目的 为了验证CodA基因是否适宜作为有效的青蒿基因打靶负选择标记.方法 用PCR法扩增大肠杆菌胞嘧啶脱氨酶基因CodA,经克隆和测序后插入植物基因表达载体pROK,并导入根癌农杆菌LBA4404(pAL4404)中.以共培养法转化青蒿叶盘,在添加25μg/mL卡那霉素(Kan)的N6培养基上选择青蒿转化愈伤组织并再生绿芽.将Kan抗性转基因青蒿芽转植到含有500μg/mL5-氟胞嘧啶(5-FC)和25μg/mLKan的MS培养基上继续培养2周.结果 转基因青蒿芽全部死亡,而未转化青蒿芽仍正常生长,表明导入CodA基因的青蒿细胞已赋予其预期的负选择表型.经RT-PCR检测,转基因青蒿芽显示CodA阳性扩增带,而未转化青蒿芽无此特异扩增带.这一结果显示,CodA基因已在青蒿细胞中转录生成相应的mRNA,从而进一步印证了表型检测结果.结论 CodA基因可作为有效的青蒿基因打靶负选择标记.

Objective To explore the feasibility of utilizing the cytosine deaminase A(CodA)gene as an effective negative selectable marker in Artemisia annua for gene targetingMethods The PCR procedure was employed to amplify CodA gene from Escherichia coli.After being cloned and sequenced,the gene was inserted into a plant expression vector,pROKⅡ,and then introduced into Agrobacterium tume-faciens LBA4404(pAL4404).The 1ear disks of A.annua were transformed by the CO-cultivation protoeol,after which the transformed calli were selected and green shoots of A.annua were regenerated on N6 medium supplemented with 25μg/mL Kanamycin(Kan).Then the Kan-resistant transgenic shoots were transplanted onto the MS medium containing 500μg/mL 5-fluocytosine(5-FC)plus 25μg/mL Kan and continuously cultured for up to two weeksResults The transgenic shoots have totally died while untransformed shoots still kept normal growth,indicating that A.annua cells introduced into the CodA gene had conferred an expected negative selection phenotype.When detected by RT-PCR,the transgenic shoots displayed a CodA-positive amplified band,but untransformed shoots gave no such CodA-specific amplified pattern.This result suggested that CodA gene had transcribed into corresponding mRNA in A.annua cells with furtherly verifying the result of phenotypic assayConclusion The CodA gene can be utilized as an effective negative selectable marker in A.annua for gene targeting.

国家自然科学基金新技术探索项目(30271591);国家中医药管理局基础研究项目(02-03ZP43);广东省自然科学基金重点项目(020799);健桥科研基金项目(JQ0205).