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Studies on genetically engineered bacterium and its introduction into the rumen

基因工程菌及其瘤胃导入研究

基本信息

批准号：
03454100
负责人：
HOSHINO Sadao
金额：
$ 4.03万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The present experiment aimed at enhancing fiber-degrading activity of rumen bacteria through a recombinant DNA technique and at successful establishment of the recombinant in the rumen. Attempts to transform rumen bacteria and to inoculate the transformant into the rumen have benn made and the results obtained are as follows : 1)Of 9 plasmids tested, only pRRI207 transformed Ruminococcus albus by electroporation. However, the results were not reproducible. 2)Sequencing of a cryptic plasmid from Butyrivibrio fibrisolvens allowed the replication region to be identified for construction of cloning vector for the host B.fibrisolvens. 3)Suitable markers have been explored to allow easy screening of transformant in the rumen. Genes coding for resistance to at least 3 antibiotics (Rif, Str and Erm) were chosen as the markers. 4)Filter mating procedure was established for a successful conjugal transfer of pAMbeta1 into R.albus. Rif and Str were given to this transconjugant through a spontaneous mutation. 5)The transconjugant resistant to 3 antibiotics was inoculated into goat rumen via a fistulae and ruminal sample was successively taken to determine ruminal level of the transformed R.albus. Untill 37hr after the inoculation the transformant was detected at a level of 10^3/ml of ruminal fluid, though none was found after then. Possible excuses are death and outflow of the transformant, and/or loss of the plasmid from the host. Positive manipulation of bacterial ability to adhere to feed particle as well as more stable vector within a host would be necessary for ecological establishment of engineered bacterium in the rumen.

本实验旨在通过重组DNA技术提高瘤胃细菌对纤维的降解能力，并在瘤胃内成功构建重组菌。将pRRI207转化瘤胃细菌并将其接种到瘤胃内，结果如下：1)在所检测的9个质粒中，只有pRRI207通过电穿孔转化了白色瘤胃球菌。然而，结果是不可重复性的。2)对分离自纤维丁状弧菌的隐匿质粒进行测序，确定其复制区域，为构建克隆载体奠定了基础。3)探索了合适的标记，以便于在瘤胃中筛选转化子。选择对至少3种抗生素(Rif、Str和Erm)耐药的基因作为标记。4)为成功地将pAMbeta1基因转入白色红曲霉建立了筛选交配程序。RIF和StR是通过自发突变给该接合子的。5)将对3种抗生素具有抗药性的接合子通过瘘管接种到山羊瘤胃内，连续采集瘤胃样品，测定转化后白头翁的瘤胃水平。直到接种后37小时，在瘤胃液中检测到10^3/ml的转化子，但此后没有检测到转化子。可能的借口是转化子死亡和外流，和/或从宿主中丢失了质粒。为了在瘤胃中建立工程菌的生态环境，需要积极操纵细菌附着在饲料颗粒上的能力以及宿主内更稳定的载体。