Leggy Seedlings Characterisitics, The Cko Gene Cloning And Expression In Cucumber

Cucumber (Cucumis sativus L.) an important worldwide vegetable, is cultivated in protection field, which plays a significant role in the supplying market and consumer market. When the plants are in an abnormal environment such as high temperature and humidity, light deficiency as well as water stress, the seedlings express the tendency of spindling during during the process of raising seedlings. Under these conditions, cucumber seedling quality decline is induced, and vegetive and reproduction growth will be affected which might finally lead to significant losses in the product’s yield and quality. Ent-Kaurene oxidase (KO), a critical enzyme in the pathway of gibberellins biosynthesis, was cloned from the apical stem of’Deltastal’(Cucumis sativus L.) in this experiment. The expression level of CKO was determined by the fluorescent quantitative RT-PCR. By agrobacterium-mediated, early transgenic plants gained, which provide the theoretical basis for the mechanism of cucumber seedling leggy. The major results were as follows:1. Using cucumber variety’Deltastal’ as material, the effect of shading and shading combined with spraying paclobutrazol on cucumber seedlings and ultrastructure of hypocotyl cells were studied. The results showed:compared with control, hypocotyl length was significantly increased; stem diameter, dry weigh and seedling index were significantly reduced under shade conditions. Spraying placlobutrazol can effectively control seedlings leggy and improve seedling index.Ultrastrucrural observations showed that:under low light, the size of chloroplast and the number of grana as well as lamella increased, starch grains disappeared, plastoglobulus reduced. Mitochondria increased and was more mature, but the endoplasmic reticulum had a slight swelling.Chloroplast stroma lamellae loose and swelling, plastoglobulus increased, mitochondrial slight swelling,the endoplasmic reticulum grow well when spraying paclobutrazol under shading.2. With homologous cloning and RACE technology, ent-Kaurene oxidase(KO), a key enzyme in the pathway of gibberellins biosynthesis, was cloned from the shoot tip of ’Deltastal’(Cucumis sativus L.), and named CKO(GenBank accession number: JN792591). The full cDNA was1895bp in length with an open reading frame(ORF)encoding a protein of519amino acids. The protein molecular weight and isoelectric point were predicted to be59.211kD and8.45respectively. Amino acids homology analysis indicated that the sequence had84%similarity with that of Momordica charantia. Sequence analysis showed that CKO belonged to cytochrome P450superfamily and contained cysteine hemeiron ligand signature (FXXGXRXCXG) and transmembrane region. The fluorescent quantitative RT-PCR result showed that the expression level of CKO was the highest in leggy seedling under shading treatment, being5.18times of the normal one. While the expression of CKO significantly reduced after spraying paclobutrazol at shading.3. CKO gene was cloned from cucumber cultivar’Deltastal’cDNA using RT-PCR method, the was inserted into the plant expression vector of pCAMBIA1301-1, subsequently the plasmid was transferred into agrobuterium strain EHA105. The recombinant vector of pCAMBIA1301-1-CKO was transformed into tobacco by leaf disc method. Transgenic tobacco plants were generated by screening of hygromycin, and through callus and differentiation periods further analysis of PCR and GUS activity were carried out.The determinations of the GUS activity assay indicated that the CKO gene could drive the expression of GUS reporter gene in leaves and callus.And the PCR detection showed a positive result. All These data provided a support for CKO gene further biological function analysis and genetic engineering application.