大豆疫霉多聚半乳糖醛酸酶pspg1基因毕赤酵母表达载体的构建Construction of Expression Vector of Polygalacturonase Gene pspg1 from Phytophthora sojae
孙文秀;
摘要(Abstract):
[目的]构建大豆疫霉多聚半乳糖醛酸酶pspg1基因毕赤酵母表达载体。[方法]利用PCR方法从大豆疫霉菌cDNA中扩增多聚半乳糖醛酸酶pspg1基因,用限制性内切酶EcoRⅠ和NotⅠ分别酶切此基因和毕赤酵母表达载体pPIC9K,然后用T4连接酶将目的基因克隆到pPIC9K载体中构建此载体。[结果]扩增的大豆疫霉菌pspg1基因成熟肽片段大小为1 250 bp。用基因特异性引物扩增阳性重组子,可得到大小为1 200 bp的片段。而用载体特异性引物扩增阳性重组子,可得到大小为1 200和1 700 bp两条带,多出来的400 bp恰好符合载体部分的大小。将此特异性片段PCR扩增回收,序列测定发现大豆疫霉pspg1基因已经成功地插入到表达载体中。[结论]该研究为进一步研究大豆疫霉菌pspg1基因在毕赤酵母中高效表达奠定了基础。
关键词(KeyWords): 大豆疫霉菌;多聚半乳糖醛酸酶;毕赤酵母表达载体;构建
基金项目(Foundation): 长江大学博士创新基金项目
作者(Author): 孙文秀;
Email:
DOI: 10.13989/j.cnki.0517-6611.2009.19.058
参考文献(References):
- [1]SCHMITHENNERAF.Problems and progressingin control ofPhytophtho-ra root rot of soybean[J].Plant Disease,1985,69:462-468.
- [2]沈崇尧,苏彦纯.中国大豆疫霉根腐病的发现及初步研究[J].植物病理学报,1991,21(4):289.
- [3]MARTELMB,LETOUBLONR,FEVRE M.Purification and characteriza-tion of two endopolygalacturonases secreted during the early stages of thesaprophytic growth ofSclerotinia sclerotiorum[J].FEMS Lett,1998,158:133-138.
- [4]SAKAMOTOT,BONNINE,QUEMENERB,etal.Purification and charac-terisation of two exo-polygalacturonases fromAspergillus nigerable to de-grade xylogalacturonan and acetylated homogalacturonan[J].Biochimica etBiophysica Acta,2002,1572(1):10-18.
- [5]SALCHA,NEVINMF,EBTSANANH,etal.Biochemical characterizationof an extracellular polygalacturonase formTrichderma harzianum[J].Jour-nal of Biotechnology,2006,127:54-64.
- [6]SHIEHMI,BROWNRL,WHITEHEADMP,etal.Moleculargenetic evi-dence for the involvement of a specific polygalacturonase,Pc2,in the inva-sion and spread ofAspergillus flavusin cotton bolls[J].Appl Environ Mi-crobiol,1997,63:3548-3552.
- [7]ISSHIKI A,AKIMITSUK,YAMAMOTOM,et al.Endopolygalacturonase isessential for citrus black rot caused byAlternaria citribut not brown spotcaused byAlternaria alternata[J].Mol Plant-Microbe Interact,2001,14:749-761.
- [8]LI J,GOODWIN P H.Expression ofcgmpg2,an endopolygalacturonasegene ofColletotrichum gloeosporioidesf.sp.Malvae,in culture and duringinfection ofMalva pusilla[J].J Phytopathology,2002,150:213-219.
- [9]COTTONP,RASCLEC,FEVREM,etal.Characterization ofPG2,an earlyendo PG produced bySclerotinia sclerotiorum,expressed in yeast[J].FEMS Microbiology Letters,2002,213:239-244.
- [10]GOTESSON A,MARSHALL J S,HARDHAMA R.Characterization andevolutionaryanalysisofa large polygalacturonase gene familyinthe oomy-cete plantpathogenPhytophthora cinnamomi[J].MPMI,2002,15(9):907-921.
- [11]巩振辉,ARVIDG,DAVIDAJ.樟疫霉多聚半乳糖醛酸酶Pcpg1、Pcpg2和Pcpg4基因的克隆、测序及其遗传转化[J].农业生物技术学报,2003,11(5):477-482.
- [12]巩振辉,ARVIDG,DAVIDAJ,等.樟疫霉多聚半乳糖醛酸酶基因9和10的克隆、测序及其遗传转化研究[J].西北农林科技大学学报,2004,32(8):1-6.
- [13]TORTOTA,RAUSERL,KAMOUNS,etal.Thepipg1 gene of the oomy-cetePhytophthora infestansencodes a fungal-like endopolygalacturonase[J].Curr Genet,2002,40:385-390.
- [14]YANHZ,LIOURL.Cloningand analysisofpppg1,aninducible endopo-lygalacturonase gene from the oomycete plant pathogenPhytophthoraparaistica[J].Fungal Genetics and Biology,2005,42:339-350.
- [15]ROGERS LM,FLAISHMANMA,KOLATTUKUDY P E.Cutinase genedisruption inFusatiumsolanif.sp.Pisi decreses its virulence on pea[J].Plant Cell,1994,6:935-945.
- [16]SHIEHMI,BROWN R L,WHITEHEAD MP,et al.Molecular geneticevidence for the involvement of a specific polygalacturonase,Pc2,in theinvasion and spread ofAspergillus flavusin cotton bolls[J].Appl EnvironMicrobiol,1997,63:3548-3552.