Preliminary Study Of Mannose Oligosaccharides On Plant Resistance Induction And Cloning And Expression Of A β-mannanase Gene From Bacillus Subtilis Z-2

Mannose-oligosaccharides (MOS) are carbohydrates that consist of 2-10 glucose and mannose. The elicitor activity of MOS on plant was investigated in a melon-powdery mildew disease system. Results showed that all the elicitors tested, including benzothiadiazoles (BTH), culture extract of Flammulina velutips (JZG) and MOS, resulted in lower disease index on the treated plants compared to the water control. Inoculation with inducers led to an increase in activities of chitinase, P -1, 3-glucanase, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (PO) in melon leaf. The peaks of chitinase and β -1, 3-glucanase activities were observed 13 and 16 days after the treatment, respectively. The activities of PAL increased all along, and the maximum activity was observed 6 day after elicitors treatments. They also stimulated the activities of PPO and PO.β -mannanases (endo-l,4-D-mannanase, mannan endo-1,4- β-mannosidase, EC3.2.1.78) are endohydrolases that catalyze the random hydrolysis of β -1,4-mannosidic linkages in the main chain of galactomannan, glucomannan, galactoglucomannan and mannan, yielding oligosaccharides that consist of 2-10 monosaccharides. This paper described a mannanase-producing bacterial strain Z-2 isolated from soil samples in different districts. The strain Z-2 was identified as Bacillus subtilis by the homologous analysis of 16S rDNA sequence, and the morphological and physiological characterization. The culture supertant of Z-2 showed mannanase activity at below 55℃ under pH 5.0-8.0, with an optimum at 55℃ and pH 6.0. The enzyme hydrolyzed Konjak powder and locust bean gum and produced oligosaccharides, but not monosaccharide.A genomic library of Z-2 was constructed by cosmid pLARF-5 and man gene was obtained from the library by using PCR-mediated screening method. The man gene with wild-type and pelB signal peptide were constructed in a expression vector pET22b(+), resuited in piasmids pET-Nco13 (pelB signal peptide) and pET-NdeI18 (intact signal peptide), and expressed in E. coli BL21 (DE3) by IPTG induction for 4 h. The extracellular activity of Man expressed by pET-NcoI3 showed two-fold higher than that of pET-Ndel 18. The culture conditions, including media composition, culture temperature, inducer concentration and different cell densities were optimized to improve the enzyme production. A final enzyme activity of 55.33 U/mL was detected when the E. coli BL21 containing pET-Ncol3 was cultured in TB medium at 32℃ to the A_600nm of 0.75 and induced with 0.01 mM IPTG for 4 h.Wild-type man gene from B. subtilis Z-2 does not express or shows a very low activity in Pseudomonads. To improve the Man expression in other bacteria, its native RBS (GGGGAG), translation initiation codon (TTG) and intact signal peptide of man gene were changed into AAGGAG, ATG and pelB signal peptide of pET22b(+). The modified man gene was inserted at the downstream of lac promoter of the shuttle vector pRK415 to obtain pMAN-TTG (wild man), pMAN-BE (pelB) and pMAN-NA (wild signal sequence). In Pseudomonas fluorescens 2P24 containing pMAN-BE andpMAN-NA, both the extracellular and endocellular mannanase could be detected under the induction by Konjack powder. The extracellular activity was 1.55 U/mL and the endocellular activity was 3.8 U/mg. In greenhouse, the Man-expressing P. fluorescens 2P24 was applied on tobacco leaves and could reduce number of lesion caused by TMV compared with the wild type 2P24 treatment.Taken together, MOS showed defense induction activity on both melon and tobacco and could be regarded as an effective eh'citor. Man gene from B. suktilis Z-2 has a potential to be used in MOS production and biological control of plant diseases, and its expression is possible to be strongly improved by genetic modification.