| Rape, as one of the important oil crops in China, greatly contributes to national agricultural economy. Due to its characteristics of infinite infloresce and limited leaves, the longivity for effective photosynthesis and the accumulation of photosynthesis production are serciously influenced by the process of rape leaf senescence. All kinds of adversity, including drought, water logging, plant diseases, insect pests etc, may lead to leaf presenescence in some rape varieties, which is known as one of the important factors to limit the seed production.Our studies have been directed at delaying leaf senescence and enhancing stress-resistance by regulating endogenous cytokinins biosynthesis in Brassica napus. The obtained results are as follows:1. By examining some physiological indexes in the course of leaf senescence in Brassica napus, the change patterns of the levels of chlorophyll, endogenous cytokinins, SOD activity and MDA accumulation are obtained.2. 2130bp-long 5vuntranslation sequence of SAG12 gene in Arabidopsis thaliana was separated by polymerase chain reaction, which is composed of 5" UTR of 55bp and 5V untranscripition sequence of 2075bp, and ipt gene was cloned fromAgrobacterium tumefaciens for cytokinin synthesis key enzyme, which contains the full open reading frame about 720bp. The 2130bp-long DNA sequence contains the -1 ?-1345 functional region of senescence-specific promoter and the enhancer region of -1345?1181, but with none of the typical TATA box and CAAT box observed by PC-GENE. pAGT, the plant expression vector with the chimeric PsAGi2-ipt, was successfully constructed by intermediary vector pBAG.3. The rape transformation system was set up by using Agrobacterium tumefaciens LBA4404 and EHA105, and by using cotyledon and hypocotyl as explants. The chimeric PsAoi2-ipt was transferred into rape and the fully regenerated plantlets with kanamycin resistance were obtained. Eleven transgenic plantlets were identified by double PCR and genomic DNA Southern blotting technique. The results show that the target gene has been integrated to the genome of the 11 regenerated plantlets.4. The growth of 11 transgenic plants and 2 controls were observed in a systematic way. The results prove that only 2 transgenic plants, numbered line 5 and line?, grew with morevigor than the others, and their leaf senescence was delayed. Long-stem leaf senescence at the base of line 5 and line? was delayed 20-30 days, and their short-stem leaves in the main stem delayed 30-40 days, while their no-stem leaves delayed 20-30 days.The results also show that the 2 transgenic plants with the characteristics of delayed leaf senescence have been obtained.5. In different leaf-positions of line 5 and line7, the levels of chlorophyll and cytokinins were examined. In the lower second from full-extended leaf, the contents of chlorophyll and cytokinins were 1.5 times those of the control, respectively, and in the lower sixth leaf, the amount of cytokinin contents was about 3-6 times that of the control. These results prove that the 2 ipt transgenic plants with promoter PSAGH obtained distinctively manifest their functions in cytokinin biosynthesis and its accumulation, and in delaying leaf senescence.6. SOD activity and the amount of MDA accumulation were also examined in different leaf-positions of line 5 and line7. The results show that SOD activity in the lower second from full-extended leaf is a little more than that of the control, but in the lower sixth leaf, its activity is 2.5 times that of the control, and the amount of MDA accumulation in the lower second is only half of that in the control, while in the lower sixth, it is two third of that in the control. The obtained data bring out experimental proofs of relationship between the content of endogenous cytokinins and the ability of stress resistance. |
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