The Preliminary Study On Cloning And Expression Of CDNA Fragments Of Related Enzymes Gene Of Chlorophyll Degradation Of Cucumber During Senescence

Chlorophyll is an important pigment in the plant chloroplasts being concerned with photosynthesis.Chlorophyll degradation is an important phenomenon in the senescence process. Chlorophyll is degraded in plants as a result of natural or induced senescence.Loss of green color is the most obvious sign of plant senescence.In this study, the cultivar Cucumis sativusL.var.Huahuang 5 and the wild species Cucumis sativus L.var.xishuangbannanesis Qi etYuan with different senescence were used as experient materials.The physiological and biochemical characteristics were preliminary studied during senescence in the wild species and the cultivar. Meanwhile, Two key enzymes gene of Chlorophyll degradation (Pheophytin Pheophorbide Hydrolase and Pheideaoxygenase) were cloned. Expression of these two genes during leaf and stem senescence was quantified through real-time PCR technique. The relationship between the expression model of PPH and PAO genes and anti-senescence were discussed. Major results are as follows:1. The physiological and biochemical characteristics of cucumber leaf during senescenceAs xishuangbannanesisQietYuan and Huahuang 5 became senescent, chlorophyll content in leaves increased first,but decreased later. The Chlorophyll content was the highest at 15d. The ageing symptoms of leaf in Huahuang 5 appeared at 35d, the contents at 45d were 45% at 15d. on the contrary, chlorophyll content in xishuangbannanesisQi etYuan was always a higer level. The activities of SOD in Huahuang 5 decreased dramatically first, but increased faint later, decreased at 35d again. The activities of SOD of xishuangbannanesisQi etYuan at 35d was the highest, decreased later, The activities of SOD in the wild species started to decrease 20 days later than the cultivar, Maybe the senescent of xishuangbannanesisQietYuan start at 35d.The activities of POD of Huahuang 5 all raise as cucumber became senescent, POD showed two peaks in the wild species, The activities of POD was higher than Huahuang 5 except at 5d. The activities of POD of Huahuang 5 and xishuangbannanesisQi etYuan was the highest at 45d. the ability of the wild species which cleaned out active oxygen was stonger than the cultivar at the later stage of cucumber growth. The soluble protein content of xishuangbannanesis Qi etYuan was higher than Huahuang 5, The Chlorophyll content was the highest at 35d. The soluble protein content of Huahuang 5 was at 45, meanwhile, the variation laws of the soluble protein was not significantly.2.The physiological and biochemical characteristics of cucumber stem during senescence As xishuangbannanesis QietYuan and Huahuang 5 became senescent, The Chlorophyll content in stem of xishuangbannanesisQi etYuan was the highest at 45d. unlike leaf, the variation trends of the Chlorophyll content in stem of xishuangbannanesis Qi etYuan was not significantly. Maybe the stem didn't enter the senescent.The Chlorophyll content in stem of Huahuang 5 was the highest at 25d. chlorophyll content in stems increased first, but decreased later.Thecontent of chl of xishuangbannanesisQi etYuan was higher than Huahuang 5.The activities of SOD of xishuangbannanesisQi etYuan and Huahuang 5, showed two peaks. It were the highest at 35d.The activities of POD of xishuangbannanesis Qi etYuan all raise as cucumber became senescent, it showed two peaks in the cultivar, The activities of POD of Huahuan 5 and xishuangbannanesisQi etYuan was the highest at 45d. The activities of POD was higher than Huahuang 5.The soluble protein content of xishuangbannanesis Qi etYuan and Huahuang 5 were the highest at 35d, overall, the variation trends of the soluble protein was in stem increased first,but decreased later.the lowest were at 45d.3. Cloning of PPH and PAO genes.The length of cloned PPH and PAO genes were 265 base pairs and 230 base pairs, Analysis of the nucleotide sequenee by BLAST on line indicated that homology of PPH gene was up to 87% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 88% between it and Brassica oleracea L. var. botrytis L, up to 80% between it and Vitis vinifera, up to 78% between it and Nicotiana tabacum. homology of PAO gene was up to 90% between Cucumis sativusL.var. and Arabidopsis thaliana (L.) Heynh, up to 86% between it and Brassica oleracea L. var. botrytis L, up to 85% between it and P. trichocarpa, up to 82% between it and Ricinus communis.4. Relative quantification of PPH and PAO genes during leaf senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.It is different that expression of both PPH and PAO genes in the leaf of xishuangbannanesisQi etYuan. The relative expression of PPH gene basically up-regulated first, down-regulated later. It was the highest expression at 35d. The minimum expression of PAO gene at 15d and 25d.The maximum at 35d. Expression of PPH and PAO genes between cultivars and wild species was quite similar. The relative expression of PPH gene basically up-regulated first,down-regulated later in stem of Huahuang 5. The maximum at 35d too, the minimum expression of PAO gene at 15d and 25d, the maximum at 45d. There was a positive correlation between both PPH and PAO genes in the leaf of xishuangbannanesis Qi etYuan and Huahuang 5.5. Relative quantification of PPH and PAO genes during stem senescence of Cucumis sativus L.var.xishuangbannanesis Qi etYuan and Huahuang 5.Unlike the expression modle of leaf. It is different that expression of both PPH and PAO genes in the stem of xishuangbannanesis Qi etYuan. Expression of PPH and PAO genes between leaf and stem was quite similar. The relative expression of PPH gene basically up-regulated first, down-regulated later too.it was the highest expression at 35d. The expression mold of PAO gene basically up-regulated first, down-regulated later, then up-regulated. The maximum at 45d. Unlike the expression modle of stem of xishuangbannanesis Qi etYuan, expression of both genes basically down-regulated first, up-regulated, down-regulated, up-regulated last, it showed two peaks in the cultivar,there was a higher expression than the value of at 5d. The maximum expression of PPH at 35d, PAO at 15d. There was a positive correlation between both PPH and PAO genes in the stem of Huahuang 5. negative correlation in the stem of xishuangbannanesis Qi etYuan.