NP-1在小球藻硝酸还原酶缺失突变体中的表达与活性检测  

Expression and Bioactivity Characterization of NP-1 in Transgenic Nitrate Reductase Deficient Mutant of Chlorella ellipsoidea

作　　者：安洋[1] 赵世民[2] 孙勇如[2] 白丽莉[2] 尹维波[2] 陈宇红[2] 李元广[1] 胡军[2] 胡赞民[2] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]中国科学院遗传与发育生物学研究所分子农业研究中心,北京100101

出　　处：《农业生物技术学报》2008年第4期635-641,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2006AA22143);国家自然科学基金(No.30671193)资助。

摘　　要：研究构建了兔防御素基因NP-1的植物表达载体,NP-1由Ubiquitin启动子控制,含NPTⅡ基因和硝酸还原酶基因2个筛选标记。以椭圆小球藻(Chlorella ellipsoidea)硝酸还原酶缺失突变体nrm-4为受体,采用原生质体转化的方法将目的基因NP-1转入该突变体中。用含G418的硝酸盐培养基筛选出阳性藻落,经PCR和RT-PCR检测,证明获得了转基因藻株。转基因藻株在无抗生素筛选压力的硝酸盐液体培养基上继代培养后进行抑菌活性检测。结果表明,转基因椭圆小球藻表达的NP—1具有正常的生物活性,并在无抗生素的培养基中稳定遗传。研究的结果为小球藻表达外源基因提供了新的技术途径,也使NP-1的规模化生产成为可能。The vector containing NP-1 gene controlled by Ubiquifin promoter with two selective marker genes NPT Ⅱ and nitrate reductase gene was constructed. The NP-1 gene was transformed into the nitrate reductase-deficient mutant nrm-4 of C. ellipsoidea via a protoplast transformation method. The transformed nrm-4 cells were screened on the medium with nitrate and G418. And the transgenic lines were characterized by PCR and RT-PCR amplification of both the NP-1 gene and NPTⅡgene from genomic DNA isolated from transformants. Then the transgenic lines were further cultured in the liquid medium with nitrate alone.In vitro assay results showed that the NP-1 protein of the transgenic C. ellipsoidea had effective bacteria- resistance activities and was stable without G418 selection stress. The research results provide a new way to express alien genes in Chlorella and make producing NP-1 lamely possible.

关 键 词：椭圆小球藻 硝酸还原酶缺失突变体 NP-1 

分 类 号：S188[农业科学—农业基础科学]