Study On The Cloning And Expression Of Bpti Gene In Tobacco

Bovine Pancreatic Trypsin inhibitor (BPTI) is a nonspecific andbroad-spectrum serine protease inhibitor,which affects known serine proteasessuch as trypsin,chymotrypsin,plasmin and kallikrein. BPTI is a single, 58amino-acid polypeptide with three pairs of disulfide linkages, and its isoelectricpoint is 10.5. BPTI has been made use of in the therapy of the acute pancreatitisfor a long time.In 1990s, following the England and Canada, American hadauthorized to apply BPTI to the operations of the chest surgery. It can defendblood platelet against bleeding, treat the patient with cruor impediment, inwhich situation the hemorrhage may occur. BPTI can be used in cardiac surgeryas being extracorporal circulated to decrease the incidence of bleeding and theinflammatory reaction caused by the surgery, and it can protect the cardiacmuscle and lung as well.BPTI is an effective substance to prevent patients fromintestinal adhesion and treat hemorrhage of gastric mucous membrane.Theclinical research indicated that BPTI is a safe medicine with little side effect.BPTI is used as a biochemistry medicament,whose clinical requirementand market scale are huge.BPTI is currently purified from bovine pancreas andlung. There are several problems associated with extracting BPTI from bovineorgans,such as the cost of storing the raw material is high. And more importantfor BPTI as a therapeutic agent is that there are more concerns about it beingcontaminated with prions.The better method of solving above mentioned problems is recombinantexpression of BPTI. It has been reported that expression of recombinant BPTI can express inE.coli. However the product is inactive in this method because of it's containingthree pair disulfide linkages. The disulfide linkages may mispair incidentallywhen it is renaturing in vitro. And the output of the secreted expressive BPTI islow. Also the expression of recombinant BPTI in Saccharomyces cerevisiae hasbeen done by other scientists. But the αfactor signal peptide digestive enzymein Saccharomyces cerevisiae can not correctly digest the N-extremity sequence.And the output is unfavorable. Tobacco as a bioreactor has many advantages over other expression systems.Because of its high propagation coefficient and the high output of per plant,many researchers think that tobacco is the best bioreactor to produce exogenousproteins. The plant cells are similar to the animal cells which possess the wholeeukaryotic expressing system responsible for regulating the transcription andthe translation of genes. They also can perform post-translation processing andstore the expressed exogenous proteins in organelle or secret them outside thecells. The cost of producing BPTI in tobacco is much lower than other methods.The situation of culturing tobacco is very simple and the seeds of transgenictobacco are easy to store and transport. There are BPTI and analog in tobacco.So far, the recombinant expression of BPTI in tobacco has not been seen. So we choose the tobacco to express the recombinant BPTI as thebioreactor. In order to obtain the tobacco which can express bpti gene with highefficiency, we did the following experiences: 1. preparation of the transgenic tobacco (1)Clone the BPTI gene and construction of the di-expression vector. Extract genomic DNA from the bovine lung, and then obtain the bpti genevia PCR using the specific primers and clone into the pBluescript vector. Afterconfirmated by endonuclease digestation assay and gene sequencing, the bptigene with XbaI and SacI target sites was subcloned into the vector pBI121 andwas transformed into E.coli. The recombinant plasmid extracted wasauthenticated via the endonuclease digestion assay. (2)Introduce the plant expression vector with the bpti gene pBI121 toagrobacterium tumefaciens EHA105 by way of ice-thaw-media. Preparecompetent cells of the agrobacterium tumefaciens, to which introduce BPTIplant expression vector pBI121-bpti though ice-thaw -media. And theagrobacterium tumefaciens transformers was assayed via PCR. (3)Obtain Kan resistant-plant by introducing the BPTI gene into tobacco bythe way of agrobacterium tumefaciens mediation. The tobacco transformation: the tobacco was transformed by the method ofthe leaf disc transformation , then the tobacco leaf discs which were infected byagrobacterium tumefaciens with bpti gene was put into MS cocultured media.After 2 days at 25℃, they were transferred into the selective media withkanamycin(200mg·L-1) and carbenicillin(500mg·L-1), and the leaf disc withoutbpti gene were transferred into the selective media as the compare group.Choose the callus around the leaf disc and maintain culture in the subculturemedia. When the bud is more than 2 centimeters, it was cut and transferred intoMS rhizogenic media. After 20 days the young rooted bud was displayed intosoil. The 157 Kan fastness transgene plants were obtained successfully. (4)The results of PCR test and bioactive assay of BPTI showed that weobtained the tobacco transfected with the bpti gene firstly in the world. ①Identification bpti gene by PCR Extracted the genomic DNA of tobacco with modified CTAB method, thenperformed PCR with specific primers and the electrophoresis result showed thatthe majority of the transfected tobacco group(135 individuals) obtained aspecific band of 180bp, and there wasn't the band in the negative group(untransfected group). ②The bioactive assay of BPTIWe cut the 2nd to the 4th expanding leaves in the base of the transgenictobacco when the tobacco grows out 7～8 leaves. The leaves weight about 2gwere skived in liquid nitrogen. We join 200ml physiological saline,centrifugated at 1000rpm, and the supernatant was used for the bioactive assayof BPTI. The aforementioned results showed that bpti gene has been integrated inthe tobacco genome and expressed in it. So we obtained the transgenic tobaccocontaining bpti gene for the first time. We obtained 157 individuals which could resist kanamycin, and 135individuals in those were transfected with bpti gene which was tested by PCR.92 individuals could express BPTI confirmed via the bioactive assay, and 37individuals high expressed the BPTI with 2000KIU in 1g fresh plant tissue. 2. Kinetic characteristic of expression of BPTI in tobacco (1) seeding and adult We detected the expression activity of the transgenic tobacco in seedlingstage and adult stage. The quantitative results showed that the proteaseinhibiting activity of BPTI was not obvious difference in seeding stage andadult stage of the same tobacco. (2)Distribution in diverse positions The protease inhibiting active assay displayed that there were not obviousdifference in various leaves of the same plant. We detected the proteaseinhibiting activity of BPTI extracted from the root, stem, diachyma andnervation respectively, which indicated that the activity in root was lower, andthe activity was not existed in the stem and nervation. The reason of thesediscrepancy was unknown. It is may be because of the distinct physiologicalfunctions in all parts of a plant. (3) Research of genetic stability We researched the transverse genetic stability, and there were activity in thenewborn shoots that were similar to the stock plant. The evidence stated clearly...