植酸酶根特异表达载体的构建及大豆转化  

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

作　　者：李桂兰[1] 乔亚科[1] 李明刚[2] 崔姗姗[1] 乔潇[3] 乔坤艳[2] 

机构地区：[1]河北科技师范学院生命科技学院,河北昌黎066600 [2]南开大学生命科学学院,天津300071 [3]上海大学,材料科学与工程学院,上海200072

出　　处：《核农学报》2011年第5期892-897,共6页Journal of Nuclear Agricultural Sciences

基　　金：国家科技部转基因生物新品种培育重大科技专项(2009ZX08004-004B,2009ZX08004-001B);河北省自然科学基金(C2009000868)

摘　　要：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,通过5个中间载体将3段DNA片段连接构成2.9kb的表达单元Ppyk10-S-phJA(KSA),将KSA片段插入初始载体pC-GENERAL,构建成植酸酶根特异表达载体pPC-KSA。利用农杆菌介导法将无花果曲霉植酸酶基因phyA转入到栽培大豆品种吉林35中,在大豆转基因植株中正确表达,产生有活性的植酸酶,且能分泌到根外。T3代植株根系分泌植酸酶活性比未转化植株提高了2～4倍。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 thalianagenomic DNA.A cDNA of phyAgene 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 phyAgene,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 phyAgene from Aspergillusficuumwas 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 Aspergillusficuumresulted in about 2~4-fold increase of phytase activity secreted from roots in the T3generation transgenic plants compared to the non-transgenic control.

关 键 词：外展蛋白信号肽 pyk10启动子 PHYA基因 大豆 转基因 植酸酶 根特异表达 

分 类 号：S565.1[农业科学—作物学]