Transformation Of T-PA Gene Into Cucumber And Analysis Of Its Expression Efficiency In Different Plants And Its Codon Reconstruction

Tissue-type plasminogn activator(t-PA) is a kind of high efficient and specific thrombolytic medicine and is used widely to cure blood vessel embolism diseases. At present, t-PA has been expressed in E.coli. successfully and reached industrialization, but its operating cost is very expensive. Take transgenic plant as bioreactor to produce t-PA will come to hot point of research because of its low costing, high safety of product, convenient for producing, saving and transport on a large scale. However, there is a low expression problem in plant expression system which restrict the application of transgenic plant in medicine gene engineering production.Take cucumber as bioreactor to express t-PA in this study. Aiming at low expression of exogenous gene, t-PA gene was designed. The designed t-PA gene was used to transform cucumber and the transgenic plants was obtained. At the same time, codon usage of potato, cucuber and strawberry was analyzed and forecasted the expression of t-PA gene in 3 organisms and designed codon of t-PA gene to fit for expression in plants. The primary conclusions as follows:1. Designation of t-PA gene(1) At gene transcription levelThe plant expression vector pBET and pBEMT were constructed, of which t-PA gene was regulated under the enhancer E12 and promoter CaMVSSS. The upstream region-419-90 of promoter CaMV35S was repeated 2 times manually, and then became enhancer E12which can improve promoting efficiency of promoter CaMVSSS to 70 folds.(2) At translation levelPlant mutual sequence of starting translation AACA was added to start codon of t-PA gene by PCR ampliation and plant expression vector pBET was constructed. Plant mutual sequence of starting translation and Kozak sequence in animals have the same function namely accelerate translation starting of mRNA.(3) At post-translation levelPlant mutual sequence of starting translation AACA and eukaryotic secretory signal peptide sequence was added to 5'-flanking region of t-PA gene and KDEL(sequence located to endoplastic reticulum ) to 3'-flanking region by PCR amplication and plant expression vector pBEMT was constructed. The new synthesized protein was led to endoplastic reticulum cavity by eukaryotic secretory signal peptide sequence and then anchored to innerwall of endoplastic reticulum by KDEL sequence, which interdicted the process of protein entering Golgi body andcytoplasm, and then avoided heterogeneous glycosylation modification of foreign protein and prolonged the disappearance of half life of protein in organism.2. Transformation of cucumberPlant expression vecor pBET and pBEMT were transferred to cucumber and PCR positive transgenic plants was obtained.(1) Plant regeneration system of cucumber contyledonary node was established.(1) The cotyledonary node was regarded as optimal explant by comparing the differentiation frequency of 3 kinds of explants(cotyledon, cotyledonary node and hypocotyl).(2) By discussing the effect of PGR(NAA and 6-BA)concentration and combination on differentiation frequency of adventitious buds, we concluded that the optimal shoot induction medium is MS basal medium with 1mg/L 6-BA(pH5.8).(2 ) Transformation of cucumber(1) The suitable selection concentration of Km is30mg/L through discussing the effect of Km concentration on differentiation frequency of adventitious buds.(2) 56 resistant plants were obtained which transformed with LBA4404(pBET)î–²LBA4404 (pBET) (The rate of resistant shoots is up to 25.0%).(3) 59 resistant plants were obtained which transformed with LBA4404(pBEMT) LBA4404 (pBET) (The rate of resistant shoots is up to 33.5%).3. Molecular biology detection of kanr plants(1) 12 of 32 kanr plants transformed with pBET detected by PCR were positive plants(Rate of PCR positive plants is37.5%).18 of 36 kanr plants transformed with pBEMT detected by PCR were positive plants(Rate of PCR positive plants is50.0%).(2) 1 of 3 plants transformed with mt-PA detected by Sonthern blot were positive plants and integ...