The Over-Expression Vector Construction Of Bnaimyap And Transformation Of Brassica Napus&Arabidopsis Thaliana

Rape is one of the four major oil crops in the word. It is not only the main source of edible plant oil, but also the energy source of biofuel. So how to improve the content and the flavor becomes the goal of many scientists. Inducible Myrosinase-associated Protein (iMyAP) occur in complexes with Myrosinases which degradate glucosinolate plays important roles in the flavor, it is also closely related to the stress response and defense response. The research cloned iMyAP gene and transformated into plant by use cell engineering and molecular biology methods. The mian results are as follows:1,Primers were designed according to iMyAP9gene (Y10155.1) and iMyAP12gene (Y10156.1) in GenBank, Two cDNA were cloned from'XY15'wounding-leaf by RT-PCR.2^Whole CDS of iMyAP9gene and iMyAP12gene were connected with pMD19-T vector followed by sequencing determination. The recombinant plasmids were isolated, double digested with BamH I and Xba I, ligated into pBI121vector which double digested with BamH I and Xba I.Transformed into DH5a and obtained CaMV35S::iMyAP9and CaMV35S::iMyAP12over-expression vector.3. Transformed CaMV35S::iMyAP9and CaMV35S::iMyAP12into GV3101. Genetic transformation of Arabidopsis was performed by floral dip method. The To seeds were screened on medium with kanamycin and the anti-kanamycin plants were tested by PCR to comfirm if anti-Kan plants were transgenic plants67transgenic plants were obtained. The results of RT-PCR showed that iMyAP9and iMyAP12genes efficiently expressed in the transgenic arabidopsis plants.4,Transferred the iMyAP over-expression vector into Brassica napus 'XY15'by Agrobacterium_mediated Transformation Methods, and the anti-PPT plants were obtained.Extracted various growth stages on The anti-PPT plants DNA for PCR analysis, the results were positive, which indicated that the stable transgenic plant.was obtained. Extracted transgenic plants non-wounding leaf'RNA, the iMyAP gene RT-PCR expression analysis showed that the over-expression vector of iMyAP gene have expressed in the transgenic plants.5,Wounding'XY15', the iMyAP gene RT-PCR expression analysis showed that iMyAP gene expressed largest in1h wounding time; iMyAP gene expression have reduced in12h and24h wounding time; iMyAP gene expressed least in48h wounding time.