植物防御素D1基因种子特异表达载体构建及在玉米中的转化

以绿豆基因组DNA为模板，通过PCR技术扩增了绿豆防御素D1基因序列，与GenBank中绿豆防御素基因的相应片段有99.08%同源性，克隆片段长为355bp。将种子特异启动子sbeIIb和D1基因插入到带有耐盐基因的表达载体pCAMBIA1301-BADH中，构建了无抗生素选择标记的种子特异表达载体pSBEIIb-D-B，该载体是利用耐盐基因BADH替代抗生素基因作为筛选标记，消除了对环境及肠道微生物的潜在威胁。将该载体利用超声波辅助农杆菌介导法转化了2个玉米自交系丹598、988的愈伤组织，经分化诱导出苗后进行PCR检测，证明得到转基因阳性植株，相对转化率最高达到7.8%。

Constructions of Seed-specific Expression Vectors for D1 Gene and the Genetic Transformation in Maize

Defensin as a main antibiotic peptides had broad-spectrum bactericidal activity.A fragment of 355bp defensin D1 gene was isolated by PCR technique from genetic DNA of Vigna radiata.The sequence of D1 gene which cloned in this experiment shared 99.08% identity with the one that announced in the Genbank.By inserting sbeIIb gene and D1 gene into pCAMBIA1301-BADH with salt-tolerance BADH gene,the author constructed an expression vector pSBEIIb-D-B,which possessed disease-cum-salt resistant,antibiotic free selective and seed-specificity characteristics.This expression vector without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes.Two new varieties of maize inbred lines were transformed by Sonication-assisted Agrobacterium tumefaciens-mediated method.The differentiated transformed calli were examined by PCR,which indicated that the genes had been transferred into maize successfully.The ratio of relative transformation reached 7.8%.