Weng Li-Xing, Deng Haihua, Xu Jin-Ling, Li Qi, Wang Lian-Hui, Jiang Zide, Zhang Hai Bao, Li Qiwei, Zhang Lian-Hui
Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673.
Pest Manag Sci. 2006 Feb;62(2):178-87. doi: 10.1002/ps.1144.
Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.
对五个优良甘蔗育种系进行了胚胎发生和植株再生效率的测试。它们都产生了可再生的胚性愈伤组织，但效率各不相同。为了培育具有强抗虫性的甘蔗，按照甘蔗密码子使用模式，将编码Cry1Ac杀虫δ-内毒素活性区域的截短cry1Ac基因的GC含量从原来的37.4%提高到47.5%。将合成的cry1Ac基因（s-cry1Ac）置于玉米泛素启动子的控制下，并通过微粒轰击法导入甘蔗品系YT79-177和ROC16的胚性愈伤组织中。Southern杂交分析表明，多个拷贝的s-cry1Ac已整合到转基因甘蔗品系的基因组中。免疫印迹分析鉴定出18个表达可检测水平s-Cry1Ac的转基因系，其表达量估计在1.8-10.0 ng mg(-1)总可溶性蛋白范围内。在叶片组织饲养试验和温室植株试验中，对4个转基因系和2个亲本系进行了甘蔗螟虫抗性测定。结果表明，未转化的对照系在叶片和茎中均受到严重损害，而表达高水平s-Cry1Ac蛋白的转基因甘蔗系对甘蔗螟虫的攻击具有高度抗性，接种后1周内接种的昆虫全部死亡。
