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).

黑芥子酶是一类催化水解芥子油苷的同工酶，Pyk10是在拟南芥根和下胚轴中特异表达的黑芥子酶基因，Pyk10基因的启动子具有根组织特异性。根据已发表的序列，用PCR方法从拟南芥基因组中扩增并克隆了Pyk10基因启动子片段。序列分表明：该片段全长1454bp，与已发表序列同源性达到98%。该启动子片段中含有多种在其他植物启动子中发现的通用启动子元件，并含有器官和组织特异性转录因子的结合位点有ACGT-、CANNTG-、GATA-以及I盒等顺式元件。Pyk10根特异性启动子的成功克隆，为培育抗根部病虫害和营养高效利用型转基因植物奠定了基础。

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.

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.

In this study we purified both acid phosphatases (Apase) secreted from tomato and lupin roots, and compared the properties of these two enzymes. The secretory Apases from tomato and lupin showed the following similar properties. 1) Both enzymes were homodimers consisting of two identical subunits, each with a molecular weight of approximately 68 kilodaltons (kD) in tomato and 72kD in lupin. 2) The enzymes were stable in the pH range of 4-9. 3) The enzymes showed an optimum temperature of 37-40℃ for their activity and were stable at temperatures below 60℃. 4) The enzymes exhibited a omparable affinity for p-nitrophenyl phosphate (the apparent Km values were 2.7-3.0×10-5M). On the other hand, there were some slight differences in the isoelectric point, optimum pH, specific activity, substrate specificity, and inhibitory effect of metal ions between the two enzymes. herefore, it was considered that the most important difference in the root ability of the two plants to hydrolyze organic phosphorus may depend mainly on the amount of Apase secreted from a unit amount of roots.