Phosphorus (P) deficiency increased the secretion of phytases from roots of various plant species. The secretory phytases were collected with a dialysis membrane tube for 24 hours from roots of sixteen plant species grown with low or adequate supply of P in nutrient solutions. The activity of not only secretory phytase, but also acid phosphatase, increased with the low P treatment in all of the plant species examined. Secretion of phytase by the roots under P-deficient conditions was highest in Brachiaria decumbens CIAT 606, Stylosanthes guianensis CIAT 148 and tomato, moderate in Brachiaria brizantha CIAT6780, Stylosanthes guianensis CIAT 2950, Alfalfa, white clover and orchard grass, and lowest in Andropgon gayanus CIAT 621, Stylosanthes capitata CIAT 10280, upland rice, timothy, redtop, alsike clover, red clover and white lupin plants. SDS-PAGE and immunoblotting analysis showed that the phytases with molecular weight of 35-40 kD could be detected in seven of the sixteen plant species tested. These results indicate that the secretory phytase may provide an efficient mechanism for certain plants to utilize inositol hexaphosphate in soil.

The distribution of secretory acid phosphatase and organic acids enhanced by phosphorus deficiency in lupin rhizosphere was investigated using a rhizo-box system which separated the rhizosphere soil in 0.5mm fractions. In the soil fraction closest to the root surface, the lupin exudates displayed an acid phosphatase activity of 0.73ug dry soil-1 and citrate concentration of 85.2μmole⋅g dry soil-1, respectively. The increase of the acid phosphatase activity induced an appreciable depletion of organic P in the rhizosphere, indicating that lupin efficiently utilized the organic P from soil through the enzyme activity. The sterile treatments demonstrated that the acid phosphatase in the rhizosphere was mainly derived from lupin root secretions. The secretory organic acids enhanced considerably the solubility of the inorganic P in three types of soil and a sludge. However, the secretory acid phosphatase and organic acids from lupin roots were only detected in a considerable amount in 0-2.5mm soil fractions from root surface.

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.

采用异硫氰酸胍法从发芽3-4天的番茄幼苗中提取出总RNA，用Oligo（dT）纤维素亲和层析分离纯化mRNA。以mRNA为模板，Oligo（dT）为引物用逆转录法合成双链cDNA。将cDNA与EcoRI接头连接后克隆到表达载体λgt11的单一EcoRI位点，经体外包装后感染宿主菌Y1090，成功地构建了完整的番茄幼苗cDNA文库。用颜色筛选法筛选重组子，然后用植酸酶抗体做探针进行免疫筛选，得到两个阳性克隆。挑取阳性克隆噬菌斑扩增后纯化DNA，经琼脂糖凝胶电泳鉴定，显示确有外源DNA存在。该插入片断序列测定正在进行。

An intact insulin gene was synthesized by half-enzymic method and cloned into expression vector pPIN9K. After transferring the expression vector pPINS319 into Pichia pastoris GS115, two Pichia pastoris strains secreting high-level human insulin were obtained. The expressed human insulin proteins of recombinant 4 and 9 were 76 and 62 times higher than that of the negative control, respectively. Moreover 88%-90% of the insulin was secreted out of the cells. The actual insulin products of recombinant 4 and 9 calculated according to the sample volume reached 0.563g/l and 0.441g/l, respectively.