农杆菌介导的DnMADS2基因转化金钗石斛初步研究

Agrobacterium-mediated Transformation of Dendrobium nobile with DnMADS2 Gene

为建立稳定有效的金钗石斛转化体系,进一步研究花发育相关基因的功能,为石斛兰分子育种提供基础,本研究以金钗石斛类原球茎(PLBs)为转化受体,通过农杆菌介导的方法将DnMADS2基因转入金钗石斛。结果表明:用20 mg/L潮霉素筛选4个月后获得抗性PLBs,对其进行HPT基因和DnMADS2基因的PCR检测表明,目的基因已成功整合到PLBs中。获得的转化PLBs在抗性培养基上分化为不定芽后,再经生根壮苗培养获得转化植株。对转化植株进行Southern检测,结果表明,DnMADS2基因已整合到5株金钗石斛转化植株基因组中。据此建立的稳定高效的金钗石斛转化体系和获得的转基因植株,将为DnMADS2基因功能的进一步研究和金钗石斛转基因育种奠定基础。

To construct a stable and effective genetic transformation system of Dendrobium nobile,for further study the functions of genes related to flower development,and provid basis for molecular breeding,the DnMADS2 gene was transformed to protocorm-like bodies （PLBs） of D.nobile mediated by Agrobacterium tumefaciens.The results showed that resistant PLBs were obtained after screening with 20 mg/L hygromycin for 4 months,which were proved to be successfully transformed by PCR amplification of HPT and DnMADS2 genes.Adventitious buds were regenerated from the resistant PLBs in differention media,and transformed plants were acquired after rooting and seedling strengthening.The results of southern blot indicated that DnMADS2 gene was successfully integrated into the genome of 5 transformed seedlings.The stable and efficient genetic transformation system constructed,and the transformed seedlings obtained in this study would provide a basis for further investigation of the function of DnMADS2 gene,and for transgenic breeding of D.nobile.