Function Analysis Of STAMDS11 And FLC Subfamily Genes In Petunia And The Genetic Mechanism Research Of Sterile Mutant

Petunia hybrida is a genus in the family Solanaceae.Perennial herbs,now in an annual or biennial cultivation.Huge flowers with rich colors and various types.Important pot and bedding plant widely used in urban landscaping,one of the most important plant of molecular biology research at present as well.MADS-box gene refers to a class of genes with highly conserved MADS-box domain,which is a kind of important transcription regulatory factor in eukaryotes,playing an important part in the growth and development regulation and signal transduction.At present,related research on Petunia MADS-box gene family mainly concentrated in ABCDE genes,involved in the development of floral organs of plants and flowering time regulation.STMADS11 and FLC subfamily genes belong to the MADS-box gene,STMADS11 sufamily genes express mainly in nutrition growth stage,which is regulated by multiple flowering pathways,such as autonomous pathway,and FLC subfamily genes are the key genes of flower inhibition,mainly involved in the vernalization pathway which negatively regulated the levels of FLC transcript and protein so as to promote plants flowering.As an important tool to taking molecular research,CRISPR-Cas9 is used in many species.Now it is applied in Petunia and the gene silencing effect is preferble.At present,transposon is the most valid molecular device in the research of gene function.Transposon Display Technology is based on the transposon character,and it is the important tool in insolating and coloning target gene.Petunia hybrida with different gene types have a different number of transposons.The high copy dTph1 transposable elements were found in W138 strain,which provide conditions to study gene function.(1)Constructing PDS-CRISPR-Cas9 vector and obtaining the transgenetic plants which the leaves are albescent.(2)This experiment mainly studied the subfamily genes function of Petunia STMADS11 and FLC.Take wild-type petunia W115 cDNA as template,amplifying AGL27-like,PMADS15-like,PMADS17-like genes and constructing overexpression vectors and CRISPR-Cas9 vectors.Constructing PGBKT7 and PGADT7 vectors of genes AGL27-like,PMADS15,PMADS15-like,PMADS17,PMADS17-like,FBP13,FBP25,PMADS20 respectively.Constructing PGBKT7,PGADT7 vector,performing yeast two-hybrid experiments to verify the interactions of the two genes,so as to analyze the protein regulation function.(3)It is concluded that,by means of yeast two-hybrid experiment,strong interactions both existed between PMADS15,PMADS 15-like proteins of the FLC family with FLC subfamily and STMADS11 subfamily proteins;in addition,other genes also existed different degree of interaction phenomena.(4)Transformed Arabidopsis and Petunia,observing and analyzing phenotypic and statistical data.Arabidopsis phenotype transformed through 35S:AGL27-like was obvious compairing with wide type.Rosette and cauline leaves increased significantly,stout stem,bolting and anthesis time was delayed clearly.Calyx became large with pistillody of petals.Pistil increased,distorted;the main top phenotype reversed with new normal inflorescence and flower organs.Transformations of Arabidopsis through the other genes were not obvious and the flowering time was slightly delayed.Transformation of petunia through 35S:FBP13 had the characteristics of delayed flowering time,smaller flowers,sagging stigmas,slender fruits,etc.Transformation of petunia through 35S:PMADS17-like had the characteristics of stout stem,short internodes,dark green and round leaves,and bigger petals,calyxes,stamens,stigmas and ovaries.(5)The sterile mutant that emerged in the multi-generation self-crossing of W138 strains were collected to the phenotypic observation and statistics.The mutant had the following character:The internode was short,leaves were small and dense,stalk was harder;The leaves were curly and became more curly from bottom to top;Flowers became smaller;The chapiter was white small and the pollens were little;It could not obtain seeds by self-crossing.The mutant and plants with background of W138 and W115 were crossed,then self-crossed to obtain the F3 generation.Through the statistics to the mutant' seperation from F1,F2 and F3 generation,we can confirm it was single gene recessive phenotype.(6)According to the Transposon Display Technology,the mutants and homozygotes with stable genetically in F3 genetration were sampled(12 mutants and 12 homozygotes).The DNA were extrated to take Transposon Display Technology,then they were imaged in PAGE.The fragments that existed only in mutant samples or existed just one or two in mutant samples were recoved.The target genes were confirmed by the sequencing to the recoved fragments.