葡萄糖氧化酶基因转化小麦的研究

Transferring Glucose Oxidase Gene into Wheat by Biolistic Particle

葡萄糖氧化酶(glucoseoxidase,GO)在植物抵抗真菌和细菌感染时起重要作用,但将GO基因转入小麦的研究目前还未见报道。本研究利用基因枪介导法将来源于黑曲霉的GO基因转入优良小麦品种扬麦10号、鲁麦22和鲁麦23,从轰击的3300块幼胚愈伤组织中,获得了246棵再生植株,经PCR鉴定,其中的31棵含有GO基因,PCR阳性植株比例为0.94%。T1代经Southernblotting分析,证明GO基因已整合到小麦基因组中。T2代植株淀粉碘化钾染色结果证实GO基因已在蛋白质水平表达。对T2代植株进行了白粉病抗性鉴定,结果在34个转基因株系中有23个株系的病情指数低于相应的非转基因对照。T1代转基因植株经PCR分析,筛选到48株含有GO基因而不带有筛选标记基因(bar)的植株。

It is difficult to prevent and control wheat diseases only by traditional disease-resistant breeding technique. In such case, plant genetic engineering might provide a new approach to make use of disease-resistant resource from different species. Some foreign genes such as Glucose Oxidase(GO)plays a very important role in plant disease resistance caused by bacteria and fungus pathogens, while, no study has been reported on the transfer of GO gene into wheat. In addition, biosafty of transgenic crops has been strengthened in these years by farmers and scientists worldwide. Therefore, it will be very necessary to introduce GO gene isolated from Aspergillus nige into wheat and create new marker-free germplasm for its breeding. 13-14 days after pollination, wheat immature embryos were collected and pre-cultured on callus-induction medium for 7 days, and then these calli were bombarded by gold particles coated with mixed plasmids of pUbi-GO and pAHC20 at the ratio of 1∶1, in which GO gene and bar gene were contained respectively. The bombarded calli were selected on the medium with 3-5 mg/L bialaphos for several times. At last, 246 regeneration plantlets were obtained from 3300-bombarded calli, and the ratio of PCR positive plants to calli bombarded was 0.94%. By PCR analysis and southern blotting analysis, it was proved that GO gene was integrated into the host's chromosomes in T_1 plants(Fig.3, Fig.4). The result of starch-KI stainning in T_2 generation lines indicated that GO gene was expressed and showed enzyme activity. To identify GO gene's function in wheat, powdery mildew was artificially inoculated onto T_2 plants, the statistics result indicated that the disease index of some transgenic plants were lower than wild type plants, among which line 86, line 105 and line 113 were identified to be with available resistance to powdery mildew. At the same time, 48 plants with GO gene but without bar gene were screened in T_1 generation, this work paved the way of removing bar gene from transgenic plants for their environment release.