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cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli

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Abstract

Powdery mildew, caused by Uncinula necator Burr, is one of the most seriously damaging diseases of grapevine all over the world. To gain the novel gene and investigate the resistance mechanism in Chinese Wild Vitis pseudoreticulata clone Baihe-35-1, mRNA differential display was employed to study the differential expression of the resistant gene to the disease of it when inoculated by Uncinula necator under natural field conditions, 5′ RACE and 3′ RACE have been used to clone the whole cDNA sequences of VpAPX, the novel gene related to Ascorbate Peroxidase which involved in resistant to the disease, is composed of specific sequence 1077 bp and has an open reading frame of 750 bp coding for 250 amino acid residues with a molecular weight of 27.566 kDa. The VpAPX gene was obtained by polymerase chain reaction (PCR) with the special primers synthesized according to the sequences of cDNA, and further cloned it into the pGEM-T easy vector. The cloned VpAPX gene was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector pGEX-4T-1, then transferred into E. coli BL21. As result, GST-VpAPX fusion protein was successfully expressed by induction of IPTG and purified by GST affinity resin. After injecting rabbit, the polyclonal antibodies were produced. Western blot analyses showed that the antibody reacted specifically to GST-VpAPX fusion protein and the titer for this antibody is 105. This research made the foundation to transform the VpAPX gene into grape plants for follow research in processing.

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Abbreviations

APX:

ascorbase peroxidase

DDRT-PCR:

differential display reverse transcript polymerase chain reaction

GST:

glutathions-S-transferase

IPTG:

isopropyl β-d-thiogalactoside

5′ RACE:

rapid amplification of 5′ cDNA end

3′ RACE:

rapid amplification of 3′ cDNA end

References

  1. JM Lenne D Wood (1991) Ann. Rev. Phytopathol. 29 35–63 Occurrence Handle10.1146/annurev.py.29.090191.000343

    Article  Google Scholar 

  2. YJ Wang Y Liu PC He J Chen DW Ramming J Lu (1995) Vitis 34 159–164

    Google Scholar 

  3. YJ Wang PC He (1997) Sci. Agr. Sinica 3 19–25

    Google Scholar 

  4. PC He YJ Wang Z Ren C He (1991) Sci. Agr. Sinica 24 50–56

    Google Scholar 

  5. G Allewwldt JV Possingham (1998) Theoret. Appl. Genet. 75 669–673

    Google Scholar 

  6. JJ Zhang YJ Wang XP Wang KQ Yang JX Yang (2003) J. Fruit Sci. (in Chinese) 20 178–189 Occurrence Handle1:CAS:528:DC%2BD3sXmtVOiurw%3D

    CAS  Google Scholar 

  7. P Liang AB Pardee (1992) Science 257 967–971 Occurrence Handle1354393 Occurrence Handle1:CAS:528:DyaK38Xls1Cqt70%3D

    PubMed  CAS  Google Scholar 

  8. BJ Bassam G Caetano-Anollés PM Gresshoff (1991) Anal. Biochem. 196 80–83 Occurrence Handle1716076 Occurrence Handle1:CAS:528:DyaK3MXks1Ortr8%3D Occurrence Handle10.1016/0003-2697(91)90120-I

    Article  PubMed  CAS  Google Scholar 

  9. Wang XP (2004) Cloning and Analyzing for the Gene Sequences of Resistance to Uncinula necator in Chinese Wild Vitis Species. Dissertation for Doctoral Degree (Graduation) of Northwest A&F University

  10. WR Patterson LT Poulos (1995) Biochemistry 34 4331–4341 Occurrence Handle7703247 Occurrence Handle1:CAS:528:DyaK2MXks1Grs7o%3D Occurrence Handle10.1021/bi00013a023

    Article  PubMed  CAS  Google Scholar 

  11. R Mittler BA Zilinskas (1992) J. Biol. Chem. 5 21802–21807

    Google Scholar 

  12. C Bowler MV Montagu D Inze (1992) Annu. Rev. Plant Physiol. 43 83–116 Occurrence Handle1:CAS:528:DyaK38XltVyjsb0%3D Occurrence Handle10.1146/annurev.pp.43.060192.000503

    Article  CAS  Google Scholar 

  13. MJH Jimenew MM Lucas MR Felipe (2002) Plant Physiol. Biochem. 40 645–647 Occurrence Handle10.1016/S0981-9428(02)01422-5

