Rice Apx Gene Cloning And Tobacco Genetic Transformation Research

Ascorbate peroxidase(APX,EC1.11.1.11), which was also called vitamin C peroxidase, was belonged to one type of terminal oxidase. It was mainly found in higher plants, eukaryotic algae, cyanobacteria and even some insects.APX was one of the key enzymes that scavenging H202, which was indispensable for keeping chloroplasts and other cell components away from the destroied of H202 and its hydroxyl radical。When the H202 accumulated in plants, H202 can rapidly induce the expression of APX, APX can catalyze the reaction of ascorbic acid and H202 , so H202 was decomposed. APX played an important role in anti-oxidative stress of plant.In higher plants, APX was a multi-gene family, and its isoenzyme located in four different regions: the stroma of chloroplast in the APX (sAPX), the thylakoid of chloroplast APX (tAPX), micropropagation APX (mbAPX) and cytoplasmic APX (cAPX). APX gene family of rice had three types including eight members, that were cytoplasm APX (APX1, 2); peroxisome APX (APX3, 4); chloroplast APX (APX5, 6,7,8). The research of cytoplasm APX (APX1, 2) was studied well,so the peroxisome APX (APX3, 4) as the main research object.In this research, the cDNA of japonica rice was used as a template, specific primers of APX3(AY382617),APX4(NM₀01068974was designed according to Genbank database. After PCR amplication of APX3, 4 gene; connection the target sequence with pMD18-T vector; transformation of E. coli DH5α; enzyme digestion identification as well as sequencing, the result showed that APX3 was 1038bp and APX4 was 997bp, which had the similarity of 99% with original sequence.Bioinformatics analysis results indicated that APX3 encoded the protein of 32.075KD, while APX4 encoded the protein of 31.738KD, the difference was only 0.337 KD. Isoelectric point of APX3 was 8.26, so APX3 was basic protein; isoelectric point of APX4 was 7.74 and APX4 was neutral protein. The hydrophobic average of APX3 was -0.371 and APX4 was -0.297,the two proteins are both hydrophilic. The instability coefficient of APX3 was 46.22, it was an unstable protein, and the instability coefficient of APX4 was 34.55, it was a stable protein. The difference of the protein's secondary structure of them was larger, only theα-helix contained 101, while the random coil andβ-helical spiral were more difference; but the difference of protein tertiary structure was only two hydrogen bonds and one angle. These results showed that although there was tiny difference between APX3 and APX4 gene as well as their protein, the APX gene family had the same function.In order to validate the function of APX gene, we constructed the expression vectors. According to the sequence of APX3, APX4 gene, forward and reverse primers with XbaⅠ,SacⅠrestriction site were designed. Full-length cDNA of APX3, APX4 with restriction sites were amplified, then the cDNA had been connected with expression vector pBI121 whose GUS gene was missing. The forward and reverse expression vector was called pBI121-APX3,pBI121-APX4,pBI121-AsAPX3,pBI121-AsAPX4 respectively. And then, the vectors were transformed into Agrobacterium tumefaciens EHA105.APX3 and APX4 were transformed into mode plant with leaf disc transformation respectively. According to the PCR result of the transgenic tobacco genomic DNA, which developed under the pressure of kanamycin selection, APX3 and APX4 had been transformed into tobacco genome successfully and the transformation efficiency was high. We had got 10 transformed plants of APX3 gene, 11 transformed plants of APX4 gene. The reverse expression vectors BI121-AsAPX3,pBI121-AsAPX4 were used for the further research of gene function in the future.