Abstract

Valsa mali var. mali is a causal agent of apple tree canker disease, that causes serious economic losses in China. The lack of understanding of the pathogenic genes and mechanisms of V. mali, which poses difficulties to control the disease. Whereas, previous studies have indicated that xylanase produced by V. mali is a key pathogenic factor. In this study, Xylanase gene was cloned and obtained recombinant expression vector pET-Xylanase, in addition, a prokaryotic expression system for Xylanase protein was established and the expression conditions were optimized. Xylanase protein expressed in Escherichia coli reached a maximum level after induction with 0.5 mmol/L isopropyl ß-thiogalactopyranoside (IPTG) for 12 h at 30°C. The recombinant protein was confirmed by Western blot analysis using Anti-His mouse monoclonal antibody and the result showed that the induced ~63 kDa band in E. coli was Xylanase protein. The results presented will help to better reveal the xylanase gene functions in the pathogenicity of V. mali.

Keywords:

Condition optimization, Prokaryotic expression, SDS-PAGE, Western blot, Xylanase protein,

References

1. Abe, K., N. Kotoda, H. Kato and J. Soejima, 2007. Resistance sources to Valsa canker (Valsa ceratosperma) in a germplasm collection of diverse Malus species. Plant Breeding, 126: 449-453.
   CrossRef    
2. Abe, K., N. Kotoda, H. Kato and J. Soejima, 2011. Genetic studies on resistance to Valsa canker in apple: Genetic variance and breeding values estimated from intra-and inter-specific hybrid progeny populations. Tree Genet. Genomes, 7: 363-372.
   CrossRef    
3. Bai, S., C. Dong, B. Li and H. Dai, 2013. A PR-4 gene identified from Malus domesticais involved in the defense responses against Botryosphaeria dothidea. Plant Physiol. Biochem., 62: 23-32.
   CrossRef    PMid:23178481    
4. Cao, K.Q., L.Y. Guo, B.H. Li, G.Y. Sun and H.J. Chen, 2009. Investigations on the occurrence and control of apple canker in China. Plant Protect., 35: 114-117.
   
5. Chen, C., M.N. Li, X.Q. Shi, J.G. Guo, Z.F. Xing, X.W. Zhang and Y.X. Chen, 1987. Studies on the infection period of Valsa mali Miyabe et Yamada, the causal agent of apple tree canker. Acta Phytopathol. Sin., 17: 65-68.
   
6. Chen, C., 2009. Research on the Occurrence Regularity and Control of Apple Tree Canker. China Agricultural Scientech Press, Beijing, China, pp: 9-17.
   
7. Chen, X.L., C.W. Niu, B.H. Li, G.F. Li and C.X. Wang, 2012. The kinds and activities of cell wall-degrading enzymes produced by Valsa ceratosperma. Acta Agr. Boreali-Sin., 27: 207-212.
   
8. Gao, J., Y.B. Li, X.W. Ke, Z.S. Kang and L.L. Huang, 2011. Development of genetic transformation system of Valsa mali of apple mediated by PEG. Acta Microbiol. Sin., 51: 1194-1199.
   
v
9. Hannig, G. and S.C. Makrides, 1998. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol., 16: 54-60.
   CrossRef    
10. Heegaard, E.D., C.J. Rasksen and J. Christensen, 2002. Detection of parvovirus B19 NS1-specific antibodies by ELISA and western blotting employing recombinant NS1 protein as antigen. J. Med. Virol., 67: 375-383.
    CrossRef    PMid:12116031    
11. Koganezawa, H. and T. Sakuma, 1982. Possible role of breakdown products of phloridzin in symptom development by Valsa ceratosperma. Ann. Phytopathol. Soc. Jpn., 48: 521-528.
    CrossRef    
12. Li, B.H., C.X. Wang and X.L. Dong, 2013. Research progress in apple diseases and problems in the disease management in China. Plant Protect., 39: 46-54.
    
13. Li, C., B.H. Li, G.F. Li and C.X. Wang, 2014. Pathogenic factors produced by Valsa mali var. mali and their relationship with pathogenicity of different strains. North. Hortic., 13: 118-122.
    
14. Liu, F.C., M.N. Li and Y.Q. Wang, 1980. Preliminary study on the virulence factor (pectinase) of valsa ceratosperma. China Fruits, 4: 45-48.
    
15. Munjal, N., K. Jawed, S. Wajid and S.S. Yazdani, 2015. A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production. PloS One, 10: e0119917.
    CrossRef    PMid:25768292 PMCid:PMC4358894    
16. Natsume, H., H. Seto, S. Haruo and N. Otake, 1982. Studies on apple canker disease: the necrotic toxins produced by Valsa ceratosperma. Agr. Biol. Chem. Tokyo, 46: 2101-2106.
    CrossRef    
17. Pei, D.L., H.Y. Zhang, M. Li, Z.L. Duan and C.W. Li, 2014. Optimization of prokaryotic expression of ShSTU1 gene in tomato resistant to powdery mildew. J. Henan Univ., Nat. Sci., 44(2): 202-207.
    
18. Sørensen, H.P. and K.K. Mortensen, 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol., 115: 113-128.
    CrossRef    PMid:15607230    
19. Wang, C., C. Li, B. Li, G. Li, X. Dong, G. Wang and Q. Zhang, 2014. Toxins produced by Valsa mali var. mali and their relationship with pathogenicity. Toxins, 6(3): 1139-1154.
    CrossRef    PMid:24662481 PMCid:PMC3968381    
20. Wang, C.X., N. Hong, G.P. Wang, J.K. Zhang and L. Hui, 2007. Molecular analysis of a Chinese citrus tristeza virus isolate showing anomalous serological reactions. J. Plant Pathol., 89(3): 377-383.
    
21. Wang, C.X., X.L. Dong, Z.F. Zhang, G.F. Li and B.H. Li, 2012. Outbreak and the reasons of apple valsa canker in Yantai apple production area in 2011. Plant Protect., 38: 136-138.
    
22. Wang, C.X., X.N. Guan, H.H. Wang, G.F. Li, X.L. Dong, G.P. Wang and B.H. Li, 2013. Agrobacterium tumefaciens- mediated transformation of Valsa mali: An efficient tool for random insertion mutagenesis. Sci. World J., 2013: 11.
    CrossRef    
23. Wang, J., Q. Ma, X. Zhuang and L.P. Yang, 2009. Determination of pectinase in fungi secretion of apple tree canker. Inner Mongolia Agric. Sci. Tech., 4: 39-40.
    
24. Zhang, Q., C. Wang, D. Yong, G. Li, X. Dong and B. Li, 2014. Induction of resistance mediated by an attenuated strain of Valsa mali var. mali using pathogen-apple callus interaction system. Sci. World J., 2014: 201382.
    
25. Zhao, H., C.X. Wang, X.R. Chen, H.Y. Wang and B.H. Li, 2012. Methods of promoting porulation of Valsa ceratosperma. Chin. Agric. Sci. Bull., 28: 151-154.
    

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