    Article  Google Scholar 

  14. CL Ma PP Wang ZY Cao YX Zhao H Zhang (2002) J. Plant Physiol. Mol. Bio. 28 261–266 Occurrence Handle1:CAS:528:DC%2BD2cXksVWqsL4%3D

    CAS  Google Scholar 

  15. FK Teixeira LM Benavente R Margis MM Pinheiro (2004) Mol. Evol. 59 761–770 Occurrence Handle1:CAS:528:DC%2BD2cXhtVKrurvL Occurrence Handle10.1007/s00239-004-2666-z

    Article  CAS  Google Scholar 

  16. WH Chao (1994) Plant Physiol. Commun. 30 452–458

    Google Scholar 

  17. K Yoshimura Y Yabuta T Ishikawa S Shigeoka (2000) Plant Physiol. 123 223–232 Occurrence Handle10806239 Occurrence Handle1:CAS:528:DC%2BD3cXjsFemsrk%3D Occurrence Handle10.1104/pp.123.1.223

    Article  PubMed  CAS  Google Scholar 

  18. WB Shen LQ Huang LL Xu (1997) Life Sci. 17 24–26 Occurrence Handle1:CAS:528:DyaK2sXmvFOmtr4%3D

    CAS  Google Scholar 

  19. A Kubo H Saji K Tanaka N Kondo (1992) Plant Mol. Biol. 18 691–701 Occurrence Handle1558944 Occurrence Handle1:CAS:528:DyaK3sXitVygsbY%3D Occurrence Handle10.1007/BF00020011

    Article  PubMed  CAS  Google Scholar 

  20. CR Caldwell FJ Tarano MB McMahon (1998) Planta 204 120–126 Occurrence Handle9443387 Occurrence Handle1:CAS:528:DyaK2sXnvVygtb8%3D Occurrence Handle10.1007/s004250050237

    Article  PubMed  CAS  Google Scholar 

  21. S Kawakami Y Matsumoto A Matsunaga S Mayama M Mizuno (2002) Plant Sci. 163 829–836 Occurrence Handle1:CAS:528:DC%2BD38XnsVSgsbw%3D Occurrence Handle10.1016/S0168-9452(02)00232-7

    Article  CAS  Google Scholar 

  22. Kim WI Chung (1998) Plant Sci. 133 69–77 Occurrence Handle1:CAS:528:DyaK1cXis1yis78%3D Occurrence Handle10.1016/S0168-9452(98)00029-6

    Article  CAS  Google Scholar 

  23. M Ding JF Yu RD Ding (1998) Biotechnology 8 5–8

    Google Scholar 

  24. KG Ford AJ Whitmarsh DQ Hornby (1994) Methods Mol. Biol. 30 185–197 Occurrence Handle8004194 Occurrence Handle1:CAS:528:DyaK2cXmsleisLY%3D

    PubMed  CAS  Google Scholar 

  25. Sambrook & Russell DW (2001) Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor laboratory press, Cold Spring Harbor, NY pp. 1217–1220 and 1224–1225.

  26. JT Aatsinki HJ Rajaniemi (2005) Protein Express. Purif. 40 287–291 Occurrence Handle1:CAS:528:DC%2BD2MXit1Cjsrk%3D Occurrence Handle10.1016/j.pep.2004.11.012

    Article  CAS  Google Scholar 

  27. W Cao YX Zhou S Ma QP Luo DZ Wei (2005) Protein Express. Purif. 40 404–410 Occurrence Handle1:CAS:528:DC%2BD2MXit1CjsL4%3D Occurrence Handle10.1016/j.pep.2004.12.007

    Article  CAS  Google Scholar 

  28. XP Yin DZ Wei LN Yi XY Tao YS Ma (2005) Protein Express. Purif. 41 259–265 Occurrence Handle1:CAS:528:DC%2BD2MXjslKit7k%3D Occurrence Handle10.1016/j.pep.2004.10.014

    Article  CAS  Google Scholar 

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Correspondence to Yuejin Wang.

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Ling Lin, Xiping Wang: These two authors contributed equally to this work.

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Lin, L., Wang, X. & Wang, Y. cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli . Mol Biol Rep 33, 197–206 (2006). https://doi.org/10.1007/s11033-006-0008-5

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