Functional Characterization Of MiR319 And Its Targets TCPs In Petunia

Roles of miR319 in leaf growth and development have been validated in various species,even though loss-of-function mutants of miR319 and role of miR319 in regulating anthocyanin biosynthesis are rarely reported.CRISPR/Cas9 system that is an effective genome editing tool has been exploited in many specious,which in turn improved breeding and studies of gene function.In this thesis,we utilized CIRSPR/Cas9 system to induce targeted mutagenesis in petunia genome.Furthermore,using the CRISPR/Cas9 system,we uncovered the functions of miR319 targets in controlling venation pattern in flower tube.The research results are listed as follows:(1)To evaluate gene editing efficiency of CRISPR/Cas9 in petunia,two sg RNAs were respectively designed to target exons of Ph PDS,and then cloned into constructs containing Cas9.After transformation,transgenic shoot lines with albino phenotype accounted for 55.6%–87.5% of the total regenerated T0 Basta-resistant lines.Meanwhile,sequential transformation of Cas9 and sg RNA were used to edit PDS,transgenic lines with albino phenotype accounted for 55.6% of the total transgenic lines regenerated from these h Cas9 plants.In addition,chromosomal fragment deletion could be induced by sequential transformation or cotransformation of CRISPR/Cas9 system,the deletion was close to 1 kb in length.(2)To compare efficiency of CRISPR/Cas9-mediated multiplex gene editing between traditional way and polycistronic t RNA-g RNA(PTG)way.sg RNA5610(traditional way)and t RNA-sg RNA5610(PTG)constructs were designed to simultaneously edit TCP5,TCP6 and TCP10.In the sg RNA5610 construct,three sg RNAs were transcribed by individual promoters,while the three sg RNAs were transcribed by a single promoter.In t RNA-sg RNA5610 transgenic lines,targeted mutations were detected in TCP6 locus,but neither TCP5 locus nor TCP10 locus.In sg RNA5610 transgenic lines,various mutation types were detected in TCP5,TCP6 and TCP10 locus.Among the transgenic lines,loss-of-function mutations of TCP5,TCP6 and TCP10 were found in sg RNA5610-16 and sg RNA5610-17 lines.(3)Based on sequence of miR319 family members in petunia parent species and the transcriptome of MD in NCBI database,members of miR319,including miR319 a,miR319 c,miR319 d,miR319 e,miR319 h and miR319 i,were identified in the genome of MD.Transgenic lines overexpressing of miR319 c was achieved.According to phenotypes of these transgenic lines,miR319 can increase leaf area and length of flower tube,decrease cell size,improved venation pattern in flower tube and anthocyanin content.Comparing with MD,the expression levels of anthocyanin biosynthetic genes,CHS-A,CHI-A,F3 H,F3’5’H,DFR,3RT,5GT and GST,were enhanced in the lines overexpressing miR319.These results indicate that miR319 promote leaf growth and development and improve venation pattern in flower tube.(4)To study the functions of miR319 members,CRISPR/Cas9-mediated gene editing was used to mutate miR319 members.After transformation,mutants of miR319 a,miR319 c and miR319 h were achieved.Mutations of miR319 a and miR319 h,but not miR319 c,could be readily transmitted into the next generation.New mutation types of miR319 c were detected in the next generation.The length of flower tube become shorten in the mutant of miR319 c.No obvious phenotype changes were observed in the mutants of miR319 a and miR319 h.(5)According to sequences of miR319 targets in petunia parent species and the transcriptome of MD in NCBI database,6 predicted miR319 targets,Ph TCP5,Ph TCP6,Ph TCP9,Ph TCP10,Ph TCP11 and Ph TCP12 were isolated from MD.Corresponding Genebank accessions are KC297683,KJ175239,MH711923,MH711924,MH711925 and MH711926,respectively.Phylogenetic tree revealed that they also belong to CIN clade of TCP family.Comparing with MD,expression levels of Ph TCP5,Ph TCP6,Ph TCP9 and Ph TCP10 were dramatically decreased in the lines overexpressing miR319,however,transcript levels of Ph TCP11 and Ph TCP12 were not reduced.