Cloning And Function Analysis Of Heat Shock Factor Binding Protein 1 From Cucumber

Cucumber (cucumis sativus L.) ,that is to say Hugua or Wanggua, originated from the North of India, the region of Kuman or Sikkim of Hymalays. It is a kind of cultispecies in melon, Cucurbitaceae. Cucumber prefer warm surroundings to cold ones, whose optimum temperature of growth and development is 25-30℃ and its growth is limited above 30℃, its growth is in stagnancy exceed 35℃, its breath is stop at 50℃ for 1 hour, its tissues will be dead at 60℃ for 5 or 6 minutes. Cucumber is of highly nutrition, various species, high output, long supplying stage and cultured widely in both open fields and greenhouse, so it is an important vegetable crop of the South and the North of our country. But high temperature stress has caused serious losses of cucumber production, and is particularly severe in the valley of the Changjiang River and its south areas. It is the basic ways to solve this problem to cultivate the excellent heat-resistant species; however, there is lack of heat-resistant varieties or inner-specific resources in cucumber. It is important on clarifying the molecular mechanism of (non) heat-resistant or heat shock reaction to heat-resistant research at the level of physiological regulation or molecule improvement.Heat shock factor-binding protein (HSBP) is a highly conserved and specific structure family of protein, which involves with the negative regulation of heat shock protein expression in vivo. The text has cloned CsHSBP 1 gene, analyzed gejiome copies via Southern hybridization, expression by RT-PCR and the putative amino acid sequence and CsHSBP 1 gene structure, which provided the foundation for further CsHSBP 1 gene functional studying in heat shock reaction from cucumber in the future.1 Cloning of CsHSBP 1 geneAfter transfecting the cDNA primary library of cucumber young fruit to the host cell XL-Blue, the mixture was poured onto LB plates containing appropriate antibiotic and incubated at 37℃ overnight to get bacteriophage plaque. Picking a single colony to inoculate degradation buffer, Aphage was released and transfected to host cell BM 25.8, sub-cloning was get after being incubated at 31℃ .Selecting some single plaques at random to detect positive colonies by colony PCR, plasmid was extracted and decided the size of plasmid and inserted fragment by plasmid PCR. Sequencing with 5'terminal of cDNA was submitted to company. After EST sequence analysis, HSBPl gene related to heat shock reaction was found, named CsHSBPX .According to the known sequence of CsHSBPX gene to design PCR primers, the target products was obtained by nested PCR via Pfu DNA polymerase, then the fragment was reclaimed, tailed "A" via Taq DNA polymerase , and linked to the pMD18-T vector by the method of T-A Cloning in end. After detecting positive colonies and inserted direction of the target gene by PCR amplification, the sub-cloning of CsHSBPX gene coding region was obtained, which was favor of function analysis of CsHSBPX gene by methods of genetic engineering in the future.2 Similarity sequences analysis of putative amino acid from CsHSBPX geneAfter the amino acid sequence can be deduced according to the known cDNA sequence, the sequence similarity among HSBPl homologues from various species was analyzed by BLASTp program. The result revealed the similarity is higher between CsHSBPl protein and HSBP from Arabidopsis thaliana in plants, which is up to 76%; but it drops to 30-34% between CsHSBPl protein and that of animal or microorganism.3 Structure analysis of CsHSBPl gene and putative protein3.1 Structure analysis of CsHSBPl geneUsing genome DNA and plasmid DNA containing full-length cDNA sequence as templates respectively in nested PCR, the size of CsHSBPl gene can be compared. The result of electrophoresis showed the size of target fragment was the same and was 340 bp. It was concluded there may not introns in CsHSBPX gene.The size of full-length cDNA of CsHSBPX gene was 535bp, containing 69 bp-length 5'non-translation region sequence, 270 bp-length coding region sequence and 196 bp-length 3'non-translation region sequence. In the sequence, there were digestion sites of restriction endonuclease such as Taq K Sau 3A, Alu I et al., lacking of those of EcoR V, Hind ILU BamH I et al.3.2 Advanced structure analysis of CsHSBPl proteinThe result of phylogenesis tree analyzing protein relatives in diverse species cleared CsHSBPl protein belonged to one of three large branches with Arabidopsis thaliana, Zea mays, Oryza sativa,et al., and proved CsHSBPl protein had closer relatives with HSBPl protein from plants.The result of secondary structure analysis of CsHSBPl protein by Coils 2.1 found the hydrophobic heptad repeat pattern lied in the region of 34 to 66 residues, the formation probability of coiled-coil structure is the largest at the site of 36 residue. The advanced structure and biochemistry characteristic prediction result through Vector 9. 0 and Anthewin V 5. 0 implied CsHSBP protein had the higher proportion of α -helix and no transmembrane region. The hydrophobic residues focused on relatively one profile of coiled-coil structure in tertiary structure, which could form the wide hydrophobic surface extanding.