Rice Genetic Transformation Of Osa-miR319 And Osa-miR164 Precursors And Analysis Of Developmental Role In Leaf Morphology

Plant miRNA widely participate in the regulation in plant body, such as development, organ formation,biological or abiotic stress process. miRNA319 and miRNA164 plays a vital role in control plant leaf formation and organs boundary. When Rice ragged stunt virus (RRSV) infect Rice, Rice appear symptoms,such as roll leaf, incision, dwarf, leaf vein swell. To detect the expression quantity of miRNA319 and miRNA 164 in Nipponbare which infected Rice Ragged Stunt Virus by means of Northern Blot, the results show both of their expression amount raised 2.4 times.Through the fluorescence quantitative PCR to detect miRNA319 target genes expression level in Nipponbare which infected Rice Ragged Stunt Virus.Compares with healthy Nipponbare in the same growing period,Os01g59660 expression quantity rise by 2 times; Os12g42190 expression level rise by 1.4 times; Os03g57190 and Os07g05720 expression quantity dosen’t change. MiRNA164 target genes Os02g36880, Os04g38720, Os06g23650, Os06g46270, Os12g05260, Os12g41680 expression quantity are all decreased. The Os12g05260 expression quantity only 0.05 times compares with health rice; Os06g23650, Os06g46270 and Os12g41680 expression quantity was 0.25 times compares with that in health rice;the expression amount of Os04g38720 and Os02g36880 in infected Nipponbare is only 0.6 times and 0.4 times compares with that in health rice respectively.Using agrobacterium-mediated method to caltivate transgenic Nipponbare, we obtain miRNA319a and miRNA319b and miRNA164a, miRNA164b, miRNA164c and miRNA164d over express transgenic Nipponbare plants. Through the PCR amplify of resisitance gene NPT Hand the miRNA precursors to detect transgenic positive plant.The transgenic rice of miRNA319a show abnormal earing.The comparison result of health rice with the miRNA319b transgenic rice, miRNA319b transgenic rice appear dwarf symptoms. In forty days, miRNA319b transgenic rice is short 12.8 cm comparews with health rice in the same growing period, in 55 days, than the transgenetic group is 12.3 cm shorter, in 90 days transgenetic group is 12.9 cm shorter comparews with health rice in the same growing period.At the same time, the transgenetic group’s show earing anomaly, seeds form variation.Through the fluorescence quantitative PCR technology to dectect miRNA319 target genes Os01g59660, Os03g57190, Os07g05720, Os12g42190 expression level in miRNA319 trangenetic rice, the expression level of EF is internal reference. In miRNA319a precursor transgenetic rice, Os01g59660 expression quantity rise 2.2 times; The expression quantity of target genes Os03g57190 is extremely low; Os07g05720 expression quantity is the same level of health rice; Os12g42190 expression quantity only 0.2 times of health rice. In miRNA319b precursor genetically modified rice, Os01g59660 expression quantity go up 2.2 times; The expression of target genes Os03g57190 and Os12g42190 amount is 0.2 times of the normal rice; Os07g05720 expression quantity does not change much. In miRNA164a, miRNA164b and miRNA164d transgenic plants, Os06g23650 expression quantity go down, Os06g46270 expression quantity increased, Os12g05260 expression quantity do not change much, Os12g41680 expression quantity increased.