Study On The Cloning And Expression Of Cucumber Lipid Hydroperoxide Lyase Gene(Cs HPLs)

Hydroperoxide lyase(HPL) combined with hemoglobin is a kind of membrane proteins that can cleave hydroperoxides of polyunsaturated fatty acids(HPOT or HPOD) into short chain of volatile aldehydes and ω-oxoacids. The products of HPL are important constituents of volatile aldehydes. Firstly we cloned CsHPLs in the materials of two cucumbers of NO. 26 and NO. 14 which had quite different aroma components by PCR amplification technology, secondly analyzed CsHPLs expression pattern in the different developmental processes of both cucumber fruits and different treatments of both cucumber seedings, finally successfully constructed 9-CsHPL prokaryotic expression vector and induced 9-CsHPL protein expression. The study preliminary discussed the role and function of CsHPLs which would provide a theoretical basis for the formation of aldehydes in molecular mechanism. In addition the lipoxygenase propadiene(allene oxide synthase, AOS) that had the same subfamily with HPL was also successfully cloned and analyzed in both cucumbers. The main conclusions of this test as follows:1. Cloned CsHPLs gene from cucumber CsHPL gene primers were designed according to released cucurbitaceae figures and cloned using cucumber germplasm material of NO. 26 and NO. 14, two CsHPLs homology were 99.9% in two cucumber germplasms through DNA man multiple sequence aligning. Sequence analysis showed that, the CDS region of 9-CsHPL was 1476 bp, 13-CsHPL was 1478 bp; Bioinformatics analysis showed that two CsHPLs encoding amino acids were 478 and 481 respectively, molecular weight was 54.34 kD, and 54.15 kD, CsHPLs had four typical family characteristics of Cyt P450 domain, belonging to 9-HPL and 13-HPL. Cs AOS gene CDS region was 1599 bp, relative molecular mass was 60.00 kD, belong to Cyt P450 family.2. CsHPLs gene expression patterns in different tissues of NO. 26 cucumber 9-CsHPL expression was lower in the root, stem, leaves than tendrils, the amount of gene expression in the tendrils was four times than the leaves; 13-CsHPL had the highest expression in the stem, followed by tendrils, the lowest expression was in the roots and leaves; 913-HPL expression successively increased in roots, stems, leaves and tendrils; CsAOS expression had little difference with 9-CsHPL in roots and stems, the lowest expression in leaves. In different leaf the expression of 9-CsHPL, 13-CsHPL and CsAOS were old leaves> leaves> functional leaves, 913-HPL was leaf> old leaves> leaves.3. CsHPLs gene expression patterns in different cucumber seedlings 9-CsHPL expression increased after damage, MeJA, SA and gray mold processing in NO. 26, but had little changes when NO. 14 subjected to gray mold and SA treatment; 13-CsHPL were significantly increased after damage, MeJA and SA treatment in two cucumber seedling, after infection by the botrytis cinerea, 13-CsHPL gene expression increased in NO. 26, had little change in NO. 14; 913-HPL were increased when two cucumber seedlings subjected to the same treatment. In addition CsAOS expression significantly increased in 26 cucumber seedling after SA and gray mold treatment, CsAOS expression had no significant changes after injury and MeJA treatment in NO. 26, In addition to SA, CsAOS gene expression decreased in NO. 14 seedlings, in the other treatment its expression levels increased.4. CsHPLs gene expression patterns in NO. 26 cucumber seedlings The genes of 9-CsHPL expression increased after treatment of drought, MV and H2O2 in NO. 26 cucumber seedlings, but 9-CsHPL gene expression did not change significantly after the NaCl treatment; 13-CsHPL expression levels increased in the same treatment of NO. 26, 913-HPL had no changes in drought, in the other treatment it increased. CsAOS gene expression increased under the same treament.5. CsHPLs gene expression pattern in fruit development process Quantitative and semi-quantitative analysis showed that, 9-CsHPL gene expression was high in the peel and flesh of NO. 14 and NO. 26 cucumber fruit; 13-CsHPL expression significantly increased in the peel and had no significant changes in the pulp and fruit cavity of NO. 26 cucumber fruit, while 13-CsHPL was elevated in the peel and pulp and had small changes in the cavity of NO. 14 cucumber; 913-HPL expression increased in different parts of NO. 26 cucumber fruit, large changes in the peel of NO. 14, in the other parts had small changes. CsAOS expression increased in peel and pulp parts of NO. 26, CsAOS had similar expression levels with 13-CsHPL in NO. 14.6. CsHPLs gene expression patterns in different treatment of NO. 14 cucumber fruits All genes expression levels were significantly higher after injury treatment in NO. 14, but after MeJA treatment 13-CsHPL, 913-HPL and CsAOS expression increased except 9-CsHPL which decreased at first.7. 9-CsHPL prokaryotic vector induced and protein expression The cloned gene 9-CsHPL was connected to different prokaryotic expression vector pMAL-c4 x and pGEX-4t, after induced by IPTG in E.coli BL21(DE3), SDS-PAGE showed noticeable protein in the 94 kD and 54 kD bands, the molecular weight of the recombinant protein was about 54 kD, results in line with expectations.