Molecular Cloning And Eukaryotic Expression Of Brassica Napus PGIP9and PGIP15

When Brassica napus was infected by phoma stem canker pathogens, they would release a series of Polygalaeturonases (PGs) to destruct the plant natural barriers-cell walls, while many resistance genes were reduced by pathogens to defense the infection. Polygalacturonic acid-inhibiting proteins (PGIPs) can react with PGs spectificity, which inhibiting the activity of endo-polygalaeturonase (endo-PG) and enhancing the resistance of plants. So PGIPs were effective proteins for the disease resistance in plant. To separate and purification PGIPs from plants had many weak points, so we hoped to construct an engineering strain which can produce PGIPs. Two pgips were cloned and transformed into the host cell Pichia pastoris PMAD16and induced expression with0.5%methanol. Then we detected their inhibition effect on PG from NM-1. It was very significant for the research on improving resistance of Brassica napus. The main research contents are as follows:1、The cotyledon leaves of Brassica napus Qingza5inoculated spore suspension of L. biglobosa NM-148h. The total RNA of cotyledon leaves around spots was extracted, and pgip9/pgip15CDS sequence were amplified. The cloned vectors pMD-19-T-pgip9/15were constructed. 2、Expression vectors pPICZaA-pgip9/15were buided to transformate into the host cell Pichia pastoris PMAD16to construct the engineering strain. Proteins PGIP9and PGIP15were detected by SDS-PAGE and Western blot. The results showed a singal expression band appeared at the place where expected molecular weight36-37KDa by SDS-PAGE and Western blot analysis.3、The protein concentration of PGIP9and PGIP15in the supernatant were detected. The protein concentration of PGIP9was12.356μg/μL and PGIP15was11.976μg/μL, DNS method and agarose diffusion method were used to detect the inhibition effects of PGIP9and PGIP15on PG of L. biglobosa NM-1. These results indicated that the proteins PGIP9and PGIP15can inhibit PG...