Identification And Functional Verification Of MADS-box Transcription Factors Related To Flowering Time Of Iris Laevigata

Iris laevigata is a perennial herb of Iris genus,Iridaceae,with unique flower color and flower shape and high ornamental value.However,its flower viewing period is short,which seriously affects its popularization and application.It is an effective method to prolong the flowering period of population by regulating the early or delayed flowering of plants through molecular breeding,so the mining of flowering regulation gene resources is very necessary.Many members of the MADS-box transcription factor family are involved in the regulation of plant flowering time,but there are few reports on related studies in I.laevigata.In this study,the MADS-box transcription factors related to flowering time were mined from the transcriptome of the perianths of I.laevigata,and the interacting genes Il SEP3(Gen Bank:ON398430)and Il SVP(Gen Bank:ON39843)were cloned and transformed into Arabidopsis to analyze the regulatory function of flowering time.Also,the specific internal reference genes of I.laevigata were identification,which laid a foundation for the study of I.laevigata gene expression characterization using RT-q PCR.The main results are as follows:1.41 MADS-box transcription factors were identified from the transcriptome data of I.laevigata.Through phylogenetic analysis,8 of them were type I MADS-box genes(5 belonged to Mαgroup,3 belong to Mβgroup);33 genes are type II MADS box genes,of which 2 are MIKC*group and 31 genes are MIKC~Cgroup(including seven major gene clades:SEP-like,AGL6-like,SQUA-like,AG-like,TM3-like,DEF+GLO-like,and STMADS11-like).Through gene function prediction,two target genes that may be related to flowering time were selected,named Il SEP3 and Il SVP.2.Il SEP3 and Il SVP were amplified by PCR.The sequences of the two genes were 720bp and 705bp respectively,encoding 239 and 234 amino acids.The molecular weights of the proteins were 27.64kda and 26.26kda,and the theoretical isoelectric points were 8.99 and 6.24.They were unstable hydrophilic proteins without transmembrane structure and signal peptides;Protein secondary structure analysis found thatα-Helix accounted for 51.88%,extended chain accounted for 9.21%,β-Corner accounted for 5.02%,and random curl accounted for 33.89%Il SEP3 protein;α-Helix accounted for 56.84%,extended chain accounted for 11.97%,β-Corner accounted for 3.42%,and random curl accounted for 27.78%Il SVP protein;The protein tertiary structure model was successfully constructed;Il SEP3 and Il SVP proteins are contains mainly serine phosphorylation sites,and the subcellular localization prediction protein is located in the nucleus;Homology comparison found that they all have typical MADS and K-box domains.Phylogenetic analysis found that Il SEP3 has the closest genetic relationship with Freesia hybrid cultivar and Crocus sativus,and Il SVP has the closest genetic relationship with Crocus sativus.Through homologous sequence function analysis,it is speculated that Il SEP3and Il SVP may regulate flowering time,flower organ development,pollen development and so on.3.p GBKT7-Il SEP3 vector and p GADT7-Il SVP vector were constructed to carry out yeast two hybridization.It was found that Il SEP3 protein could interact with Il SVP protein.4.Il SEP3 was identified as a gene regulating early flowering,and Il SVP had no obvious regulation on flowering time:The overexpression vectors p CAMBIA1300-Il SEP3-GFP and p CAMBIA1300-Il SVP-GFP were constructed,and the transgenic T3 lines were obtained by heterologous transformation of Arabidopsis.And the detection of flowering-related genes by q RT-PCR showed that Il SEP3 may directly or indirectly promote the expression of FT and SOC1 and other genes through the multi-flowering pathway to make the plants bloom earlier,and the flowering period was about 3 days earlier than WT.However,there was no significant change in flowering time of Il SVP transgenic lines.In addition,Nicotiana benthamiana leaves were transiently transformed with Agrobacterium.It was determined that Il SEP3 and Il SVP proteins were located in the nucleus,which was consistent with the prediction results.5.Eight reference genes commonly used in plants were selected,and their expression stability was identificated in 11 different tissues of I.laevigata(four sample groups).The comprehensive ranking of eight genes in four sample groups was obtained by using reference gene stability analysis software ge Norm,Norm Finder,and Best Keeper.Also,the optimal number of reference genes in each group was determined by ge Norm.The optimal reference genes in the group of all sample(11 tissues were compared simultaneously)were PP2A,AP-4and CYP,the optimal reference genes in the group of different floral development stages(flowers at bud stage,early flowering stage,full flowering stage and final flowering stage)were PP2A and GAPDH,the optimal reference genes in the group of different floral organs(outer perianth,inner perianth,stamen,pistil and ovary)were GAPDH and PP2A,and the optimal reference genes in the group of different tissues(leaves,scapes and flowers at full flowering stage)were AP-4 and PP2A.