Transgenic Prove Of Maize S-CMS-associated Mitochondrial Gene Segment Orf77/orf17

Maize is one of the main three crops,it's significant to study cytoplasmic male sterility(CMS).A CMS-associated mitochondrial DNA sequence from maize,orf77,and its mutant orf17 were introduced into the maize,rice and Arabidopsis nuclear genome.The coding sequence of orf77,orf17 was fused to a mitochondrial targeting presequence(leading sequence of ATP synthase subunit beta),placed under the control of a pollen specific promoter(zm13 promoter).The plasmid vector we used to reconstruct is pCAMBIA1303.Two methods were used to introduce extrinsic gene into maize: Particle-bombardment-mediated transformation and Agrobacterium tumefaciens-mediated transformation.The screen power of hygromycin is 25 mg/L, Agrobacterium strains LBA4404 was used for maize transformation.And Agrobacterium strains EHA105 for rice,GV3101 for Arabidopsis.The power of hygromycin is 50 mg/L while screening rice and Arabidopsis.We got resistant callus,but failed to get alive transgenic maize.The reason may be that hygromycin is not optimal screen marker for maize,the screening effect is not very good;also acceptor material is not very good,the regeneration efficiency and transformation frequency of callus tissue is very low.The gene orf77,orf17 were successfully introduced into rice and Arabidopsis.Fertility of the plants was affected in TO generation transgenic rice and Arabidopsis. Maybe that's because of the introduction of orf77 or orf17;Or because of the process of transgene and tissure culture.The transgene orf77,orf17 were segregated in T1 generation plants,but with or without the gene orf77,orf17,the pollen of T1 generation transgenic rice and Arabidopsis is fertile.RT-PCR result showed that the gene orf77 and orf17 were both transcribed in T1 generation.Then why the pollen fertility was not affected.The reason may be:gene orf77 comes from maize,and orf17 is editing product of orf77,because different sepcies may has different fertility mechanism,so orf77,orf17 cann't cause sterility of transgenic rice and Arabidopsis;The expression quantity of ORF77,ORF17 is insufficient to cause male sterility;Mitochondrial targeting sequence may have some problem,it failed to lead the ORF77,ORF17 into mitochondrial,so the protein ORF77,ORF17 didn't work;The expression period of the protein is also very important,different phase of pollen period affected the function of the protein ORF77, ORF17,cann't cause cytoplasmic male sterility.