Functional Characterization Of Tae-miR9663 And Tae-miR5062 Highly Expressed In Wheat Flag Leaves

MicroRNA?miRNA?regulates various developmental processes including plant growth,biological or abiotic stress,and plays important roles.In recent years,a large number of wheat miRNAs have been identified by high throughput sequencing.However,their biological functions are unknown.tae-miR9663 and tae-miR5062 are novel miRNAs highly expressing in wheat seedling,flag leaf and seed.In order to explore the functions of novel miRNAs,precursors of tae-miR9663 and tae-miR5062 were obtained by cloning respectively.STTMs?short tandem target mimic?of tae-miR9663 and tae-miR5062 were synthesized respectively.They were constructed into barley stripe mosaic virus?BSMV?vector to obtain BSMV:tae-miR9663,BSMV:STTM9663,BSMV:tae-miR5062 and BSMV: STTM5062.The 3rd⁵th leaf of Ningchun 16 were inoculated with these recombinant virus to overexpress or silence miRNA.The phenotypes of inoculated plants were observed.The expression of tae-miR9663,tae-miR5062 and their targets was detected by real-time quantitative PCR.Nine plants were infected with each recombinant virus.Three independent biological replicates were included in this experiment.At the same time,tae-miR9663 and tae-miR5062 was constructed into pART27 overexpression vector respectively.Then the transgenic Arabidopsis were obtained by Agrobacterium-mediated method to explore biological function of targeted miRNAs.The main findings were as follows:1.The transient overexpression and silence of tae-miR9663 in wheat leavesThe 3rd or 5th fully expanded leaf was inoculated by empty vector BSMV:00 and BSMV:PDS with 185 bp PDS fragment at 3-leaf or 5-leaf stage respectively.The phenotypes on leaves were observed at 15³0 d post-inoculation.The photobleaching appeared on the4th⁵th or 6th⁷th leaves of BSMV:PDS plants,a positive control.That result showed wheat leaves were infected with virus successfully.Compared with BSMV:00,the expression of tae-miR9663 was higher by 16.42 times in BSMV:tae-miR9663 plants,and the expression of target Traes₆BL_DF39217B8.1 was lower by 95.24%,showing that BSMV could overexpress miRNA effectively.The white dots or white stripes appeared on the 4th leaf,and twisting existed between the 4th and 5th leaf.The lengths and widths of mature seeds were higher than BSMV:00 plants by 6.50% and 4.54% respectively.Compared with BSMV:00,the expression of tae-miR9663 was lower by 90.67% inBSMV:STTM9663 plants,and the expression of target Traes₆BL_DF39217B8.1 was higher by 17.28 times,showing that overexpression of STTM could silence endogenous miRNA effectively.The white dots or white stripes appeared on the 6th leaf and the flag leaf?the 7th leaf?,serrate margins appeared in the 6th leaf,and shrinking existed on flag leaf blade tips.The lengths and widths of mature seeds were lower than BSMV:00 plants by 3.75% and6.23% respectively.2.The transient overexpression and silence of tae-miR5062 in wheat leavesCompared with BSMV:00,the expression of tae-miR5062 was higher by 4.91 times in BSMV:tae-miR5062 plants,and the expression of targets,Traes₅DL_DF690B97B and Traes₅AL₆0D577E34.1,was lower by 38.25,22.60% respectively.The white dots or white stripes appeared on the 4th leaf,and curves appeared on the 5th leaf.The lengths and widths of mature seeds were higher than BSMV:00 plants by 5.07,8.89% respectively.Compared with BSMV:00,the expression of tae-miR5062 was lower by 57.15% in BSMV:STTM5062 plants,and the expression of targets,Traes₅DL_DF690B97B and Traes₅AL₆0D577E34.1,was higher by 2.03,4.56 times.The white dots or white stripes appeared on the 6th leaf and the flag leaf,and curves appeared on flag leaf.The lengths and widths of mature seeds were lower than BSMV:00 plants by 4.55,13.14% respectively.3.The transgenic overexpression of tae-miR9663 and tae-miR5062 in ArabidopsisThe overexpression vectors,pART27-tae-miR9663 and pART27-tae-miR5062,were overexpressed in Arabidopsis respectively.The transgenic Arabidopsis was obtained by Agrobacterium-mediated method,and then the phenotypes were observed.The results showed that compared with the wild type,the expression of tae-miR9663 in rosette leaves and siliques of three transgenic lines was increased by real-time quantitative PCR,and the expression of tae-miR9663 target AT1G22610.1 was decreased.The leaves were enlarged,bolting time was about five days late.The seed weights of per 100 mature seeds were higher than the wild type by 19.1,12.1,13.9% respectively.Compared with the wild type,the expression of tae-miR5062 in rosette leaves and siliques of three transgenic lines was increased,and the expression of tae-miR5062 targets,ATCG00070.1 and AT1G48410.1,was decreased.The leaves were enlarged.The seed weights of per 100 mature seeds were higher than the wild type by 23.5,29.4,41.1% respectively..