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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%。
The mung bean defensin D1 gene sequence was amplified by PCR from the mung bean genomic DNA as a template, with 99.08% homology with the corresponding fragment of mung bean defensin gene in GenBank. The cloned fragment length was 355 bp. The seed-specific promoter sbeIIb and D1 genes were inserted into the expression vector pCAMBIA1301-BADH with salt tolerance gene to construct the seed-specific expression vector pSBEIIb-DB without antibiotic selection marker. The vector was constructed by using salt tolerant gene BADH instead of antibiotic gene As a screening marker, it eliminates potential threats to the environment and gut microbes. The vector was transformed into callus of two maize inbred lines Dan 598 and 988 by using ultrasound-assisted Agrobacterium tumefaciens method. After differentiated induction and emergence, PCR was performed to confirm that the transgenic plants were obtained with the highest relative conversion rate of 7.8% .