Research On Prokaryotic Expression, Protein Function Of Brassica Napus PGIP2 And Its Genetic Transformation

The mycelium must pass through the cell wall of rapeseed firstly,When rapeseed was infectied by Sclerotinia sclerotiorum so it secreted a series of hydrolases which degraded the cell wall.the first enzyme was poly-galacturonic acid enzyme (PG) which secreted by Sclerotinia sclerotiorum.It can degraded the pectin of rapeseed cell wall,so PG was an important pathogenic substances of Sclerotinia sclerotiorum.Poly-galacturonic acid-inhibiting protein (PGIP) can react with PG spectificity,which preventing rapeseed cell wall to degrade.So the research on PGIP was very significant.1、In this paper, from the mechanism of rapeseed which infected by Sclerotinia sclerotiorum to start research.according to the expression of pgip2 increased after the resistant Sclerotinia sclerotiorum Brassica napus Xiangyou 15 inoculated Sclerotinia sclerotiorum 18 hours.So total RNA was extrated from leaves of rapeseed Xiangyoul5 which were induced with Sclerotinia sclertiorum 18 hours.CDS was amplified from cDNA by reverse transcription PCR (RT-PCR) with a pair of specific primers based on the Brassica napus pgip2 (accession No.EU 142024).Ribonucleotide and amino acid sequence of Xiangyou 15 PGIP were anaylsed. the protein structure were predicted by biological information software.The results showed a homology of the nucleotide and amino acid sequences were 99.7% and 99.4% respectively,which reported NCBI bank.CDS of Xiangyoul5 pgip2 code area was 101 lbp. PGIP2 protein encoding 331 amino acid open reading frame with a molecular mass of 37.1 kDa and isoelectric point of 8.6.1～22 amino acid was predict to the N-terminal signal peptide with stronger hydrophobic region. The deduced amino acid sequence contained 5 potential N-glycosylation sites and contained 4 cysteine residues on N-terminal and C-termianl respectively, which involved in constituting the disulfide linkages.PGIP2 was a typical hydrophobic protein by the hydrophobicity prediction.Secondary and tertiary structure displayed it contained 11α-helices,12β-extended and 24 random coils.The concave structure of protein was composed of P-sheets/β-turns,and convex structure was composed ofα-helices,the center LRR structural domain was composed of 5 tandemly LRRs motifs, LRR consisted of aβ-strands and a-helices connected by loops and forming a horseshone-shaped molecule with the common sequence "xLxxLDLSxNxLTGxIPxxLxxL" form the 121st amino acid,it reacted with PG.It will provided a theoretical basis of PGIP2 protein biological function.2、CDS of PGIP2 was subencoded into the prokaryotic expression vector pET-32a (+) constructing recombinant expression plasmid pET-32a-pgip2 successfully.It started to the initiation codon of pET-32a (+) including the Trx,6×His-Tag, S-Tag of N-terminal pET-32a (+) vector and pgip2 gene sequence.it stopped the stop code of pgip2 gene at C-terminal.The recombinant plasmid was transformed into Escherichia coli BL21 (DE3).The fusion protein pET-32a-pgip was successfully expressed under final concentration 0.2mmolL-1 and 0.5mmolL"1 IPTG at 37℃,25℃iuducing for 2 hours.4 hours,6 hours,8 hours respectively.Expression bands appeared at the place where expected molecular weight 52KDa by SDS-PAGE analysis.It mainly appeared as inclusion bodies,not appeared as soluble protein by testing the solubility of fusion protein. It laid the foundation of the further research about pgip gene of plant prokaryotic expressed in Escherichia Coli.3、Using for ultrasonic crushing methods to broken the cell of pET-32a-pgip2 inclusion bodies and low concentrations of denaturant washing buffer 2mol/LUrea, 0.5% TritonX-100, 1mmol/LEDTA were used to wash it.we obtain high purity inclusion bodies.Then.using the high concentrations of denaturant 8mol/LUrea dissolved inclusion bodies.Using the Ni2+-NTA resin purified it,because the fusion protein containing 6×His-Tag at N-terminal which can combine with the Ni2+-NAT resin, eluting with different concentrations of imidazole, and finally we obtained high purity of the pET-32a-pgip2 fusion protein. the purified protein was analysised by Western bolt,the results show that pET-32a-pgip2 fusion protein can be immune react with anti-His antibody occurring at a specific band at about 52KDa place, further illustrateing that the pET-32a-pgip2 fusion protein was correctly expressed in E.coli,and the reading frame was correct.The purified fusion protein was digested by enterokinase, then was purified by Ni2+-NTA,the effluent liquid was collected to be PGIP2 target protein.Finally,the bioactive PGIP2 protein was obtained by gradually reducing the urea concentration in dialysate and target protein was renatured by the method of dialysis refolding.4、Sclerotinia mycelial was inhibited to infect rapeseed by dropping the PGIP2 protein on the detached leaves and inoculated Sclerotinia mycelial.The research results showed the leaves lesion area was smaller the PGIP2 protein treated leaves than on the sterile water and PBS treated in different days treatment,which demonstrated there was the PGIP2 resistance to Sclerotinia mycelial infected rapeseed. DNS method was used to determine the inhibition effects of PGIP2 on PG.the date showed that with the amount of PGIP2 protein increased,the amount of D-galacturonic acid reduced gradually, which PG degraded poly-galacturonic acid to generate D-galacturonic acid.It adequately proved that PGIP2 protein can inhibit PG.Agarose diffusion method was used to detect the inhibition effects of PGIP2 on PG.The experiment results indicated that with the amount of PGIP2 protein increased in a certain amount of PG, the area of the transparent zone around the plate with poly-galacturonic acid as a substrate lessened gradually.It proved that PGIP2 protein can inhibit PG strongly which Sclerotinia secreted.Further demonstrated PGIP2 fusion protein which was expressed in Escherichia Coil had bioactive after purification,enzymes digestion and renaturation.5、The cloned pgip2 inserted into the palnt expression vector pRI01-AN with CaMV35S strong promoter and NOS terminator.constructing the super-expression vector of pRI101-pgip2. then transferred to Brassica napus 98c40 by Agrobacterium-mediated transformation, about 300 resistant seedlings were got by Kan resistance screening.They were detected for PCR with the CaMV35S promoter-specific primers and 34 plants were amplified 422bp of the target band. This results showed that we got the transgenic rapeseed of pgip2.Four of them were detected by semi-quantitative RT-PCR.The results found that pgip2 gene expression increased compared in two plants with the non-transgenic plants.The detached leaves of the two transgenic rapeseeds were inoculated Sclerotinia mycelial for the resistance indentification.We found that the leaves lesion area of transgenic was smaller than non-transgenic rapeseed leaves lesion, and the invasion was also slight.The results suggested that we transfered the pgip2 into 98c40 and over-expressed successfully.They could improve the resistance to Sclerotinia sclerotiorum than non-transgenic.