基因枪法介导转人工合成Rs-AFP2基因小麦的获得和检测

Transferring of An Antifungal Protein Gene(Rs-AFP2) of Radish into Wheat Mediated with Biolistic Particle

萝卜（Raphanus sativus）抗菌肽Rs—AFP2在体外强烈抑制小麦赤霉病菌（Fusarium graminenrum）、黄色镰孢菌（Fusarium culmorum）、烟草赤星病（Alternaria longipes）的菌丝生长。为了研究Rs-AFP2在小麦抗病育种上的应用潜力，人工合成了Rs-AFP2基因。通过基因重组技术（包括限制性内切酶酶切和连接），将人工合成的R以FP2基因替代单子叶高效组成型表达栽体pAHC25中的GUS基因，构建了R￡AFP2基因的单子叶高效表达栽体pUAFP2。该栽体除携带受Ubiquitin启动子控制的Rs-AFP2基因表达盒外，还具有1个受Ubiqutin启动子控制的Bar基因表达盒，后者可为后续利用除草剂Bialaphos筛选转化再生植株提供抗性；采用基因枪法轰击小麦品种扬麦12、济麦19、晋麦47和豫麦34幼胚共4042个，经过2～3次Bialaphos筛选，最终获得扬麦12再生植株316株；利用高效表达栽体pUAFP2的Bar和Rs-AFP2基因的特异引物对上述成活的转化植株进行PCR检测，获得Bar和Rs-AFP2基因均为阳性的植株58株，转化率为1．43％。

Antifungal protein-2 of radish （Raphanus sativus） （Rs-AFP2） can strongly suppress tne growth of Fusariurn grarninearum , Fusariurn Culrnorurn and Alternaria longipes etc. Based on this property of Rs-AFP2, we expect to develope a new wheat germplasm with resistance to some fungal diseases, especially Fusariurn grarninearurn throught transformating Rs-AFP2 gene into a wheat variety. Therefore an expression vector pUAFP2 containing the synthetic antifungal protein gene（Rs- AFP2） of radish and Bar gene was constructed,in which Rs-AFP2 and Bar gene was controlled by maize ubiquitin promoter that could constitutively express with more efficiency than 35S promoter in monocot plant. Then,4042 embryos of Yangmai 12, Jimai 19, Jinmai 47 and Yumai 34, used as the transformation receptor of plasmid pUAFP2, were bombarded by biolistic particle of BIO-RAD corporation. 316 regenerated plants with resistance to Bialaphos were achieved on the selection medium by Bialaphos. 58 transgenic plants with Bar and Rs-AFP2 genes were identified by PCR assay in the To generation with the transformation frequency of 1.43%.