| Lower expression level is one of bottlenecks for application of molecular pharming.It is an important scientific issue to improve recombinant protein expression level for molecular pharming.The strategies of previous researches to improve recombinant protein expression level were focused on increase of transcriptional and translational level.Those included to use strong promoter,optimize 5’ or 3’ UTR to stabilize m RNA,increase specific transcription factor expression to enhance translational level and use the sorting signal to improve the protein trafficking.However,it is not obviously improving recombinant protein expression level at all.Extensive researches indicate that the ER stress becomes more serious when overexpression of recombinant protein in cells.As consequence,the recombinant protein is degraded due to the cell to promote UPR pathway,which is significantly impeded further improvement of recombinant protein expression level.It is a scientific issue that how to further improve recombination protein expression level in spite of rice endosperm cell is demonstrated as realistic platform for recombinant protein production.This study focused on how to alleviate the ER stress induced by overexpression of recombinant protein in rice endosperm cell to improve the expression level of recombinant proteins.The strategy is to reduce main endogenous storage protein content to decreasing total protein contents in rice endosperm cell,which would be provide more “space” of translational machinery for recombinant protein in rice endosperm cells.The loadmap is site-specific insertion of human serum albumin(HSA)gene expression cassette into a Glu A1 locus via TALEN-mediated site-specific insertion(Knock-In).As consequence,it is to express HSA and simultaneously knock-out Glu A1 locus.The study will verify whether Osr HSA expression level could be significantly increased via the strategy of alleviation of the ER stress that induced by overexpression of Osr HSA in rice endosperm.The main results as follows:1.The first exon of a main storage protein locus,Glu A1,was chosen as a target for site-specific knock in.A pair of TALENs was designed based on the first exon of Glu A1,and was synthesized using rice-preferred genetic codons(Gt-TALEN).The cleavage activity of Gt-TALEN was tested by single strand annealing(SSA)assay.The result showed the cleavage efficiency for single target site was 0.79,showing the prefer cutting activity of the TALEN.2.Three Agrobacterium-based binary vectors,pOs PMP623,pOs PMP624 and pOs PMP625 were constructed.Construct,pOs PMP624 was for random integration(RI)and constructs pOs PMP623/ pOs PMP625(TALEN expression cassette)were for site-specific knock-in(KI)to Glu A1 locus through Agrobacterium-mediated transformation via non-homologous end joining(NHEJ)pathway.PCR and sequencing confirmed that three out of 11 transgenic events were HSA expression cassette knock-in(KI)with 27.27% frequency.Twenty transgenic events for random integration(RI)were obtained.3.The Osr HSA expression levels in rice endosperm of transgenic lines were measured by Enzyme-Linked Immunosorbent Assay(ELISA).The expression level of Osr HSA in three KI lines ranged from 4.94-5.31 mg/g brown rice,with average of 5.13±0.15 mg/g brown rice.The efficiency of variation(CV)between KI transformants was 2.93%.The expression level of Osr HSA in 20 RI lines ranged from 0.13-2.39mg/g brown rice,with average of 1.03±0.60 mg/g brown rice.The CV between RI transformants was 58.3%.The average expression level of Osr HSA in KI lines was4.98 folds of that in RI lines and the highest exoression level of Osr HSA in KI lines was 2.22 folds of that in RI lines.The results indicated that the knock-In strategy not only significantly improved Osr HSA expression level,but also overcame the position effect of random integration on foreign gene expression.4.We found that overexpression of Osr HSA affected the transcriptional level and protein accumulation of endogouse storage proteins in rice endosperm cells.Comparison with TP309,the m RNA level of Glu A,except Glu A1 locus,and Glu(B+C+D)in KI line 623-35-16 were significantly down-regulated,especially at 8days of pollenation(DAP)and 12 DAP,it decreased by 85.4%(P<0.01)and 59.0%(P< 0.01)respectively,while the m RNA level of prolamin and globulin increased by112.8%(P<0.01)and 102.3%(P<0.01)respectively.The protein contents of glutelin and globulin in KI line 623-35-16 were significantly decreased,indicating that globulin was affected at post-translational level.However,prolamin was significantly increased.The results indicated that there was a strong compensation mechanism of total protein contents in rice endosperm cells.5.The m RNA level of a molecular chaperone Os Bi P1,and an endoplasmic reticulum(ER)stress marker,Os Bi P5 were analyzed.Compared with TP309,we found that the m RNA level of Os Bi P1 in KI line 623-35-16 at 4 DAP,8 DAP and 12 DAP was increased by 54.8%(P<0.01),480.1%(P<0.01)and 315.7%(P<0.01)respectively.Compared with RI line 624-7-9,the m RNA level of Os Bi P1 was increased by 37.4%(P<0.01),94.5%(P<0.01)and 6.8%(P>0.05)respectively.Compared with RI line624-7-9,the m RNA level of Os Bi P5 in KI line 623-35-16 at 4 DAP,8 DAP and 12 DAP,was increased by 13.8%(P>0.05),50.1%(P>0.05)and 58.2%(P<0.05)respectively.The results indicated that serious ER stress was induced by overexpression of Osr HSA in rice endosperm cells.However,the ER stress in KI line623-35-16 did not become more serious with the increase of Osr HSA level,suggesting that decrease of endogenous glutelin contents in rice endosperm cells could alleviate ER stress.6.The expression profiles of the ER stress pathway genes,Osb ZIP50 and Osb ZIP60 at different endosperm cell developmental stage were analyzed.Comparison with RI line 624-7-9,we found that the m RNA levels of Osb ZIP50 and Osb ZIP60 in KI line 623-35-16 were significantly up-regulated at 12 DAP,which were increased77.3%(P<0.01)and 39.7%(P<0.01)respectively.However,the m RNA level of Os Bi P1 was no significant difference.Furthermore,the m RNA splicing of Osb ZIP50 via IREI pathway was analyzed.The results showed that m RNA splicing of Osb ZIP50 were obviously difference between KI line 623-35-16 and RI line 624-7-9.The m RNA splicing of Osb ZIP50 in KI line 623-35-16 was more obviously at 12 DAP.It indicated that the alleviation of the ER stress in KI line 623-35-16 was via IRE1/Osb ZIP50 pathway.7.Furthermore,we observed the morphology of protein bodies(PBs)by transmission electron microscopy(TEM).The results showed that the normal PB morphology was observed in KI line 623-35-16,which was almost the same as that of non-transgenic rice TP309.8.We found that the precursor of glutelin in KI line 623-35-16 was obviously increased.It suggested that overexpression of Osr HSA in rice endosperm cells also affected the glutelin processing.Taken together,the expression level of Osr HSA was significantly increased via TALEN-mediated site-specific knock-in of the HSA expression cassette into Glu A1 locus.The mechanism of improving Osr HSA level was through lowering glutelin content,in turn to alleviate the ER stress that induced by overexpression of Osr HSA in rice endosperm cells.Our results revealed that the ER stress was induced by overexpression of Osr HSA in rice endosperm cell,and then the molecular chaperon Os Bi P1 was upregulated to help refolding of unfolded proteins.Furthermore,the ER stress marker Os Bi P5 was upregulated;finally,the ER stress was alleviated via activating IRE1/Osb ZIP50 pathway.Our results further demonstrated that rice endosperm cell had strong capacity of compensation to keep total protein content homostasis,which would eliminate the concerns that decreasing endogenous storage protein contents could affect rice life circle. |
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