新疆甜瓜组培体系的优化及抗病转基因研究

Optimization of Tissue Culture System and Anti-fungal Transfection of Xinjiang Muskmelon

选用新疆优良甜瓜未成熟子叶切块作为外植体 ,经过组织培养获得再生植株 .以 MS为基本培养基 ,取3日龄子叶块外植体 ,选用 Ms+ 6-BA0 -2 .5 mg/ L诱导丛生芽 ,最高诱导率可达 89% .在整个再生体系建立过程中 ,6-BA的浓度逐渐降低 .在生根培养培养基中 ,附加活性炭和硝酸银有利于根的诱导 .在甜瓜高效再生体系基础上 ,用根癌农杆菌介导法将几丁质酶基因和 β-1 ,3-葡聚糖酶基因导入新疆甜瓜中 .经卡那霉素的抗性筛选 ,获得转化的再生植株 .用 PCR反应、点杂交及 SDS-PAGE等手段 ,进一步研究了再生植株的基因整合与表达情况 .经 PCR扩增反应和琼脂糖电泳检测 ,在转化植株中扩增出了特异片段 ,点杂交检测转化植株有阳性反应 .SDS-PAGE分析表明 ,转化植株比对照在 35 .5 KD处有一条明显的蛋白质谱带 ,其分子量与几丁质酶基因表达产物分子量相符 .初步确定该基因在转化植株内得到正常表达.

In order to establish the transgenic plant system,the cotyledons of muskmelon as exp|ants,regenerated plantlets were used in culture.Using three-day explants,shoots regeneration are induced on basic medium MS medium supplemented with O-2.5mg/L6-BA.The highest frequency shoot regeneration observed is 89%.The induction frequency of shoot of melon varieties depends on different concentration of hormones in the medium.To achieve the high rate of rooting,the concentration of hormone 6-BA is gradually decreased during the whole growth period.The 1/2 MS medium with AgNO3 and active carbon was used for root inducing.On the basis of regeneration protocol of Xinjiang muskmelon,two genes are transferred into muskmelon plant and selected of kanamycin in the medium.Afterkanamycin resistant plants are obtained,transgenic plants are determined by PCR and confirmed by Southern dot blotting.The expression of chitinase was observed by using SDS-PAGE analysis.