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| Prediction, Prokaryotic Expression and Related Functions Analysis of Azorhizobium caulinodans ORS571 T3SS Effector Proteins in Sesbania cannabina |
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Abstract Rhizobium type Ⅲ secretion system (T3SS) effector proteins play important roles in nodulation, nitrogen fixation and decision of host range. In order to express and analysis the type Ⅲ-like secretion system effector proteins of Azorhizobium caulinodans ORS571, the whole proteome of ORS571 was analyzed by bioinformatics software BPBAac and Effective T3 and related literature reports. Seven candidate effector proteins of ORS571 were obtained, and they were AZC_0308, AZC_0649, AZC_0667, AZC_2699, AZC_2928, AZC_3165 and AZC_4087, respectively. Specific amplification primers with restriction enzyme cutting sites were designed and synthesized, and whole gene sequences of the 7 effector proteins were obtained by PCR. Than the 7 gene fragments were cloned into the prokaryotic expression vector pMAL-c4x, respectively. Through optimizing the expression conditions, a large amount of soluble fusion proteins were obtained. Amylose Resin affinity chromatography was used to purify fusion proteins, and the results indicated that purification effect was better. Detection of fusion proteins expression were completed by Western blot using anti- maltose binding protein (MBP) monoclonal antibody (horse radish peroxidase conjugated), and the results implied that these 7 fusion proteins expressed correctly and the effect of protein expression was better. AZC_2928 gene deletion mutant was constructed and infected wheat (Triticum aestivum) XiaoYan 22, and the results showed that AZC_2928 gene had inhibitory effect on the growth of wheat. In conclusion, this research lays a foundation for the application of the Azorhizobium caulinodans ORS571 type Ⅲ-like secretion system effector proteins in symbiotic nitrogen fixation of non-leguminous plants.
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Received: 30 September 2016
Published: 02 March 2017
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