Study On The Feeding Value Of Rape Straw Increased By White Rot Fungi And Expression Of Laccase Gene

This experiment was aimed to study feed utilization method of rape straw. First,through the research of solid state fermentation of the rape straw by white-rot fungi,investigating the effects of white rot fungi on the quality of rape straw and ruminal degradation. Second, laccase gene of L. edodes was put into yeast expression to obtain the degradation ability to lignin by using molecular experimental techniques ?Recombinant yeast can be used for fermentation of rape straw or producing enzyme preparation used to ferment rape straw.Experiments I: This experiment was aimed to study on the effects of white rot fungi on chemical composition and enzyme activity of rape straw. In this experiment,Solid state fermentation of the rape straw was carried out for 10 days,20 days,30 days to determine changes in chemical composition and changes of the lignin degradation enzyme activity. The results showed that: After 30 days of fermentation, P.chrysosporium and L. edodes degradation of about 45% of lignin,55% of cellulose. C.subvermispora and P. acerina partially degraded rape straw lignin and cellulose(<30%).The chitin was improved significantly in rape straw incubated with fungi.Thus, P.chrysosporium and L. edodes can significantly improve the nutritional composition of rape straw.Experiments II: The experiment was aimed to study on the effects of in vivo rumen digestibility of rape straw incubated with white rot fungi.the effects of white rot fungi on in vitro rumen fermentation parameters of rape straw.This test using in vitro batch culture method tested the 30 days experimental group and the control group rape straw. Testing the effects of in vitro rumen fermentation parameters after fermentation 48 h. The results show that: The maximum of TVFA content(P = 0.020)and Acetic acid content(P <0.001)is P. chrysosporium. IVOMD of L. edodes and P. chrysosporium were similar, larger than the other groups.Thus, IVOMD of L. edodes and P. chrysosporium was improved,C.subvermispora and P. acerina rumen fermentation was suppressed.Experiments III: This experiment was aimed to study on the effects of in vivo rumendigestibility of rape straw incubated with P. chrysosporium,L. edodes and control groups.Tests Carried out on three head of jinjiang cattle with fistula using rumen nylon bag method. The results show that: The rapid degradation portion of NDF in L. edodes group rape straw increased relative to the control group(P =0.068). P. chrysosporium has the largest possible degradation part(P = 0.004) and the minium Kd(P = 0.004). Compared with the control group, non-biodegradable part of L. edodes and P. chrysosporium was respectively reduced by 7.9% and 12.7%(P <0.001). The residence time(Lag) of two treatment groups ares not affected. Effective digestion of L. edodes and P. Chrysosporium fermentation of rape straw NDF increased significantly(P <0.001).Experiments ?:This experiment was aimed to study the heterologous expression of lignin degrading enzymes of white rot fungi.In this experiment,the L. edodes laccase gene was expressed in K. lactis by molecular techniques.According to the reported of laccase gene(Lac1) sequence characteristics on NCBI,primers designed for l.edodes gene was amplified by PCR.Laccase gene was cloned into the expression vector pKLAC2 and electroporation to obtain recombinant pKLAC2/Lac cells.After testing, the recombinant K. lactis has laccase activity.