813.
[5]张文玲,李凌,刘凡.农杆菌介导羽衣甘蓝转化体系的建立[J].西南大学学报(自然科学版),2010(4):61-65.
[6]高航,高玉亮,李葵花.羽衣甘蓝下胚轴农杆菌介导遗传转化体系的优化[J].生物技术通报,2015,31(6):111-115.
[7] REN J,FU W,DU J,et al.Identification of a candidate gene for Re,the factor determining the red leaf phenotype in ornamental kale using fine mapping and transcriptome analysis[J].Plant Breeding,2017,136(5);738-748.
[8]段岩娇,张鲁刚,何琼.紫心大白菜花青素积累特性及相关基因表达分析[J].园艺学报,2012,39(11):2159-2167.
[9]焦淑珍,张正言,王林.植物二氢黄酮醇-4-还原酶基因的研究进展[J].南方农业,2016,10(21):186-187,190.
[10]段岩娇,张鲁刚,何琼．紫心大白菜花青素积累特性及相关基因表达分析[J]．园艺学报，2012,39(11):2159-2167.
[11]熊勇,冯馨,程明明,等.几种改良CTAB法提取羽衣甘蓝基因组DNA质量的比较研究[J].中国园艺文摘,2015,31(5):47;74.
[12]廖志强,王小武,孙亮,等.甘蓝型油菜带节子叶再生体系的建立及遗传转化应用[J].中国农学通报,2017,33(34):40-45.
[13]茹磊,张鲁刚,刘学成,等.大白菜离体再生体系的建立及其不定芽对抗生素的敏感性[J].西北农业学报,2011(10):74-78.
[14]刘宏波.甘蓝型油菜抗病虫基因遗传转化及转基因植株抗性鉴定的研究[D].杭州:浙江大学,2010.
[15]许念芳,张恩慧,李殿荣,等.甘蓝CMS下胚轴和子叶的离体培养与植株再生研究[J].西北农林科技大学学报(自然科学版),2010,38(4):115-120.
[16]范爱丽,张鲁刚,惠麦侠,等.大白菜orf224基因植物表达载体的构建及遗传转化[J].西北植物学报,2008(7):1296-1302.
[17]王肖红,曾爱松,高兵,等.结球甘蓝不定芽诱导及遗传转化的影响因子[J].江苏农业学报,2013,29(4):918-920.
[18]张晓东,李利斌,宋宜现,等.大白菜高效稳定遗传转化体系的建立[J].山东农业科学,2010(2):1-7.
[19]朱小彬,于一帆,葛会敏,等.农杆菌介导的甘蓝型油菜下胚轴遗传再生体系研究[J].安徽农业科学,2013,41(31):12257-12261.
[20]杨广东,朱祯,李燕娥,等.大白菜高效遗传转化体系的建立[J].农业生物技术学报,2002(2):127-132.
[21]刘洋,徐漫,赵德刚.甘蓝型油菜花柄遗传转化体系的创建[J].贵州农业科学,2013,41(2):1-5.
[22]孙满芝,尹成涛.植物组培过程中玻璃化现象的发生与解决措施[J].山东林业科技,2001(6):19-20.
[23]何芳兰,李毅,赵明,等.影响高山杜鹃试管苗玻璃化的几个因素研究[J].西北林学院学报,2008(1):104-107.
[24]张鹏,傅爱根,王爱国.AgNO3在植物离体培养中的作用及可能的机制[J].植物生理学通讯,1997,33(5):376-379.
[25]曹家树,余小林,黄爱军,等.提高白菜离体培养株再生频率的研究[J].园艺学报,2000,27(6):452-454.
[26]王火旭,王关林,王晓岩,等.大白菜AB-81高频再生系统的建立及gus A基因瞬时表达的研究[J].园艺学报,2001,28(1):74-76.
[27]景巧丽.大白菜半胱氨酸脱巯基酶突变植株的构建[D].太原:山西大学,2017.