Silicate bacteria or Bacillus mucilaginosus, which is a special Bacillus species producing a rich variety of exo-polysaccharides and have some capability of dissolving potassium, phosphorus and silicate from minerals, may have wide potential uses in agriculture and industry. In order to increase its capability on hydrolyzing phytate phosphorus so as to make a microbial fertilizer, a transgenic Bacillus strain highly secreting phytase was constructed by the particle bombardment method using a phytase secreting expression vector pSP43 with a mini-tn5 transposon and a phytase expression cassette. In this study, the vector pSP43 was triumphantly transferred into the Bacillus mucilaginosus, and three transgenic strains with a stable copy of phytase expression cassette integrated into the chromosomal of the Bacillus mucilaginosus by tn5 transposition were selected. The phytase activity of engineering strains obtained increased 36-46times in comparison with the start strain D4B1. This is the first report made a transgenic Bacillus strain by the particle bombardment method successfully.

This paper reports a high efficient method for making cohesive end of double-stranded DNA into blunt end. Klenow fragment and Pfu DNA polymerase were used to fill-in the cohesive ends, and their illing-in efficiency and the corresponding cloning efficiency were compared. The results indicated the filling-in efficiency of Pfu DNA polymerase is 1.89 times that of Klenow fragment, and the difference was significant (p﹤0.01).

1.4-kb phytase gene amplified by RT-PCR from A. ficuum total RNA was successfully cloned into the expression vector pYES2, subsequently the recombinant expression vector pYPA1 was obtained. By the transformation with pYPA1, a transgenic S. cerevisiae that could express extracellular functional phytase was constructed. The successful construction of this engineering yeast strain made the roundwork for research on feed additive and microbial fertilizer.

A phyA gene was cloned from Aspergillus ficuum 3.4322 by reverse transcription polymerase chain reaction. The amplified fragment was cloned into the pMD18 T-vector and sequenced. Nucleotide equence analysis of the phyA gene showed that it comprised 1347bp without the signal peptide sequence and encodes a polypeptide of 448 amino acids. The phyA sequence has been deposited in GenBank (accession number: AF537344). Expression vectors pYPA1 and pYPA2 were constructed by cloning the phyA gene with and without the signal peptide sequence into the yeast expression vector pYES2. The recombinant plasmids were transformed into Saccharomyces cerevisiae INVSc1 by the method of LiAc. Phytase activity was found in pYPA2 (about 11.55IU/ml) endocellular fluid and in pYPA1 supernatant (about 11.60IU/mL) by galactose inducing. The results demonstrated that the phyA gene had been expressed in Saccharomyces cerevisiae and the signal sequence of Aspergillu ficuum3.4322 could facilitate the phytase secretion from S. cerevisiae efficiently.

Phyt1 gene was successfully transferred into soybean (Glycine max L. Merr. c.v. Jilin35, China) by Agrobacterium mediated method in this study. The results of molecular detections (Southern, Nouthern and Western) demonstrated that the phyt1 transgene was expressing in the transgenic soybean plant To and T1 generations, thereby caused a higher phytase activity and a lower phytate content in the ransgenic plants. This study is the first report about the transgenic soybean plants containing lower phytate.