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超声波辅助农杆菌介导CP基因转化番木瓜(Carioca papaya L.)的有效方法
引用本文:姜玲 MAOKATetsuo KOMORISadao FUKAMACHIHiroshi KATHidenori GAWAKazunori.超声波辅助农杆菌介导CP基因转化番木瓜(Carioca papaya L.)的有效方法[J].实验生物学报,2004,37(3):189-198.
作者姓名:姜玲  MAOKATetsuo  KOMORISadao  FUKAMACHIHiroshi  KATHidenori  GAWAKazunori
作者单位:[1]华中农业大学园艺林学学院,国家果树种质资源室内保存中心,武汉430070 [2]日本北海道,国家农业研究中心,062—8555 [3]日本盛岗,岩手大学农业生物技术系,020—8550 [4]日本农林水产省国际农业研究中心,冲绳亚热带试验站1091—1
摘    要:本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PT—NP—YKT)插入双元栽体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2MS 6%蔗糖 1%葡萄糖,pH5.7)调整至光密度OD600nm=15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP基因转化上均表现出相似的效果。试验还表明:120mg/L是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定。

关 键 词:超声波  农杆菌  CP基因  番木瓜  胚性愈伤组织  转化

An efficient method for sonication assisted Agrobacterium-mediated transformation of coat protein (CP) coding genes into papaya (Carica papaya L.).
Ling Jiang,Tetsuo Maoka,Sadao Komori,Hiroshi Fukamachi,Hidenori Kato,Kazunori Ogawa.An efficient method for sonication assisted Agrobacterium-mediated transformation of coat protein (CP) coding genes into papaya (Carica papaya L.).[J].Acta Biologiae Experimentalis Sinica,2004,37(3):189-198.
Authors:Ling Jiang  Tetsuo Maoka  Sadao Komori  Hiroshi Fukamachi  Hidenori Kato  Kazunori Ogawa
Institution:Department of Horticulture, Conservation Center of National Fruit Germ plasm Resource, Huazhong Agriculture University, Wuhan, Hubei, 430070, China.
Abstract:An efficient method for the production of transgenic papaya was developed via Sonication Assisted Agrobacterium-mediated Transformation (SAAT) of somatic embryos. The plasmid pGA482G was modified to contain gene PTi-Epj-TL-PLDMV with CP coding sequence of PLDMV Japan strain and chimeric gene PTi-NP-YKT with multiple CP coding sequences from PRSV Taiwan strain, PRSV Hawaii strain and PRSV Thailand strain, respectively. Disarmed Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pGA482G with the CP genes and nptII gene was used to transform embryo calli of papaya variety Sunset to produce transgenic papaya plants. The experiment was focused on the screening of effective transformation method. The engineered Agrobacterium grown overnight was diluted with an infection media of high osmotic pressure (1/2 MS medium contain 6% sucrose and 1% glucose, pH 5.7) and adjusted to optical density OD600nm = 0.15-0.20, embryonic calli were immerged in it for 30 min and treated with 5 s, 15 s, and 20 s sonication respectively during the infection. Results indicated that 15 s sonication treatment improved the transformation efficiency dramatically. After 15 s sonication treatment on embryo calli loaded in 15 ml sterile plastic tubes, 21 putative transgenic lines were produced from 80 pieces embryonic calli (26.3%) transformed by Agrobacterium pGA482G/CPG] and 8 putative transgenic lines was produced from 48 pieces embryonic calli (16.7%) transferred by Agrobacterium pGA482G/CPB], while only a single line came out of 64 pieces embryonic calli (1.6%) transformed by Agrobacterium pGA482G/CPG] and none from 25 pieces embryonic calli transformed by Agrobacterium pGA482G/CPB] in the non-treatment control. Results also showed that the best concentration of selection antibiotic was 120 mg/L kanamycin. A total of 42 resistant shoots were produced from 421 pieces of original embryonic calli in 9 months. The presence of the CP genes in the transgenic plants and their integration into the papaya genome were confirmed by PCR and Southern hybridization respectively.
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