Cloning Of S6PDH CDNA From Apple [Malus Pumila Mill.] And Construction Of Its Plant Expression Vector

Sorbitol is the major photosynthetic product,translocating sugar alcohol and storage carbohydrate in Rosaceae plants, the role sorbitol played is like sucrose in other plants. As low-molecular-weight osmotic compounds, it can adjust infiltrative level, so it's correlated with resistance. In addition, fruit flavor is most dependent on sugar types transformed from sorbitol. Enzyme plays the most important role in sorbitol metabolism. S6PDH is the key enzyme for the biosynthesis of sorbitol, which catalyzed the conversion between glucose-6-phosphate and sorbitol-6-phosphate. So lots of work has done by many researchers, and gain large progress. The aim of the study is to lay foundations for transferring the gene encoding S6PDH, by means of cloning and sequence analysis of S6PDH gene and constructing of the plant expression vector. At first, the three methods were adopted to extract RNA from apple leaves. The results showed that three methods were all suitable for RNA extraction. The first method is the simplest one. Second,the factor that affect RT-PCR were studied and highly efficient RT-PCR detection system was established and optimized. As for the 20μl RT system: dNTPs(2mmol/L)2.5μl; 5×Buffer4μl; RNasin(50U/μl)1μl; Oligo(dT)(10μmol/L)0.5μl; template RNA4μl; Mo-MLV Rtase(200U/μl)1μl.. As for the 25μl PCR system: dNTPs(2mmol/L)5μl; 10×Buffer2.5μl; MgCl2(25mmol/L)2.4μl; LD03(10μmol/L)2μl; LD04(10μmol/L)2μl; Taq DNA ploymerase(5U/μl)0.3μl; cDNA 4μl. Third, the special fragment of RT-PCR was used to cloning and sequence analysis. The cDNA of S6PDH cloned from apple leaves, consisted of 930bp,encoding a protein of 310 amino acids. Obtained sequence was compared with that of Kanayama's research, there were 4 different bases in 930 nucleotides. There was only one different amino acid due to code degeneracy. Simultaneity, restriction sites analysis was done. Finally, the plant expression vector pBIS was constructed containing complete S6PDH cDNA via pUCm-T and pGEm-7Zf,and combinant was transferred into agrobacterium tumefacien by direct DNA transfer and the transformant was identified with PCR.