Establishment Of Maize Genetic Transformation System And Cloning Of MDMV-CP Gene And Reserch For Transformation Of MDMV-CP Gene

Maize transformation is limited by the receipant materials.Only a few inbreds, such as A188,B73 and their derived inbreds, can regenerate well, while those elite ones that are widely used are not easy to transform successfully. On the other hand, maize dwarf mosaic (MDM) is a kind of main disease that cause huge drop in production and there is no effective pesticide now. Antisense RNA of maize dwarf mosaic virus coat protein (MDMV-CP) gene can well depress the assemble of coat protein, avoiding MDMV disease effectively. The aim of this study is to (1) establish a high-efficient transformation system including the receipant materials, culture substrate constitution and regeneration condition; and (2) clone MDMV cDNA and construct expression vector, transform and obtain finally the resistant inbreds used widely in maize commercial production.(1)Selection of optimum medium of different maize materialsIn order to decide optimum medium for different maize materials, 6 differrent mediums,Y0,Y1,Y2,Y3,Y4,YD were used for inoculation of 6 different maize materials, 178, 8160, 8108, HZ4, 501, and CIII in 2000 fall. Callus rate (typeI, typeII and typeIII) were calculated. Statistical analysis showed that YD is the best substrate for all the materials. In 2001 fall, YD medium was adopted to culture 23 maize materials including R18, which were offered by Beijing Academy of Agricultural & Forestry Sciences and Sichuan Agricultural University. The result indicated that 9 of 23 materials, including R18,4380/SZ5, 8108,3189/4380,XZ17 G53, CIII, 178, and 501 were available for regeneration.(2)Establishment of maize transformation systemMaterial R18 transformation system were established and transgentic plants of Bar gene were obtained uing gene gun. Statistical analysis showed that R18 performent well with YD culture medium in the process of conservation, screening and regeneration. After screening with barsta, 18 plants survived and 8 of which successfully produced ears. PCR and PCR-Southern of To generation indicated 17 plants were integrated with Bar gene,and genetically transmitted to Tl generation, phenotypic and molecular identification is undergoing.(3) Cloning of MDMV-CP gene and construction of expression vectorMDMV-CP gene was cloned from infectious maize leaf. Sequencing showed thatthis gene is 920 bp , with open-reading-frame encoding 219 amino acids.The homology with MDMV-KS1 (A34978),MDMV-A (A34974),MDMV-B (A34978), and MDMV-B(CN) (S77088) was 62.3%, 80.0%, 91.2% and 98.5% respectivrly. Two expression vector, p35Scpbar and p35scpfbar, were constructed. (4) Transformation of MDMV-CP geneMDMV-CP gene was successfully into R18.W18 and 501 in 2001 fall, each material being bombed 20,15,and 10 plates. 213 bastar (effective constitution PPT) resistant plants were obtained of which R18, W18 and 501 resistant plants counted 103,90, and 20 plants respectively. Molecular test was undergoing.