Studies on genetically engineered bacterium and its introduction into the rumen

本试验旨在通过重组DNA技术提高瘤胃细菌纤维降解活性，并在瘤胃中成功构建重组体。本研究对瘤胃细菌进行了转化和瘤胃内接种的尝试，结果如下：1）在9种质粒中，只有pRRI 207电转化了白瘤胃球菌。然而，结果不可重现。2)对溶纤丁酸弧菌的一个隐蔽质粒进行测序，确定其复制区，用于构建宿主溶纤丁酸弧菌的克隆载体。3)已经探索了合适的标记物以允许在瘤胃中容易地筛选瘤胃微生物。选择编码对至少3种抗生素（Rif、Str和Erm）的抗性的基因作为标记。4)建立了成功将pAMbeta 1接合转移到白葡萄球菌中的过滤器交配程序。通过自发突变将Rif和Str给予该接合子。5)通过瘘管将对3种抗生素具有抗性的转化接合子接种到山羊瘤胃中，并连续采集瘤胃样品，测定转化的白罗斯酵母在瘤胃中的水平。直到接种后37小时，才检测到瘤胃液中有10^3/ml的瘤胃液，但此后未检测到。可能的借口是死亡和流出的质粒，和/或损失的质粒从主机。要使工程菌在瘤胃内生态学建立，必须对工程菌的粘附能力进行有效调控，并在宿主体内建立更稳定的载体。

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.