Studies On Plant Expression Vector Construction Of HrpZ_psg12 Gene And Transformation Into Maize

hrpZpsg12gene is from Pseudomoas syringae pv. glycinea which can code harpin proteinand cause hyperensensitive response in the non-host plants and makes most plants systemicacquired resistance.Vectors with gene hrpZPsg12fragment were constructed and were transferred into maizeinbred line Zong31by Agrobacterium-mediated method, pollen tube method and particlebombardment, in order to acquire transgenic maize materials.being of a broad spectrumresistance to maize diseases.Main results are as follows:(1) A pair of special primers were disigned for amplification of hrpZPsg12based on its sequenceof GeneBank, and were modified with BglⅡor BstEⅡat the two ends, then were linked withpMD18-T. pMD18-hrpZPsg12plasmid and pCAMBIA3301plasmid were digested by BglⅡ andBstEⅡ, recycle target fragments. The recombinant shared vectors was successfully constructedafter target fragments linking with together by T4DNA Ligase.(2) A pair of special primers was disigned based on the sequence of gene hrpZPsg12and wasmodified with XhoⅠpartly at the two ends to amply hrpZPsg12by PCR.. pCAMBIA3301andpMD18-hrpZPsg12plasmid were digested by XhoＩand XhoＩ, recycled target fragments. Therecombinant expressed vector was successfully constructed after linking the target fragmentstogether by T4DNA Ligase.(3) Infective concentration and infective time are the important factors influencingtransformation efficiency. Studies on infective time and bacteria liquid concentration showedthat the best infective concentration was OD600=0.6-0.8and best infective time was infected for5-10min.(4) By pollen tube pathway, importing200μL with200μg/mL of plasmid DNA solution at theselected time6:00-8:30am,4:00-7:00pm after pollination8-20h, the results showed that45positive plants were detected from obtained575transgenic plants by PCR, the positive conversion rate was7.82%.for575stains of the plant in PCR detection,45were positive with the conversion rate of7.82﹪..(5) In particle bombardment process, tungsten powder consumption, plasmid concentration,callus pretreatment methods had a certain effect on transformation rate.20ng per gun oftungsten powder concentration for,2000μg per gun of plasmid concentration and callus treatedwith high permeability processing4h had the higher transformation rate,the results showd that9 positive plants from the36transgenic plants by PCR, the transformation rate was25﹪.(6) Bar regard gene as selective gene, hrpZPsg12gene were transferred into maize byAgrobacterium-mediated method. and26transform plants were tested positive by PCR..