Purification, Gene Cloning And Functional Research Of Activator Protein From Botrytis Cinerea

Activator protein is a type of protein elicitor which can induce many kinds of plants to gain systemic resistance,promotes plant growing and improve crop quality.To discover the new activator protein which has independent knowledge property right of China,an activator protein from Botrytis cinerea was isolated and purified.The protein elicitor could induce plant resistance to disease and drought.The protein coding gene was coloned and expressed in Escherichia coli and Pichia pastoris respectively.The function of expressed protein was evaluated.This research provided the genetic engineering way for obtaining an amount of activator protein quickly,which set foundation for molucular structure of protein-pesticide by analysis of relationship between protein structure and its function.The result is the following:An activitor protein named as PebCl was isolated and purified from the hypha of Botrytis cinerea BC-4-2-2-1 with the method of boiling,centrifugation,ultra-filtration and anion exchange chromatography,etc.The activator protein appeared a single band on SDS-PAGE with silver staining.Molecular weight was about 36 kD.The pI of the purified activator protein was about 4.85 based with 2-DE.Through enzyme digest in gel,the purified protein was analyzed by biological mass spectrometry(MS).Three certain peptide sequences were gotten.By blsat on NCBI,this protein has high homology to protein XP₀01551609.1 from Botrytis cinerea and comparability was 95%.Function study indicated that activator protein PebCl is able to promote wheat growth, enhance drought tolerance and increase tomato disease resisitance to gray mould.Soaking wheat seeds with different concentration of activator protein PebCl,seedling height was higher than that of the untreatment at 11 days after soaking and the best concentration of PebCl was 5μg/mL.Wheat root activity increased by 1.29 times comparing with CK when wheat seed was soaked with activator protein for 8h and coefficience of wheat drought tolerance increased from 36.53 to 57.08,which indicated that activator protein PebC1 improved wheat drought tolerance might correlate with the increasement of root activity.Tomato seeds were soaked with activator protein suspension at different concentration,then,45-day-old seedlings were sprayed with spores suspension of pathogenic fungus,results revealed that disease index of all the plants was lower than that of CK at 14^th day post inoculation with the spores of pathogenic fungus of 10⁴/mL concentration and the induced efficiency reached 31.95%～69.19%.The best concentration of PebC1 was 10μg/mL Inducing efficiency was still as high as 66.10%at 21^th day. Tnis indicated that PebC1 not only increased tomato resistance to gray mould but also kept induced resisitance for long periods.To reveal the mechanism of inducing plant disease resistance,the dynamic curve of Phenylalanine ammonia lyase(PAL),Peroxides(POD),polypheol oxidase(PPO) which related with plant resistance metabolism was measured.The activity of PAL,POD and PPO was increased after inducement.The activity of PAL increased by 46.84%at 24h.The activity of POD appeared the peak at 72h after inducement and increased 109.5%.The activity of PPO increased by 111.0%at 72h.The enhancement of the enzyme activity correlated with plant defense is one of the main physiological mechanism of protein activator inducing tomato disease resistance.With the method of reverse genetics,the PebC1 gene cDNA sequence of Botrytis cinerea was gained and which is identical to gene XM₀01551559 from Botrytis cinerea.Protein coding gene belongs to the nascent polypeptide-associated complex alpha polypeptide(alpha-NAC) protein family of Botrytis cinerea,the gene was named as PebC1.It has conserved NAC and UBA construction domains,which is same with activator protein PeaT1 from Alternaria tenuissim in structure domain.These two proteins are highly identical in amino acid composition, the similar amino acids are 79.4%and uniform amino acids were 71.0%.This cDNA is cost of 639bp and codes 212 amino acids.Predicted molecular weight is 23080.2 Dalton and its pI is 4.57.The hydrophilicity analysis revealed that it is a hydrophilic protein.The prokaryotic expression of PebC1 was accomplished by connecting the gene to vector pET-28a(+),transforming recombined vector to competent cell Rossetta(DE3) and inducing expression by IPTG..The following purification was executed by hitrap chelating column on (?)KTA explorer10.The MS analysis proved that the expressional protein was actually activator protein of Alternaria tenuissima.The biological activity research revealed that this purified recombined protein could promote tomato seedling growth and increase drought resistance of wheat.We constructed expressed vector of PebC1 Pichia pastoris and got expressed protein that has biological activity.It is the first time that PebC1 gene was amplified from cDNA of Botrytis cinerea and then was connected to the Pichia pastoris expressed vector pPIC9K to get recombination plasmid pPIC9K/PebC1.The plasmid was linearized by Bglâ…¡and then transformed to Pichia pastoris GS115 by electric shock.Through screening by MD,G418-YPD plain and PCR method,we got secreted expressed strain.After methanol inducing and SDS-PAGE determination,PebC1 was successfully secreted expressed in Pichia pastoris.The biological activity experiments revealed that PebC1 protein could induce cucumber to gain resistance against gray mould disease and have the biological function of activator protein.