Study On Expressing Recombinant Phytase(rphyA) In Fatbody Of The Transgenic Silkworm

Phytase,one of the most widely used feed enzyme additives,can catalyze the hydrolysis of phytic acid into inositol and inorganic phosphate,improve the utilization efficiency of phosphorus in monogatric animals and reduce the pollution of phytate phosphorus to the enviroment. Although phytase gene has been expressed in various systems, currently it is mainly achieved through yeast fermentation system for its low cost and high yields.The silkworm is the one of economic insects which are domesticated. Its value is not confined to the traditional Sericulture industry,it can also be used as a new bioreactor to produce useful protein with low cost. In this paper, we constructed phyA transgenic expression vectors based on piggyBac transposon vector, in which phyA were driven by BmLP3promoter. Transgenic silkworms were generated by micro-injection. The main results are as follows.1. Constructing of phyA transgenic expression vector.The mature peptide fragment of phyA was obtained from patent published and synthesized after optimizing its codon for silkworm.New gene was cloned to replace DsRed cDNA in pSL[BmLP3-DsRed] and then this expression cassette was inserted into a piggyBac-derived vector pBac[3xp3-DsRed] to produce pBac[3xp3-DsRed,BmLP3-phyA-SV40].2. The generation of germline transgenic silkwormWe injected the transgenic vector pBac[3xp3-DsRed, BmLP3-phyA-SV40] with helper plasmid into Bombyx mori7532eggs at the preblastodermal stage. Among8broods injected by BmLP3-phyA, fluorescence were detected in2broods, the G1brood positive frequency reached to25%, but only one brood survived.3. Molecular detection of transgenic silkwormRNA were extracted from tissues of G2silkworm of5th day of5-instar. qPCR assay showed that phyA specially expressed in the fat body of transgenic silkworm. Southern blot revealed that only one insertion fragment was integrated into the chromosome of silkworm. The insertion site was recovered and sequenced by inverse-PCR technique, the results showed that the insertion fragment of BmLP3-phyA transgenic line was located in the14th chromosome.4. Detection of rphyA proteinPolyclonal antibody of phyA was prepared for the detection of the recombinant phytase in silkworm. The gene was cloned into pET-28a expression vector to generate recombinant plasmid and then transformed into Rosetta strain. After the IPTG induction, recombinant protein expressed in the form of inclusion bodies. Protein was purified through affinity chromatography and ultrafiltration,and immunize rabbit to prepared the polyclonal antibody.The protein of fat body in transgenic silkworms was extracted with PBS, no significant discrepancy was observed on SDS-PAGE,wsetern blot results showed that the recombinat phyA protein were expressed successfully in transgenic silkworm.5Activity determination of phytaseTotal protein of transgenic silkworm was extracted from pupa and phytase activity was determined according to GBT18634-20. The results show that transgenic silkworm have significant enzyme activity with its highest activity up to99.05U/g.The recombinant phytase shows an optimum temperature at55℃and has two optimum pH in1.5-2.0and5.5-6.0. The research primarily confirmed that using fat body of silkworm as bioreactor to produce value-added exogenous protein is feasible.