| Potato(Salanum tuberosum L.)is one of the major food crops and vegetables in the world. Because of its fine processing characteristics, potato starch has been used extensively. Under the historical opportunity of the development of the west regions, potato processing will become the major food industry of the west.But elite processing cultivas,especially high starch content and low reducing sugar cultivas presently used have varied more or less.For meeting the rapid development of potato processing industry,the work of breeding high starch,low reducing sugar cultivas is extremely urgent.As the high starch content potato material for breeding is deficient ,what't more , potato is homology tertraploid,so use routine breeding method cultivate high content starch potato cultivar not only time and energy consuming but also having many difficulties.Gene engineering can break species limits and introduce exogenous genes into different species.With the further investigation of relevent enzymes of starch metabolism and improvement of molecular biotechnology in recent years,it makes using gene engineering technology to control the activity of relevant enzyme of starch metabolism so as to raise the content of starch and reduce the content of the reducing sugar become possible.In this study,According to known sequence of Arabidopsis thalian cold-induced promoter rd29A,we cloned a 953bp distinctive fragement.Sequence analysis indicated that the homology between this fragement and the corresponding reported sequence is 99.47%.the AcInV gene was amplified by RT-PCR from potato tubers'mRNA.the nucleotide sequence of the clone was confirmed by sequencing, the homology between this 1920bp fragement and the corresponding reported sequence is 99%.GFP gene was also cloned and sequencing indicated that the homology between this 720bp fragment and that reported sequence is 100%.Sequence analysis showed that there were two Dehydration-responsive Element(DRE) and several other main regulation domains in rd29A promoter, and TATA box located at 851-856 bp from ori.Two Plant expression vectors,pBIGF of GFP gene drive by constructive promoter CaMV 35Spromoter and pBIRDGF of GFP gene controlled by rd29A,were constructed to assay the activity of both promoters.pBIGF and pBIRDGF were introduced into epidermis cells of onion by particle bombardment.the transformed epidermis was cultured at 0℃,4℃,10℃,25℃and 28℃ for 2d.the expression of in onion epidermis was observed with. Fluorescent microscope oberservation of GFP driven by different promoters showed fluorescent of GFP have not observed in epidermis cells of both vectors cultured in 0℃;while the GFP gene drived by rd29A promoter expreesed higher than that controlled by CaMV 35S when cultured at 4℃,10℃ and 28℃.The result indicated that rd29A promoter is not only a cold-induced promoter but also a high temperature-induced promoter.Then by introducing rd29A promoter and AcInV gene(1920bp or 300bp) into plasmid pBI121,we constructed the Antisense AcInV gene plant expression vector pBIAC with CaMV 35S promoter,pBIRDAC with rd29A promoter and pBIRDAC300 with rd29A promoter. and the plant expression Vector pBIAC,pBIRDAC, pBIRDAC300 were intergarted into agrobaterium LBA4404.The transformation efficiency of potato via Agrobaterium tumefaciens system was studied,and the result showed that stem cutting is better than leaf for its high callus induction efficiency ,strong ability to differentiation and regeneration and easy to obtain. The optimal conditions for cultivating "Longshu 3"have been founded: stems and leaves are pre-cultured on the MS medium with 6-BA 2.5mg/L and 2,4-D 0.5mg/L for 2 days and 3 days,respectively, OD600 of the Agrobaterium is 0.5-0.8; infection Agrobaterium for 5 minuter ; co-cultured on MS medium for 3 days under 25℃ in darkness.After co-culture all the explants were washed by sterile water.Consequently,all of them were transferred to the medium(MS + 6-BA 2.5mg/L + 2,4-D 0.5mg/L + AgNO3 4mg/L + Kan 50mg/L + cb 300 mg/L) for inducing resistant c...
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