高羊茅和黑麦草农杆菌介导转化体系的研究  

Agrobacterium-mediated Transformation of Tall Fescue and Perennial Ryegrass

作　　者：王艳丽[1] 叶兴国[1] 董芳[2] 乔卫华[3] 陶丽莉[1] 李晓璐[4] 许兴[5] 

机构地区：[1]中国农业科学院作物科学研究所,北京100081 [2]宁夏大学农学院,银川750021 [3]山东农业大学生命科学院,泰安271018 [4]北京工商大学化工学院,北京100037 [5]宁夏农业生物技术重点实验室,银川750002

出　　处：《中国生物工程杂志》2007年第1期22-27,共6页China Biotechnology

基　　金：国家重点基础研究发展计划("973"计划)资助项目(2006CB100106)

摘　　要：利用C58C1农杆菌菌系(携带的表达载体上含GUS基因和nptII基因)感染4个草坪草品种追寻者、爱神特、腾跃和守门员成熟胚来源的愈伤组织,共培养后部分愈伤组织进行X-Gluc组织化学染色检测,其余愈伤组织在含G41810～25mg/L的MS改良培养上先后筛选抗性愈伤组织和分化抗性再生植株,对移栽成活的144棵抗性再生植株分别进行了ELISA检测、PCR检测和组织化学染色检测。愈伤组织阶段X-Gluc染色检测结果表明,4个草坪草品种GUS基因瞬间表达率8.6%～46.9%,爱神特愈伤组织对农杆菌侵染最为敏感,其次是腾跃和守门员,追寻者最不敏感;ELISA检测结果表明,45株呈现阳性,证明nptII基因已转入草坪草并已表达;PCR检测结果与ELISA检测结果一致,表明nptII基因确实已经整合到了草坪草基因组中,且没有发生沉默现象;转基因植株X-Gluc染色检测结果表明,GUS基因在43株中得到了稳定表达,在2株中发生了沉默现象。4个草坪草品种抗性再生植株分化率0%～43.5%,转化率0%～21.5%。结果还表明,GUS基因瞬间表达率与稳定转化率在草坪草上很不一致,不能作为衡量基因型转化效果的指标。Embryonic calli derived from the mature embryos of four turfgrass cultivars including Accent, Quest, Tengyue and Goalkeeper were used as target materials to be infected by Agrobacterium harboring nptⅡ selectable gene and GUS report gene. Some calli were tested by histochemical staining for GUS gene transient expression after three days co-cultivation. And the remaining calli was transferred onto G418 10.0 - 25.0mg/L containing medium for selection and regeneration. The resistant plantlets to the antibiotics were identified by ELISA, PCR and X-Gluc staining for the integration and expression of the foreign genes. The results indicated that the frequency of GUS gene transient expression in the four cultivars varied from 8.6% to 46.9%, the highest in Accent, the lowest in Quest, and the medium in other two genotypes. In 144 total regeneration plants, 45 phnflets were conformed to be positive, in which the expression of nptⅡ gene was suggested by ELISA, and among which the expression of GUS gene in 43 plants and the silence in 2 plants were demonstrated by histochemical staining. Regeneration frequency of resistant planflets to the antibiotics from the four cultivars was 0% -43.5%, and transformation efficiency of the four genotypes was 0% -21.5%. The resuhs also suggested that the both frequencies transient expression and stable transformation of the alien gene were not coincident in turfgrass, and the former frequency cannot be used to evaluate the transformation ability of turfgrass for agrobacterium.

关 键 词：草坪草 农杆菌转化法 GUS基因瞬时表达 分子检测 

分 类 号：Q778[生物学—分子生物学]