甘露糖正向筛选体系在巨尾桉遗传转化中的应用

Application of positive selection system of mannose in Eucalyptus grandis × E. urophylla genetic transformation

为了研究甘露糖正向筛选体系在巨尾桉遗传转化过程中的有效性,构建了以6-磷酸甘露糖异构酶(6-phosphomannose isomerase,PMI)为筛选标记的pCAMBIA1301植物表达载体,并将该载体通过农杆菌介导的遗传转化转入木本植物巨尾桉中。将获得的阳性植株通过氯酚红(chlorophenol red,CPR)法及PCR检测,桉树遗传转化的阳性率达到26.09%。另外,通过正交试验优化法,对巨尾桉组培快繁体系建立过程中不同浓度激素配比进行了研究,建立起良好的巨尾桉组织培养再生体系,由甘露糖筛选敏感性测试,获得了巨尾桉筛选临界浓度,蔗糖与甘露糖比例为19∶11,优化了巨尾桉遗传转化体系,为今后巨尾桉组织培养与遗传转化研究提供了重要的参考依据。

In order to study on validity of positive selection system of mannose in the transformation of Eucalyptus grandis × E. urophylla,the plant expression vector pCAMBIA1301 with 6-phosphate isomerase（ 6-phosphomannose isomerase,PMI） as selection marker was constructed and transformed into Eucalyptus grandis × E. urophylla by Agrobacterium-mediated genetic transformation method. The obtained positive plants were identified through chlorophenol red（ CPR） method and PCR testing to show that PMI gene was really introduced into the genomes of Eucalyptus grandis × E. urophylla,and the genetic transformation positive rate reached26. 09%. In addition,the process of establishing tissue culture system in different concentrations of hormones was studied and the tissue culture regenerations system and genetic transformation system was optimized by orthogonal optimization method and mannose sensitivity tests of Eucalyptus grandis × E. urophylla The ratio of sucrose and mannose was 19 ∶ 11.