PRODUCTION OF HIGH-DIGESTIBLE,BROWN MIDRIB TYPE FORAGE

高消化性棕中脉型饲料的生产

• 批准号: 07660361
• 负责人: KAWAMURA Osamu
• 金额: $ 0.32万
• 依托单位: MIYAZAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660361/
• 关键词: FORAGE; DIGESTIBILITY; LIGNIN; TRANSFORMATION; AGROBACTERIUM; ANTISENSE; CINNAMYL ALCOHOL DEHYDROGENASE; BIRD'S-FOOT TREFOIL

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.

为了生产高消化率、木质素含量较低的棕色中脉型饲料，我们进行了如下实验：1)用携带含有卡那霉素抗性基因(NTP II)的质粒载体pBI121的根癌农杆菌转化鸟足三叶草(Lotus corniculatus L.)， β-葡萄糖醛酸酶基因（GUS）。在卡那霉素选择下，接种的下胚轴插条上发育出的绿色愈伤组织高频率地再生出抗性植株。2)组织学GUS检测表明，7.0%的抗性芽在多种组织中也表达GUS基因。聚合酶链式反应(PCR)分析证实该基因的稳定整合。3)通过PCR从龙牙楤木中分离出编码CAD(肉桂醇脱氢酶)的部分基因。将PCR获得的CAD基因序列的1.2-kb部分以反义方向插入到T-DNA区的35S启动子和NOS终止子基因之间。 将构建的质粒pGAH/anti-UCAD导入鸟足三叶中。通过对卡那霉素和潮霉素磷酸转移酶的抗性筛选转基因植株。4)PCR证实反义基因在转基因植株中稳定整合。转基因植物的CAD活性显着低于对照植物，但木质素和醛的含量以及瘤胃微生物的体外消化率在转基因植物和对照植物之间没有显着差异。

项目成果

RYO AKASHI et al.: "High-frequency embryogenesis from cotyledons of bird's-foot trefoil (Lotus coniculatus) and its effective utilization in Agrobacterium tumefaciens-mediated transformation." (in press). (1997)

RYO AKASHI 等人：“鸟足三叶草（Lotus coniculatus）子叶的高频胚胎发生及其在农杆菌介导的转化中的有效利用。”

明石良・内山徹哉・坂本篤史・川村修: "遺伝子工学的手法を用いた飼料作物の品質向上に関する研究 1.アグロバクテリウムを用いたバーズフットトレフォイルの形質転換" 日本草地学会誌. (発表予定).

Ryo Akashi、Tetsuya Uchiyama、Atsushi Sakamoto、Osamu Kawamura：“利用基因工程方法改善饲料作物品质的研究1.利用农杆菌转化鸟足三叶草”日本草原学会杂志（已安排演示）。

R.Akashi et al.: "High-frequency embrogenesis from cotyledons of bird's-foot trefoil and its effective utilization in Agrobacterium tumefaciens-mediated transformation" J.Plant Physiol.(in press). (1997)

R.Akashi 等人：“鸟足三叶草子叶的高频胚胎形成及其在根癌农杆菌介导转化中的有效利用”J.Plant Physiol.（出版中）。

R.Akashi,T.Uchiyama,A.Sakamoto,O.Kawamura and: "High-frequency embrogenesis from cotyledons of bird's foot trefoil and its effective utiligation in Agrobacterium tumefaciens-mediated transformation" J.Plant Physiol.(Accepted).

R.Akashi、T.Uchiyama、A.Sakamoto、O.Kawamura 和：“鸟足三叶草子叶的高频胚胎发生及其在农杆菌介导转化中的有效应用”J.Plant Physiol.（已接受）。