Genetic Transformation Of Vgb Gene And Cry5B Gene Into Paecilomyces Lilacinus Mediated By Agrobacterium Tumefaciens

Paecilomyces lilacinus is an effectively parasitic fungal against plant pathogenic nematodes and also have a good control effect to the eggs.But it grows very slowly of agent production cycle of the bacterium is long and large energy consumption.We transfer the vgb gene of Vitreoscilla hemoglobin into P.lilacinus genome and enhance the utilization rate of oxygen in the fermentation process and lay a foundation for improving the production efficiency.At the same time we also transfer the high poison activity nematicidal gene cry5B of Bacillus thuringiensis into PR lilacinus ACSS in order to overcome the shortcomings of nematode larvae work slowly,making it become the most promising nematophagous fungi.In this study,we set up a highly efficient genetic transformation system for Paecilomyces lilacinus ACSS strain taking advantage of the Agrobacterium tumefaciens-mediated transformation(ATMT).First of all,we optimized gene promoter,codon,gene structure during the gene clone,Then,through the Agrobacterium Ti plasmid pur5750,the express components PgpdA-npt ?-vgb-TtrpC and the screen element PgpdA-npt ?-TtrpC were constructed and transferred into the P.lilacinus ACSS.In the condition of neomycin G418(300 ?g/mL),we obtained two transformants PNVT8 and PNVT18 which contained the vgb gene.the result of gene npt ? Southern blot of the two transformants indicted that target gene integrate into the genome DNA of the P.lilacinus in a single-copy and random way.The results of RT-PCR analysis about vgb transformants confirmed that the npt ? gene and vgb gene had been transcripted and the expression of VHb protein was confirmed by CO-binding test.We optimized the volume of culture in the flask and confirmed that VHb protein can most abundantly expressed when adding 40 mL culture in the 100 mL flask.Compared to the wild strain,the number of hypha ball increased 5 times of PNVT8 and 2.5 times of PNVT18.In addition,the diameter of the PNVT18 transparent circle is 150 percetage of the wild strain ACSS when cultivated in milk fermentation broth medium,while the PNTV8 increased one time.The fermentation of PNVT8,PNVT18 and wide type ACSS in the 15 L fermentor results show that the pH,ammonia nitrogen,reducing sugar of PNVT8 and PNVT18 have no obvious change when compared with the wild strains and this result is similar to the growth of the form and appearance on PDA plate without antibiotic.This shows the vgb gene has no change in physiological metabolism and structure appearance of P.lilacinus ACSS.The detection of fermented liquid protease concentration we find that PNVT18 is higher than wild strain throughout the fermentation process,and PNVT8 was higher than wild strain in the late fermentation,which is consistent with the above experimental results transparent circle;The results of detection of alpha amylase concentration in fermented liquid indicates transformants are higher than strain ACSS after fermentation of 4 d and PNVT18 was a little higher but PNVT8 an order of magnitude higher than wild strain ACSS.Moreover,chitinase tests revealed that transformant PNVT8 is higher and the PNVT18 is an order of magnitude higher than wild strain but no difference at fermentation late between the three strains;Meanwhile,the biomass of PNVT8 than wild strain increased by 11.0%and PNVT18 than wild strain increased by 6.6%and similar to the results that the the spore production rate of transformants PNVT8 than wild strain ACSS increased 3.7 times and PNVT18 increased 2.7 times after the fermentation.All above results show that the vgb genes has been integratd into the P.lilacinus genomic DNA.The recombinant improves the growth and survival ability of P.lilacinus in low oxygen conditions,improve the expression and secretion total protease,alpha amylase,chitinase and various enzymes of the host strain,and the vgb gene also plays a significant role in improvement of biomass production and the spore production rate.however,different sites of the vgb gene on the chromosome of the host strain ACSS may present a variety of specificity.Using the same method,we obtained two transformants puPCTPNT13 and puPCTPNT26 which contained the cry5B gene,npt ? gene of the Southern blot test shows that target gene integrate into the genome DNA of the P.lilacinus in a single-copy and random way.cry5B transformants further testing and infect nematodes experiment will proceed later on down the line.