Construction Of An Exogenous Gene Expression System Based On T7 RNA Polymerase In Anabaena

The foreign genes could be efficiently transcripted in T7 based promoter-diven protein expression system due to the strong specificity and efficient binding of the T7 RNA polymerase with its promoter.This system comprised a T7 polymerase,wherein the system was a host cell transformed with a plasmid comprising the T7 promoter equence.Therefore,to construct this system,T7 promoter and T7 RNA polymerase should be inclould in the host cells.Cyanobacteria are photoautotrophic prokaryotes that perform oxygenic photosynthesis.They have long been studied as model organisms of photosynthesis and the circadian rhythm.Cyanobacteria is an ideal host for genetic engineering because that it is non-toxic,nutrition,easy genetic manipulation,lower protease activity inside and cheap to culture.However,cyanobacterial genetic engineering has developed slowly these years because of lacking efficient exogenous gene expression system.The low efficiency of exogenous gene expression in cyanobacteria has been the bottle neck of the development of cyanobacteria genetic engineering,restricted the cyanobacteria genetic engineering pharmaceutical industry.Objectives:1.By use of electroporation method with integration vector containing T7 RNA polymerase pointing into the genome of Anabaena sp.PCC 7120.2.Use kanamycin resistance screening of genetically modified algae.3.Construction of shuttle expression vector containing the T7 promoter and lux AB genes and using the method of three pro-conjugation transferred to transgenic algae.Methods:1.Exogenous gene into cyanobacteria approach:electroporation transformation and three pro-conjugation methods.2.Vector construction methods:using primers with the appropriate restriction sites and promoter sequences for the PCR method to obtain a fragment containing the T7 promoter and the corresponding restriction sites.DNA ligase using the corresponding vector fragments.3.Transgenic algae screening and evaluation:resistance screening and PCR validation.Results:1.The site-specific integration vector by the HindⅢ restriction enzyme digestion,get the linear size of 9800 bp fragment,electroporation Anabaena,screening transgenic algae by kanamycin resistance.2.In order to determine whether the site-specific integration vector was integrated into the genome of Anabaena sp.PCC 7120,verified by PCR on genomic,in 900bp size kanamycin resistance gene that has bands at.3.Cloning vector by sequencing results compared with the target sequence show the same sequence,the shuttle expression vector is digested by EcoRI,Xhol,Show digested large fragment of about 11000 bp and small fragment of about 600 bp,the correct size to prove shuttle expression vector was constructed successfully.