Establishment Of Stable Expression System For Exogenous Gene In Ulva Prolifera

Genetic transformation system of seaweeds has been established extensively,however,some bottlenecks hindered the large-scale application,for example: firstly,vector elements from virus widely used in seaweed transformation were of transgenic concern.Secondly,relatively low protein content and long-time cultivation period restricted the efficient expression of exogenous protein.Thirdly,the accumulation of biomass needed open-sea area,which would increase the risk of genetically modified seaweed release into environment.Ulva prolifera is a cosmopolitan green seaweed and has characteristics of high growth rate and protein content,short life cycle period and fitness for enclosed cultivation.We believed that U.prolifera was very suitable to be developed into a new stable,effective and safe seaweed expression system to break the above bottlenecks.In this paper,on the basis of established transformation system,we tried to establish a safe,effective and stable exogenous gene expression system of U.prolifera.Firstly,we cloned three constitutive or inducible endogenous promoters of U.prolifera,and verified their activities with the methods of transient expression,deletion mutation and quantitative GUS assay.In aspect of constitutive promoter,the genomic DNA sequence and 5’ flanking region from U.prolifera actin1 and actin2 gene(Upactin1 and Upactin2)were cloned and characterized.One special 5’ UTR intron(leader intron)was detected in both 5’ UTR regions and one 5’ Py-rich stretch element was found in Upactin2 5’ UTR.The results of quantitative GUS assay showed that promoter activity of Upactin1 and Upactin2 5’ flanking region were much higher than that of CaMV35 S and SV40.In algae,this was the first report that 5’ UTR intron and 5’ Py-rich stretch were the necessary element for promoter activity.In aspect of inducible promoter,we cloned the 5’ flanking region from U.prolifera hsp70 gene(Uphsp70).One heat shock element(HSE)and one low-temperature-element(LTR)were detected.Quantitative GUS assay demonstrated that the 5’ flanking region of Uphsp70 could be activated by both heat and cold stress.In addition,for the first time,we found that HSE and LTR cooperatively respond to temperature stress.The above endogenous elements would significantly elevate the safty and efficiency of exogenous gene expression in U.prolifera.Secondly,based on the spore-mediate and in situ germination regeneration approach,the nuclear expression system of U.prolifera was established.Using particle bombardment method combined with spore-mediate regeneration approach,basta resistance gene(bar)was successfully transformed and positive transformants were obtained.Southern blot experiment showed that bar gene was stably integrated into the genome.Moreover bar gene was also stably expressed in three generations and all tested offspring were detected with positive PCR signal.Using particle bombardment method combined with in situ germination approach,the reporter gene GUS and GFP were stably expressed in germlings.With Upactin1 promoter,bar and rpa was stably integrated in cells of U.prolifera.These two approaches would pave the way for efficient and safe expression system of U.prolifera.To verify whether complicated proteins could precisely fold after translation in non-model green algae,we obtained the engineering algae strain which could stably express high-value thrombolytic protein rt-PA in green microalgae Platymonas(Tetraselmis)subcordiformis.Western blot and FAPA experiment demonstrated that the highest expression level of recombinant rt-PA was 1.91 μg·mg-1 soluable protein and which had effectively thrombolytic activity.Our experiments indicated that complicated post-translational modification of rt-PA could accomplished in cells of green algae.Moreover,100 L amplification culture system was in progress,which could provide a vital reference for systematic evaluation of U.prolifera expression system based on enclosed cultivation.At last,for further elevation of efficiency and safety,we tried to establish chloroplast stable transformation system.First,the fragment 16S-trnI-trnA-23 S from chloroplast genome was cloned and the trnI-trnA insertion site was determined.Then,the chloroplast transformation vector carrying bar gene was constructed.Using particle bombardment method combined with in situ germination approach,the vector was transformed into chloroplast of U.prolifera and the positive transformants were obtained.The molecular detection results showed that bar gene was integrated into the expected insertion site.This was the first report about chloroplast transformation of seaweed and it would lay the foundation for chloroplast expression system.