PRODUCTION OF HIGH-DIGESTIBLE,BROWN MIDRIB TYPE FORAGE

为了生产高消化率的、木质素含量较低的棕色中脊型牧草，我们进行了以下试验：1)鸟脚三叶草(莲花)将含有卡那霉素抗性基因(NTP II)和β-葡萄糖苷酸酶基因(GUS)的质粒载体pBI121转化根癌农杆菌。2)组织学GUS分析表明，有7.0%的抗性植株在不同组织中也表达了GUS基因。3)利用聚合酶链式反应(PCR)从阿拉伯木中分离到肉桂醇脱氢酶(CAD)基因，将该基因的1.2kb插入到pGAH/SL-GUS T-DNA区35S启动子和NOS末端基因之间的反义序列中，构建了pGAH/anti-UCAD载体，并将其导入鸟足三叶草中。通过抗卡那霉素和潮霉素磷酸转移酶筛选转基因植株。4)经PCR检测，反义基因在转基因植株中稳定整合。转基因植株的CAD活性显著低于对照植株，但转基因植株和对照植株的木质素和醛含量以及瘤胃微生物的体外消化率没有显著差异。

In order to produce the high-digestible, as it were, the brown midrib type forage contained less lignin, we carried out the experiments as follows ;1) Bird's-foot trefoil (Lotus corniculatus L.) are transformed with Agrobacterium tumefaciens carrying the plasmid vector pBI121 contained a kanamycin resistant gene (NTP II) and beta-glucuronidase gene (GUS). Resistant plants were regenerated at high frequency from green calli developed on the inoculated hypocotyl cuttings under the kanamycin selection.2) The histological GUS assay showed that 7.0% of the resistant shoots also expressed the GUS gene in a variety tissues. The stable integration of this gene was confirmed by polymerase chain reaction (PCR) analysis.3) A partial gene encoding CAD (Cinnamyl Alcohol Dehydrogenase) was isolated from Aralia cordata using PCR.An 1.2-kb portion of the sequence of the CAD gene obtained by PCR was inserted into an antisense orientation between the 35S promoter and the NOS terminater gene in T-DNA region of pGAH/SL-GUS.This constructed plasmid pGAH/anti-UCAD was introduced into bird's foot trefoil. The transgenic plants were selected by means of the resistance to kanamycin and hygromycin phosphotransferase.4) The stable integration of the antisense gene was confirmed by PCR in the transgenic plants. The CAD activity of the transgenic plants was lower significantly than that of the control plants, but the lignin and the aldehyde contents and the in vitro digestibility by rumen mincroorganisms were not significantly different between the transgenic and control plants.