Construction Of Gene-Modified Lactic Acid Bacteria For Cellulytic Decomposition In Silage

Silage is that,in hermetic containers,fresh green material is fermented by plant surface of naturally occurring lactic acid bacteria. The result is soluble carbohydrates is transformed into organic acids, which leads to the lower pH. With inhibiting the growing of harmful micro-organisms, the nutritional characteristics of crops can be maintained. Compared with other types of feed, silage has unique advantages. First of all, protein, soluble carbohydrates, small molecules, vitamin and mineral of raw materials are maintained, and absorption and utilization of livestock are promoted. Silage improves the digestibility, but also increases productivity. The second one, during the activities of beneficial micro-organisms, the products such as the lactic acid produced by lactic acid bacteria, can kill a lot of disease micro-organism, parasite eggs, etc. Adding some gene-modified lactic acid bacteria as additive agents furtherly degradate the cellulose component of raw materials into small molecules of simple sugars, oligosaccharides, or oligosaccharides, at the same time the specific engineering bacteria increases productivity.In this experiment, the model E. coli strains and lactic acid bacteria strains NZ9000 were used to express exogenous cellulase by transfected CBHâ…¡g ene of Trichoderma reesei. The application of REAL-TIME RT-PCR was used to analysis the transcription of foreign gene in the engineered lactic acid bacteria. Then, method of pNP is devoted to measure the enzyme activity of engineering bacteria. The experiment provided a technical route to construct gene-modified lactic acid bacteria for silage, and with methods providing by this project, associated indicators can be evaluated properly.Through the experiment we owned the following results: the cellulose of CBHâ…¡gene and expression vector pET30a constructed the recombinant plasmid pETCBH. The recombinant plasmid was transformed into Rosetta host bacteria obtained recombinant strain Rosetta (pETCBH).IPTG induced recombinant strain Rosetta (pETCBH), and through determination of the restructuring pNPC cellulase CBHâ…¡activity, its activity was 2.677U/mL. In the experiment, cellulose of CBHâ…¡gene and expression vector pNZ8148 constructed the recombinant plasmid pNZCBH. The recombinant plasmid was transformed into NZ9000 host bacteria obtained recombinant strain NZ9000 (pNZCBH). Also, the project established the method of REAL-TIME RT-PCR based on DNA subtraction for absolute quantification of gene expression in engineered lactic acid bacteria. Method with good parallelism, high reliability can be analyzed target gene expression at the transcriptional level. This method is in theory to solve the traditional quantitative method without an accurate result. The comparison of two methods to stain Polyacrylamide gel confirmes that the effect of silver nitrate staining method is much better than the traditional coomassie brilliant blue.The innovative points of this test are as follows:1. A more accurate way to detect the absolute volume of transcription and the expression of foreign genes in prokaryotic absolute volume was established.2. The project provided a method of recombinant lactic acid bacteria strain, which can draw the draft of the technical route.