植酸酶根特异表达载体的构建及大豆转化

应用PCR方法分别从胡萝卜基因组中扩增出96bp的外展蛋白信号肽编码序列片段,从拟南芥基因组中扩增出1454bp的pyk10启动子片段,用RT-PCR方法从无花果曲霉（Aspergillus ficuum3.4322）中扩增出phyA基因,长1347bp。然后,分别克隆到pMD18-T载体。应用已设计的限制酶切位点,通...

CONSTRUCTION OF THE VECTOR OF PHYTASE GENE SPECIFIC EXPRESSION IN ROOTS AND TRANSFORMATION OF SOYBEAN

A 96bp sequence encoding the extension signal peptide was cloned by PCR from carrot(Daucus、carota L var. sativus H offm) genomic DNA. It was identical to the sequence reported (GenBank accession No: XO2873). A promoter fragment of 1454bp long of Pyk10 gene was cloned by PCR from Arabidopsis thaliana genomic DNA. A cDNA of phyA gene was cloned from Aspergillus ficuum 3.4322 by RT-PCR, which comprised of 1347bp without signal peptide coding sequence. An expression cassette Ppyk10-s-phyA (KSA), comprising of the pyk10 promoter fragment, the carrot extension signal peptide coding sequence and the cDNA of phyA gene, was successfully constructed through five transitional plasmids. The expression plasmid of pPC-KSA was constructed finally by inserting KSA fragment into original vector pC-GENERAL.The phyA gene from Aspergillus ficuum was successfully transfermed into soybean (Glycine max L. Merr. c.v. Jilin 35) via Agrobacterium tumefaciens-mediated method, which was able to be expressed in the recipient soybean cultivar. The introduction of fungal phytase from Aspergillus ficuum resulted in about 2~4-fold increase of phytase activity secreted from roots in the T3generation transgenic plants compared to the non-transgenic control.