Screening Of Proteins Interacting With ATP6 And The CDNA Cloning Of The Gene

Cytoplasmic male sterility (CMS), the transmitted failure to produce functional pollen, is a widespread trait in higher plants. Brassica napus is widely grown as the oilseed crop of rape or canola, its hybrids may be enhanced by as much as 60% above that of parental lines, so it has great value in applying Brassica CMS in the production of hybrid rapeseed. At present, investigations of CMS in Brassica napus have focused on three male sterile cytoplasms: ogu, nap, and pol. Both because the "Polima" or pol cytoplasm confers a relatively temperature stable male sterility and because of the availability of restorer genotypes. This system appears to be the most advantageous for hybrid rapeseed production.Studies have shown that as the result of DNA rearrangements in the pol mitoch-ondrial genome, the gene coding for subunit 6 of the mitochondrial ATPase, atp6, is preceded by and co-transcribed with a chimeric open reading frame(or f224). The atp 6 mitochondrial gene region has also been found to be associated with the Polima or pol CMS system of Brassica napus.The yeast two - hybrid system is a powerful technique for detecting in vivo protein - protein interactions and screening cDNA libraries in recent years. We used yeast two-hybrid to screen the proteins that may have interactions with atp6 and correlate with Brassica CMS.The atp 6 was ligated with the vector pGBKT7 toconstruct recombinant bait plasmid pGBKT7/atp6. We use the double strand cDNA and the vector pGADT7 - -Rec to transform the AH109 strain transformed with recombinant bait plasmid (pGBKT7/atp6). Positive clones were obtained by the selection of autotrophic ph-enotype andβ-galactosidase activity analysis. Then we extacted the yeast total DNA, amplified the cDNAs insert by PCR, digested the PCR products. Based on the PCR and restriction enzyme analysis, we classified the cDNA inserts, the representatives were transformed into E. coli JM109 and spreaded onto the LB+Amp plates. Then the pGADT7-Rec-cDNA was isolated and transformed AH109 strain transformed with recombinant bait plasmid to validate the interactions.Five positive clones were obtained。Sequencing and Gene bank blasting demonstrated that They are all important genes in the physiological processes of Brassica napus l., such as the genes encoding the a-subunit of Sec61 complex in ER membrane, the subunit of positive ion-translocation ATPase, the glucan 1, 3-βglucanase, hydrolase, the photic system 1L subunit.According to the sequence of the hydrolase gene, we designed the primers to clone the unknown sequence by rapid amplication of cDNA ends(RACE). Based on the sequence acquired by RACE, we designed the primers to clone the 5'-end sequence by Thermal Asymmetric Interlaced PCR (Tail PCR). According the the three sequence, we performed sequence assembly and designed primers: up Primer: 5'-TAGTTTAGGGATGCTGTCCAAAAGTGT-3', down Primer: 5'-CAGAAGAATAACCAATCAAAAAACCAT C-3'. The cDNA sequence was obtained.Based on the sequence of the amino acid, BLAST analysis against NCBI datab-ase was performed. The BLAST results indicate that putative conserved domain (G-lyco_hydro₁7) has been detected and the amino acid sequence have 84% identies with beta-1, 3-glucanase 5 [Arabidopsis thaliana]. We presumed it maybe a member of hydrolase family. Multiple biosoftwares are used for bioinformatics analysis. The comparisons indicate that the target have the following characters: (1) contains signal peptide sequence, the sequence from 1AA to 26AA is the signal peptide sequence; (2) at least two membrane-spanning domains.We designed primers: up-Primer 5'-ATAGAATTCATGCTGTCCAAAAGTGTTTTTGC, down Primer: 5'-CAGAAGAATAACCAATCAAAAAACCATC-3', amplified the fragment by using Pfu E. The PCR product was digested by EcoRâ… , then it was inserted into PSK vector that was digested by EcoRâ… and Smaâ… . Then the recombinant plasmid was digested by Xbaâ… and EcoRâ…¤, the digested fragment was inserted into pCAMBIA 2301G vector that was digested by Xbaâ… and Ecl136â…¡. The reverse plant expression vector was constructed successfully.