Effects Of Veillonella Parvula And Its Gene Deletion Engineering Bacteria On The Ruminal Microbial Fermentation

A strictly anaerobic bacterium which was capable of producing propionate was separated from ruminal fluid of health dairy cow ,named V1.Its biochemical characteristic was analyzed.According to the morphorical and biochemical characteristic ,V1 was preliminary identified Veillonella Parvula.The 16S ribosomal RNA primer was designed on the basis of the sequence of GenBank,and 16S ribosomal RNA gene was cloned.The result showed that the obtained sequence was 98% identical with those of Veillonella Parvula in GenBank.The sequence was submitted to the GenBank,the number is DQ394709.Based on the above mentioned results,it was confirmed as Veillonella Parvula.Transposon tagging method was applied to construct the engineered strain Veillonella Parvula V1. The twelve mutants were screened from the numerous transconjugants with selective culture medium containing kanamycin and fluoroacetic acid. The 16S ribosomal RNA gene and Tn5 transposon gene of the strains TnV1 were amplified by polymerase chain reaction (PCR) and sequenced. 16S ribosomal RNA and Tn5 sequence which was obtained by PCR indicated that the transposon had inserted in chromosomal DNA of Veillonella Parvula V1. It was proved that the twelve mutants (TnV1) were PTA gene deletants by measuring the specific activity of acetate kinase(AK) and phosphotransacetylase (PTA).Primary substrates (lactic acid and pyruvate) were cocultured with V1 and TnV1 for 48h ,respectively ,in order to observe the fermentation of ruminal fluid in vitro.Culture fluid was sampled for analysis of pH and VFA. The results were as follows:the lactate in the culture fluid was utilized by the strains of V1 and TnV1. Compared to strains V1, the ratio of acetate to propionate were obviously reduced in the culture fluid of the strain TnV1.The concentration of lactic acid was reduced by the inculating V1 and TnV1 into rumen of sheep.Compared to the V1,the ration of acetinate to propionate was obviously lower than that of TnV1. The effect demonstrating that the transponed engineering bacteria TnV1 belonged to a deleted strain of acetic acid-producing gene.To explain the transposition mechanism,the geneome library of Veillonella parvula was constructed,and was tried to clone the ACK or PTA gene from the library.The analysis of recombinant plasmid showed that the constructed genome library which contained the 3-4kb fragment ,the titer of the library was 105pfu/ml,and 99% genome was included in the library according to the formula N=ln(1-p)/( 1-f ), proving that the genome library was constructed successfully.The situ-hybridization and ACK probe labeled with digoxin were employed to screen the library,but only 1200bp fragment of ACK was obtained.Sequence analysis showed that the Tn5 transpon was not inserted into the ACK gene and could be inserted into the PTA gene presumedly.But now,we continue to clone the total length of PTA to understand the mechanism of the impact of Tn5 on the Veillonella parvula.