Functional Identification Of Rice ARP Gene And The Study On Establishment Of Sorghum Transgenic System

In order to verify the ARP gene function,the experiment established suoer expression vectors and gene knockout vector to transfer the ARP(auxin responsive protein)gene which is new discovery to rice nipponbare genome by Agrobacterium mediated method.The results showed that,the experiment gained 65 p CAMBIA1300-ARP transgenic plants,of which 63 strains have target gene fragments and hygromycin gene fragments by PCR amplification,and results proved the expression amount of ARP genes in rice No.2,No.8,No.50 strains higher than the wild type by semi quantitative RT-PCR,And the height of transgenic plants was significantly lower than that of wild type,growth became slower,After Arabidopsis thaliana transforming,it was found that the results were similar to that of rice,but its leaves become smaller and more,pods become smaller,root and hypocotyl length became shorter,lateral root and fibrous root to be more.Through rice nipponbare transforming gene knockout vector p CAMBIA1300-ami RNA and control vector p CAMBIA-1300,founding that comparing with control,the resistant callus of ARP gene knockout vector which were obtained by transformation were small and soft,differentiation frequency was relatively lower.Moreover,p CAMBIA1300-ami RNA transgenic seedlings compared with the control,the plant type was smaller,stems was more thin,the colour of leaves were more shallow,it was pale green,lateral root and fibrous root to be less.In addition to,the experiment took Agrobacterium mediated mathod to study sorghum transformation gene system,in order to establish a highly efficient system of sorghum.The results showed that,When immature spikes of 3 sorghum materials were used as explants to induce the callus,the response of sorghum immature spikes callus induction frequency varied with 2,4-D、ZT and genotype.When the concentrationof 2,4-D was 4.0mg/L,the concentration of ZT was 0.1mg/L,and the genotype was M81-E,the induction rate was the highest,which reached 74.2%;When immature embryos of 3 sorghum materials were used as explants to induce the callus,the response of sorghum immature embryos callus induction frequency varied with 2,4-D、ZT and genotype.When the concentrationof 2,4-D was 1.0mg/L,the concentration of ZT was 0.1mg/L,and the genotype was xin liang52,the induction rate was the highest,which reached 59.3%;Response of sorghum immature spikes callus differentiation frequency did not vary with genotypes.That is,Xin Liang 52 differentiation frequency was15.6%,M81-E was 14.9%,and San chi san was 14.1%.However,response of sorghum immature embryos callus differentiation frequency varied with genotypes.Of which,07-27 differentiation frequency was the highest,it was16.2%.The second was Xin Liang 52,it was13.1%.Differentiation frequency of M81-E was the lowest,which was 10.3%.Moreover,response of sorghum callus differentiation frequency varied with explants,and the differentiation frequency immature spikes was higher than that of immature embryos.Of which,the differentiation frequency of M81-E immature spikes callus was 14.9%,and M81-E immature embryos was 10.3%.The differentiation frequency of Xin Liang 52 immature spikes callus was 14.9%,and Xin Liang 52 immature embryos was 13.4%.In addition to,the results of callus on the regeneration medium for immature spikes,M81-E and san chi san occurred at the highest frequencies when ZT concentration is 0.0mg/L,its were 21.2%and20.0%,respectively.Xin Liang 52 showed the highest frequency of callus regeneration under the condition of ZT concentration is 1mg/L,and it was27.1%;the results of callus on the regeneration medium for immature embyros,07-27 and M81-E occurred at the highest frequencies when ZT concentration is 0.5mg/L,its were 21.9%and 14.6%,respectively.Xin Liang 52 showed the highest frequency of callus regeneration under the condition of ZT concentration is 1.0 mg/L,and it was10.2%.The transformation results of sorghum callus were: Xin liang 52 got 6 resistant callus,san chi san got 31 resistant callus.However,the resistant callus did not differentiate transgenic seedings,and all became brown to death.