Cloning Of Phenylalanine Ammonia-lyase From Caragana Korshinkii Kom, Construction Of RNAi Vector And Anaylysis Of Its Function

Caragana Korshinskii Kom. is a kind of good forage, has many useful functions such as enduring of drought , coldness and hotness etc. Beacuse its abundant rough protein and rough cellulose , it is the most important forage and paper pulping plants in the Three North areas . But it has shortcomings .Its lignin content is high, and the high content of lignin may affect the digestion of animals and add the cost of paper pulping. So how to reduce the lignin content and how to manipulate the lignin biosynthesis in Caragana Korshinskii Kom.is so important .Therefore , the research of manipulation the lignin biosynthesis in Caragana Korshinskii Kom.is more significant .Using Caragana Korshinskii Kom.as our experimental plants ,we do some reasearches about the first enzyme PAL in the lignin biosynthesis: the separation of PAL , purification of the enzyme , the enzyme activity in the different space-time , the expression of coded gene in different space-time .cloning part of pal gene , constructing the RNAi vector and transforming the model tobacco plants , at last got the transgenic plants and validating the function of transgenic tobacco plants .By using of biochemistry techniques ,we determined the enzyme PAL activity and specific activity and got the results that the PAL activity and specific activity in leaves and stems reached the highest at rapid growth stage,but in roots the PAL activity and specific activity reached the highest at really leave stage differently.With the help of ion exchange chromatograph DEAE-52 , gel filtration chromatography Sephadex G-200 and SDS-PAGE electrophoresis, we obtained the different constitutes and molecular weight of PAL enzyme ,one molecular weight is 306KD made up of 74KD and 82KD, and the other molecular weight is 222KD made up of 74KD ,respectively.According to all the conservertive sequenes of pal genes published in NCBI , we designed a pair of degenerate primers . With CTAB method , we extracted the DNA of Caragana Korshinskii Kom. genomic DNA in young stage ,used PCR technique , got the amplified fragment and cloned the PCR fragment by use of T Easy vector .By sequence analysis ,we know that ,this amplified fragment has the similiriaty of 90% in the DNA level with many other plants' pal gene published in NCBI. So we conclude that the amplified fragment is part of Caragana Korshinskii Kom.pal gene .By using of TRIZOL method ,we extracted the RNA of Caragana Korshinskii Kom. in different growth stage ,and did the northern blot with the help of dig high primers .So we know that the pal gene expression ascends with the growth of Caragana Korshinskii Kom. plant itself .On the base of DNA sequence , we designed a pair of specific primers to amplify pal gene . Using the high quality of RNA in really leaf stage .By RT-PCR , TA cloning , we got the amplified fragment . By comparasion , the fragment we got by RT-PCR shares the similiriaty of 90.78% ,92.9%85.11% and 89.72%. in Trifolium subterran* M.Sative* Manihot esculenta ^0 Phaseolus vulgaris respectively. And the differences in the DNA sequence and RNA sequence is only five bases .With the use of amplified fragment pal , pBluescript SKII , pHellsgate8 and pCAMBIA2300 , we constructed the final RNAi vector named palRNAiq.By using of agarobacterium-mediated transformation , we transformed palRNAiq into model plant tobacco, and got transgenic tobacco plants . Using different probes , we did PCR -southern blot , genomic DNA southern blot , and single enzyme digested genomic DNA southern blot .identified that the palRNAiq sequences has already been transformed into tobacco plants .With the help of dig markered pal probe and 18SrRNA probe , we did northern blot of transgenic tobacco plants , and got the conclusion: in the different transgenic tobacco plants , the pal gene expression has been restrained ,but the restrainction degree is different, ranging from 37% —67% respectively .