The Biological Function Of Amino-deoxy-chorismate Lyase In Arabidopsis Thaliana

Tetrahydrofolate (THF) and its derivatives-commonly grouped under the name of folates-are vital cofactors for enzymes that mediate one-carbon transfer reactions. Folates are involved in a wide range of key metabolic functions. The chemical structure of folate consists of three parts:pteridine, p-aminobenzoic acid (pABA) and glutamate. There are two branches in the plant folate biosynthesis pathway:pABA and pteridine branch, respectively. The Amino-deoxy-chorismate Lyase (ADCL) is in the pABA branch catalytic amino acid oxygen (ADC) aroma into pABA. In this study, an Atadcl mutant was used to investigate the biological function of amino-deoxy-chorismate lyase(AtADCL) by gene mutation, RNA interfering, over-expression and prokaryotic expression, and the results are highlighted as follows.1. AtADCL was expressed ubiquitously both in different development stages and in various tissues. We ordered the T-DNA insertion mutant SALK₁11981 from ABRC, named Atadcl. Homozygous mutant Atadcl was identified by PCR, and analyzed for transcriptional expression of AtADCL. When using the gene specific primers that span the T-DNA insertion site the transcripts could not be detected in the mutant; when using the gene specific primers that don't span the T-DNA insertion site the transcript abundance degree decreased significantly compared with the wild type. There was nearly no difference in visible phenotypes between wild type plants and Atadcl mutant plants during vegetable and reproductive growth. We calculated the rosette leaf number at different developmental stages, and found that the number of rosette leaves of mutant did not differ significantly from wild type. In the process of dark-induced senescence, the rates of senescing leaves were no significantly different between the mutant and wild type. When the mutant seedlings were grow on normal 1/2 MS and nitrogen limitations medium, the mutant grew better than wild type on both mediums. Moreover, RT-PCR analysis detected a higher expression of some of the genes involved in nitrogen metabolism in the mutant under the same growth conditions. By using the microbiological method, we found that the total folate in the mutant was 134.61%of wild type. In addition, HPLC-MS/MS analyses revealed that the tetrahydrofolate in the mutant was 179.26%of wild type, the 5-methylene-tetrahydrofolate was 125.18%of wild type, the 5-formylmethenyl-tetrahydrofolatet was 114.68%of wild type.2. Regarding the AtADCL transcript level, it was largely reduced in five independent AtADCL RNAi transgenic lines, and markably increased in four independent AtADCL overexpressors, as compared to the wild type.3. Using microbiological method to measure the total folate content of the mutant, RNAi and overexpression transgenic lines, we found that the total folate contents of the five RNAi transgenic lines were increased as much as 146.85%,172.15%,136.01%,126.12%and 132.03%, respectively, as compared to wild type, the total folate content of the four overexpression lines were increased as much as 184.27%,173.29%,136.76%and 144.76%, respectively, as compared to wild type.4. The AtADCL CDS was cloned into pET30a, and prokaryotic expression of the recombinant polypeptide was achieved by IPTG induced in Rossette (DE3) cells, followed by SDS-PAGE analysis.The results showed that the recombinant protein was expressed only in the precipitate as the form of inclusion bodies.To test whether AtADCL in Arabidopsis thaliana has enzymatic activity, soluble form of the recombinant protein has to be obtained. We cloned the CDS into vector pET-YdRb to make a fusion with a super acid sequences and carried out the induction with IPTG, followed by SDS-PAGE analysis.Consequently, the new version of recombinant protein was successfully expressed in the supernatant. In the following experiments, we will collect a large number of soluble recombinant proteins, to determine AtADCL enzymatic activity in vitro. Meanwhile, we were able to use prokaryotically expressed recombinant protein to generate AtADCL antibody which will be used to detect the expression of the ADCL in plant.The results above indicate that neither the T-DNA insertion mutation nor RNA interfering block the folate synthesis pathway in Arabidopsis, and surpsingly we found that the total folate contents both in mutant and in RNAi transgenic lines increased as compared to wild type.The speculations are:1) T-DNA insertion and RNA interfering did not silence the gene fuction completely which somewhat retain enzymatic activity; 2)when the gene AtADCL(At5G57850) mutated in plants, there must be other genes functioning and being responsible for mataining pABA synthesis normally(or even increasing pABA synthsis in turn), as pABA is a precursor for the synthsis of folate which is essencial for plant growth and development.