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根癌农杆菌介导CYP1A1转化菠萝的研究
  • ISSN号:1007-1032
  • 期刊名称:《湖南农业大学学报:自然科学版》
  • 时间:0
  • 分类:S667.8[农业科学—果树学;农业科学—园艺学]
  • 作者机构:[1]华南农业大学园艺植物生物技术研究所,广东广州510642
  • 相关基金:农业部公益性行业科研专项(NYHYZX07-30);国家自然科学基金项目(30971984);广东省重大科技专项(2006820101003)
中文摘要:

将菠萝(Ananas comosus)愈伤组织经含有植物表达重组质粒(pUHA1-CrP1A1)的根癌农杆菌(LBA4404菌株)侵染后,在MS+3.0mg/LBA+2.0mg/LNAA+100μmol/LAS+8g/Lagar上共培养3d,转入选择培养基(MS+3.0mg/LBA+2.0mg/LNAA+20mg/LKin+400mg/LCarb+8g/Lagat)上培养约10d后开始分化出不定芽;28d后将绿色的抗Km不定芽转入MS+2.0mg/LNAA+30mg/LKm+300mg/LCarb+8g/Lagar上再进行连续2轮选择,随后将绿芽转入MS+1.0mg/LIBA+30mg/LKm+8g/Lagar上生根,共获得95株Km抗性植株,转化率0.12%~2.69%.对其中部分的抗Km植株进行PCR检测,PCR阳性植株率达64.29%.经Southern杂交进~步证实,CYP1A1已整合到菠萝基因中.以琼脂为培养基凝固剂、共培养基中添加AS、增加选择次数和逐渐增加新一轮选择培养基中的Km质量浓度等是根癌农杆菌介导菠萝愈伤组织获得转基因植株的重要条件.

英文摘要:

Pineapple (Ananas comosus) callus was infected by the Agrobacterium tumefaciens (LBA4404 strain), containing a plant expression recombinant plasmid (pUHA1-CYP1A1). After infection, the callus was co-cultivated with agrobacteria for 3 d on medium(MS+3.0 mg/L BA+2.0 mg/L NAA+100 μmol/L AS+8 g/L agar) then transferred to selection medium(MS+3.0 mg/L BA+2.0 mg/L NAA+20 mg/L Kin+400 mg/L Carb+8 g/L agar). Adventitious shoots were initiated after 10 d of culture; 28 d later, the green Km-resistant shoots were transferred to selection medium (MS+2.0 mg/L NAA+30 mg/L Km+300 mg/L Carb+8 g/L agar) for the second successive selections, then were transferred to mediun (MS+1.0 mg/L IBA+30 mg/L Km+8 g/L agar)for rooting, as a result, a total of 95 Km-resistant plants were obtained, its transformation rate is 0.12%-2.69%. PCR assays were performed to some of the Km resistant plants and its positive rate is 64.29%. The results of southern hybridization further confirmed that CYP1A1 has been integrated into the genome of pineapple. Taking the agar as medium coagulant, adding AS to co-culture medium, increasing selection times, gradually increasing the concentration of Km in the new round of selection medium etc are the important conditions of obtaining transformants by Agrobacterium tumefaciens-mediated genetic transformation in pineapple callus.

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期刊信息
  • 《湖南农业大学学报:自然科学版》
  • 北大核心期刊(2011版)
  • 主管单位:湖南农业大学
  • 主办单位:湖南农业大学
  • 主编:官春云
  • 地址:长沙市芙蓉区农大路1号
  • 邮编:410128
  • 邮箱:zkb4618035@hunan.edu.cn
  • 电话:0731-84618035
  • 国际标准刊号:ISSN:1007-1032
  • 国内统一刊号:ISSN:43-1257/S
  • 邮发代号:42-157
  • 获奖情况:
  • 2006、2008、2010年连续三届全国高校精品科技期刊
  • 国内外数据库收录:
  • 美国化学文摘(网络版),英国农业与生物科学研究中心文摘,德国数学文摘,英国科学文摘数据库,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),中国北大核心期刊(2000版)
  • 被引量:17328