耐盐芦苇大片段BIBAC载体构建及水稻遗传转化

Construction of Large Fragment DNA-BIBAC Vector of Salt Tolerant Reed and Its Genetic Transformation in Rice

利用盐生植物的耐盐基因改良作物耐盐性是保障土壤盐渍化地区粮食生产和改良盐渍化土地的最经济最有效的途径。本研究以生长于西北盐盖上的一种耐盐野生芦苇为材料,制备了Mb级耐盐芦苇基因组DNA,酶切后经脉冲场凝胶电泳将其分离为不同大小的大片段DNA区段,并与酶切回收的BIBAC-S载体进行连接,成功构建了耐盐芦苇不同大小的大片段DNA-BIBAC载体,经根癌农杆菌EHA105介导,成功地转化了粳稻品种‘中花11’成熟胚愈伤组织,获得了转基因水稻植株。研究表明,不同大小的大片段DNA-BIBAC载体在转化同一受体材料时,其所获得的愈伤转化率、植株转化率、转基因植株的阳性率等存在差异,说明插入片段的大小与转化效率之间存在关系。本研究为创制耐盐水稻新种质、克隆盐生植物的耐盐基因提供了理论和技术参考。

Using the salt-tolerant genes of halophytes to improve crop salt tolerance will be the most economical and effective way to ensure food production in soil salinized areas and to improve salinized land.In this study,a salt-tolerant wild Phragmites australis grown in the saline-alkali soil of Northwestern China was used as the materials to prepare Mb class genomic DNA,which was digested by restriction enzyme,and was separated into different size of DNA segments by pulsed field gel electrophoresis,then connected with the BIBAC-S vector,and successfully constructed the salt tolerant reed large fragment DNA-BIBAC vector.The large fragment DNA-BIBAC vector was successfully transformed into the callus of the mature embryo of‘Zhonghua 11’mediated by Agrobacterium tumefaciens EHA105,and 23 transgenic positive rice plants were obtained.The results showed that there were differences in callus transformation rate,plant transformation rate and transgenic plant positive rate among different sizes of the DNA-BIBAC vector,indicating that there was a relationship between the size of inserted fragments and transformation efficiency.This study would provide a theoretical and technical reference for creating new germplasm of salt tolerant rice and cloning salt tolerant genes of halophytes.