(6)Functional analysis of miR319 targets.Mutants of Ph TCP5,Ph TCP6,Ph TCP9 and Ph TCP10 were obtained by CRISPR/Cas9 system.The mutation of Ph TCP5,Ph TCP6,Ph TCP9 and Ph TCP10 could be readily transmitted into the next generation.Leaf area was increased in the tcp5,tcp6,tcp9 and tcp10 mutants,especially in tcp6 mutant.In the tcp6 mutant,venation pattern was improved in flower tube,accompanying increase of anthocyanin content.Although no obvious change of venation pattern was observed in the tcp5,tcp9 and tcp10 mutants,the anthocyanin content was increased in flower tube of tcp5 mutant.Comparing with tcp6 mutant,venation pattern and anthocyanin content were improved in tcp5/6/10 mutant.Overexpression of TCP6 inhibited the formation of venation pattern in flower tube,resulting decrease of anthocyanin content.Comparing with MD,m RNA abundances of anthocyanin biosynthetic genes,C4 H,CHS-A,CHI-A,F3 H,F3’5’H,DFR,3RT,5GT,GST and ANS,were significantly reduced in lines overexpressing Ph TCP6.Combining two results of subcellular localization and transcriptional activity assays,we found that Ph TCP6 locates in nucleus and functions as transcriptional repressor.These results indicated that miR319 targets inhibit leaf growth and development and pigmentation of venation patterning in flower tube.(7)Spatial-temporal distribution of anthocyanin is specifically determinated by R2R3-MYB which is one of members of MYB-b HLH-WD(MBW)complex that regulates anthocyanin biosynthesis.To investigate how miR319-targeted TCPs modulate venation pigmentation in flwoer tube,we analysed the spatial-temporal relationship between formation of venation and expression pattern of two R2R3-MYBs,DPL and AN4,in flower tube.During flower development,venation pattern is gradually accentuated by the present of anthocyanin,the expression of AN4 is gradually increased,conversely,expressions of miR319 targets are gradually reduced.In flower tube,the expressions of AN4 and miR319 mature have higher expression levels in dorsal side containing venation pattern than in ventral side without venation pattern,however,miR319 targets have reverse expression pattern in ventral and dorsal sides.In addition,expression of DPL is increased in 35S:miR319 lines but not changed in 35S:m TCP6 lines,expressions of AN4 were singnificantly increased in lines overexpressing miR319,tcp6 and tcp5/6/10 mutants,but dramatically reduced in lines overexpressing TCP6.Yeast two-hybrid results indicated that AN4 and DPL can interact with AN1(b HLH)but not Ph TCP6.These results indicated that miR319 targets regulate the pigmentation of venation pattern by inhibiting the expression of AN4 rather than interacting with R2R3-MYBs.(8)To further investigate roles of AN4 and DPL in regulating venation pigmentation in flower tube,mutants of an4 i,an4ii and dpl were obtained by CRISPR/Cas9 system,respectively.Anthocyanin distribution in an4 i mutant was the same as in MD.However,anthocyanin biosynthesis was lost in abaxial vein of flower tube in dpl mutant.In the an4ian4 ii mutants,venation pattern was lost in adaxial surface of flower tube and consequently reduced anthocyanin content and expressions of F3’5’H,DFR,ANS,3RT,5GT and GST.Meanwhile,overexpression of AN4 can ectopically induce anthocyanin biosynthesis in leaves,stem,sepal,flwoer tube and limb.That indicats AN4 is anthocyanin transcriptional activator.These results suggested that DPL determines pigmentation of abaxial venation and AN4 controlls pigmentation of adaxial venation in flower tube.Taken together,CRISPR/Cas9-mediated genome editing system can be efficiently used to mutate single,multiple genes,miR319 family members,or delete chromosomal fragment in petunia.Phenotypes of mutants indicated that miR319 c promotes growth of flower tube,and DPL controls anthocyanin biosynthesis in abaxial vein of flower tube,and AN4 determines venation pigmentation inside flower tube.Our results demonstrated that miR319 improve venation pattern in flower tube by negatively regulating the expressions of its targets which repress the expression of AN4.Distribution of venation pattern in flower tube is regulated by asymmetrical expression pattern of miR319 targets,in which miR319 targets,TCP5,TCP6 and TCP10,have higher expression level in ventral side without venation,and have lower expression level in dorsal side containing venation.