Cloning And Functional Analysis Of Malus Xiaojinensis Citrate Synthase Gene MxCS2

In the present study, the RNA of Malus xiaojinensis Cheng et Jiang was extracted by Trizol method, using the degenerate primer designed from the conserved sequence of the known CS genes. Single stranded c DNA was synthesized by reverse transcriptase, then the CS gene fragment was obtained using the way of PCR. Different from the gene MxCS1 which had been cloned before, the new one was named MxCS2. MxCS2 gene has a open reading frame with length of 891 bp according to monoclonal sequencing. The gene was predicted that encoding a protein composed of 296 amino acids by the software of DNAMAN5.2. The new gene has a predicted molecular weight of 33.2k Da and theoretical isoelectric point 7.79. Analyzing its three-dimensional structure, there are three ?-helixs and three random coils in the functional domain of the protein. The result of subcellular localization revealed that the MxCS2 protein mostly exists on the plasma membrane. The phylogenetic tree was established by the similarity analysis of MxCS2's amino acids and nucleotides and other species, using the blast function of the NCBI w ebsite. MxCS2, MxCS1 and Pp CS1 were clustered together. At CS4, Dc CS and Nt CS were grouped into another cluster. Os CS was alone for the third branch.The RT-PCR analysis showed that when the normal Fe concentration supplied, the MxCS2 gene expressed richly in leaves, roots and phloems, but hardly expressed in xylems. This indicated that the MxCS2 gene plays a role in the active organs. When provided with low iron and IAA, the MxCS2 gene expression of the new leaves, phloems and roots had a uptrend with the t reat time, with maximum of 12 hours, but decreased slightly at 24 hour. When provided with high iron concentration, the expression level of MxCS2 in these parts decreased. However the gene in main leaves was opposite to the above parts under Fe stress, and had the same trend when treated with IAA. When treated with ABA, the expression of MxCS2 gene wasn't significantly affected.When operating the qualification test of the transgenic Arabidopsis T 3 generation, it showed that in the occasion of supplying normal iron concentration, the transgenic plants and wild type plants have normal phenotype. When supplying by low iron concentration, the wild type plants put up more obvious aetiolation than the transgenic plants. The transgenic plants and wild type plants have a similar phenotype to the low iron circumstance, when treated with high iron concentration. The result showed that the transgenic Arabidopsis has a better iron stress resistance ability in the case of iron deficiency or excess, compared with the wild type. By testing the relevant physical signs of the transgenic Arabidopsis, the follow results were listed: in the circumstances of iron excess or deficiency, the transgenic Arabidopsis' s fresh weight respectively was 1.9 or 2.1 times that of wild type, the root length was 1.4 or 1.9 times that of wild type. When treated with low and high iron concentration, wild type plants have more aetiolation than transgenic plants, that in accord with the chlorophyll content. The transgenic Arabidopsis' s CS activity was 3.1 or 3.8 times that of wild type in high and low iron, and the citric acid content was 3.1 or 3.1 times in high and low iron, and 2.2 times in normal iron. While the iron content was 1.3, 1.1 or 1.8 times that of wild type in high, normal, and low iron. In brief, the transgenic Arabidopsis had high fresh weight, root length, CS activity, citric acid content, chlorophyll content and iron content, compared with the wild type, especially under the low iron concentration.