Cloning Study Of Glucose-6-Phosphate Dehydrogenase Gene From Cucumis Hystrix Chakr

A valuable wild cucumis germplasm resource, Cucumis hystrix Chakr.(2n=24)(Cucurbitaceae) was the unique species which was able to interspecific hybridize with the cultivated cucumber(Cucumis sativus L.) successfully in cucumis. C. hystrix had a lot of important agronomic traits, such as tolerance to low temperature and low light, high resistance to downy mildew, gummy stem blight, root-knot nematode, that cultivated cucumber were lack of. Based on the successfully interspecific hybridization between C. hystrix and C. sativus, to fine-map and map-based clone the excellent genes/QTLs resistant to downy mildew, gummy stem blight, root-knot nematode in C. hystrix, we constructed C. hystrix genomic BAC library and stored in96-well cell plates.The oxidative pentose phosphate pathway (OPPP) was an important metabolic pathway to regulate the normal growth and improve the stress-resistance responding to different stresses in environment. Glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49) was a key rate-limiting enzyme that catalyzed the first step in OPPP to generate NADPH. In consideration of the importance of G6PDH in OPPP, we planned to clone and regulate the G6PDH gene by the genetic engineering method to promote the ability to respond to stresses and improve the stress-resistance in environment. So, we selected C. hystrix and its BAC libraries as materials to investigate, the results were as follows:1. Construction of the PCR Screening System of C. hystrix BAC LibraryIn this experiment, C. hystrix BAC library clones were picked and stored in96-well plates.457plates of the library were inoculated to culture in96-well deep plates. After the pooling of the Superpools and second pools, we extracted BAC plasmid DNA of the pools to establish the PCR screening system. The system consisted of46Superpools and457second pools. Each Superpool contained960BAC clones (ten96-well plates) and second pool BAC clones (one96-well plate). BAC plasmid DNA were extracted from Superpools and second pools and got approximately50-100ng·μL-1200μL and100μL, respectively. According to15μL PCR reaction system, plenty of the DNA sample for PCR reaction could be carried out at least1000-2000times, which was sufficient for a large number of BAC library screenings.2. Conserved Sequence Cloning of C. hystrix G6PDH gene mRNA and BAC Library ScreeningBased on the conserved sequences of G6PDH amino acids reported in GenBank, the degenerate primers were designed to isolate intermediate fragment of the G6PDH gene from C. hystrix. Conserved sequence of the G6PDH gene in C. hystrix was gotten by RT-PCR. It was807bp. High homology with other plants was indicated by online NCBI Blast and this result showed that this fragment was partial of C. hystrix G6PDH gene. Based on the conserved sequence specific primers were designed to screen the BAC library screening system. We got two positive clones,173-8F and237-1F.3. Analysis of Cytoplasmic G6PDH Gene sequence and Promoter StructureThe positive clone173-8F was sequenced. After splicing, we got full-length G6PDH gene sequence and its upstream regulatory sequence. The sequence was submitted to GenBank, the accession number:JQ771576. Sequence analysis indicated that total length of G6PDH gene was6.5kb and it consisted of15exons and14introns. The introns agreed with the structure5’-gt-ag-3’. The open reading frame (ORF) of G6PDH gene after splicing was1551bp and encoded a protein containing516amino acid residues. The identities of nucleotides of ORF and amino acids sequence sharing with cucumber reported on the web of cucumber genome database were98.71%and99.03%, respectively. We confirmed that the protein G6PDH was cytoplasmic because of the absence of N-end transferring peptide. Compared with tobacco, potato, parsley, kiwi fruit, arabidopsis, soybean, wheat, corn, grape, poplar et al, the cytoplasmic G6PDHs showed high a high similarity more than77%. Phylogenetic analysis showed that cytoplasmic G6PDH clustered with Solanaceae family firstly and the molecular evolution of cytoplasmic G6PDH was corresponding to the evolution of species. Analysis of the promoter sequence indicated that the basic elements TATA-box and CAAT-box of promoter were found. And moreover, there were abundant light responding elements, different elements involved in abiotic stress responses, and two5UTR Py-rich repeat elements increasing high transcription, CAT-box activating higher expression.