| Bovine chymosin, which was firstly obtained in rumen of unweaned calf, is widely used incheese industry. With the increasing global demand of cheese, the nick of calf derived chymosinis getting larger, and therefore it is a great challenge for researchers to find other new chymosinresources to meet demand of the market. Plant bioreactor, differing from conventionalexpression systems like Escherich coli and yeast, will be an alternative to express variousrecombinant proteins via biotechnology, and shows great potential in the future. Application ofplant bioreactor could be of remarkable significant to push cheese industry forward.In this study, a high frequency of regeneration system from leaf explants of tomato cultivar“green cherry” was established. Based on this regeneration system, the bovine chymosin genewas introduced into tomato genome via Agrobacterium-mediated transformation, expressingCYM protein in tomato cells. Before that, the bovine prochymosin gene was firstly transferredinto E. coli which has feature of prokaryotic expression mechanism to confirm the function ofchymosin gene. The aim of this study is to develop a large-scale production pipeline so as tomeet the increasing demand of bovine chymosin home and abroad. The main results are asfollowed:(1) A prokaryotic expression vector pET28a-cym, fused His-tag with bovine prochymosin,was constructed and transformed into E. coli. The transformed E. coli strain can yield a higherproduction of target protein when induced by ITPG under37℃condition; most of the targetprotein is soluble. The prochymosin with His-tag was purified by Ni+affinity chromatography,and SDS-PAGE analysis demonstrated that purity of protein eluted by elution buffer containing100mM imidazole can up to90%.(2) The leaves of four tomato cultivars were exploited as explants to establish an efficientregeneration system, depending on the optimized combination of various tissue culture factorssuch as concentration of different hormones, seed germination ratio, calli induction frequency,differentiation rate of adventitious bud. The results demonstrated that, the leaves from cultivar“green cherry” yielded the best regeneration rate in MS media containing3.0mg/l of6-BA and0.2mg/l of IAA. The induction ratio of calli and adventitious bud were96.88%and90.63%respectively and the adventitious buds rate per explant was252%. The proper medium forrooting was MS medium supplemented with0.2mg/l of IAA. (3) Twenty-six putative resistant plantlets were obtained by multiple attempts using tomatoleaves as explants transformed with a binary vector harboring bovine preprochymosin gene andbar gene. Five of them were positive tested by PCR amplification with bar gene, and four of thePCR-positive plants are also positive tasted with preprochymosin gene.(4) Collaurum labeled immune tests were carried out to determinate the expression of bargene, the results demonstrated that the bar gene was expressed in four out of the fivePCR-positive plants, and the integration of preprochymosin gene with genome of these fourPCR-positive tomato plants was reconfirmed further by Southern blot analysis. Milk-clottingactivity of total soluble protein extracted from leaves of transgenic plants was studied and themilk-clotting activity was observed only in No.3transgenic plant, indicating the amount ofrecombinant chymosin accumulation in this plant is sufficient to cause milk coagulation. |
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