National Institute for Genomics and Advanced Biotechnology, NARC, Islamabad, Pakistan.
Mol Biotechnol. 2012 Feb;50(2):129-36. doi: 10.1007/s12033-011-9426-2.
A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T(0)) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T(0)) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.
通过农杆菌介导法，将增强型 CaMV35S 启动子控制下的水稻几丁质酶-3 基因导入花生（Arachis hypogaea L.）。使用携带含有几丁质酶（chit）和潮霉素抗性（hpt）基因的二元载体（pB1333-EN4-RCG3）的根癌农杆菌菌株 LB4404。将含有目的基因的农杆菌侵染花生外植体，共培养后再生芽，筛选并培养在含有 5mg/L BAP、1mg/L 激动素和 30mg/L 潮霉素的 MS 培养基上。伸长的芽被检测到整合的水稻几丁质酶基因和潮霉素基因的存在，作为选择标记。通过 Southern 杂交分析基因组 DNA，确认了外源基因在拟转化植株（T(0))的核基因组中的整合模式。体外再生植株的成活率超过 60%，而通过农杆菌介导的水稻几丁质酶基因转移，产生了健康的、具有 42%以上转化率的拟转化（T(0))植株，所有植株均正常开花和结实。T1 植株通过接种小孢子来检测对花生褐斑病的抗性。转基因株系表现出比对照（非转基因植株）更高的抗性。与易感对照相比，高度抗性的转基因株系中的几丁质酶基因表达。观察到几丁质酶活性与真菌病原体抗性之间存在良好的相关性。
