Functional Analysis Of PhWRIL1 Gene In Petunia Hybrida

The PhWRIL1(Petunia hybrida WRINKLED Like 1)gene belongs to the basal ANT subgroup of the ANT(AINTEGUMENTA)group of the AP2/ERF(APETALA2 / ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR)family.Two major groups,euAP2 and ANT groups are identified in the AP2 clade genes,while the ANT clade contains these two insertions,the euAP2 genes lack both.The ANT group can be further divided into euANT and basalANT subgroups,the euANT group have three conserved motifs(euANT 2,3 and 4 motifs)located in the N-region in front of the R1 domain and the length of the predomain region is longer than the basalANT clade genes which contain none of these three motifs.The AP2 gene family is mainly involved in plant growth and development,regulating flower organ morphology and development,controlling the formation of inflorescence meristem and the normal development of ovule and seeds,and participating in various stress responses.The euAP2 group of the AP2 gene family mainly regulated the flower meristem characteristics and floral organ differentiation during plant flower development.The euANT group genes mainly involved in regulating cell proliferation and organ growth,flower organ development,promoting somatic embryogenesis and ectopic organ formation.basalANT clade genes are closely related to eu ANT clade genes,they have been found to function mainly in oil formation.However,few studies have been done on the influences of overexpression of the basal ANT clade gene of AP2 / ERF gene close to euANT on de novo organogenesis in tissue culture.Analysis of the function of PhWRIL1 gene in petunia basal ANT group,especially in in vitro regeneration,not only helps to understand the role of basalANT gene in the growth and development of Petunia,but also the use of genetic engineering methods for petunia has a great significance to transform the traits and improve the ornamental nature of the petunia.In this study,we constructed the overexpression and knocked out the vector of PhWRIL1 gene in basalANT group,and observed the phenotypes of T1 transgenic plants and the E1(Genome Editing 1)mutants and the results of in vitro culture to study the function of PhWRIL1 gene of the basal ANT group in Petunia.Following achievements have been obtained:1.Molecular cloning and sequence analysis of PhWRIL1 gene in PetuniaPetunia hybrida inbred line MD was used as material,and an AP2 / ERF family gene PhWRIL1 was cloned from Petunia by EST database search and RACE technology,the cDNA sequence was obtained by sequencing.The CDS of PhWRIL1 was 945 bp and encodes a protein of 314 amino acids,there were 161 bp 3'-UTR and 196 bp 5'-UTR at both ends of the coding frame.Sequence analysis and phylogenetic tree analysis indicated that PhWRIL1 belongs to the basalANT class of genes.2.Tissue specificity analysis of PhWRIL1 gene in PetuniaReal time PCR analysis indicated that the PhWRIL1 mRNA was accumulated in various organs in Petunia,and were more abundant in the roots,stems,flower buds than the shoot apexes,young leaves.3.Phenotypic observation of T1 transgenic plants transformed with PhWRIL1overexpression vector in PetuniaPetunia transgenic plants overexpressing PhWRIL1 showed heavily dwarfed phenotype,the leaves and flowers are notably smaller than the control,but the flowering time was not clearly delayed by the introduction of the transgene.In vitro culture of T1 generation PhWRIL1 overexpressing transgenic line with 2.5 ?mol/L 6-BA(6-Benzylaminopurine),and the ability of PhWRIL1 overexpressing plants to regenerate adventitious buds was strongly inhibited.4.Vector of knockout of PhWRIL1 in Petunia and phenotypic observation of E1mutantsA target was selected from the 5' end of the PhWRIL1 gene near the translation initiation site,and a plant expression vector knocked out of PhWRIL1 was constructed,Agrobacterium-mediated transformation of Petunia was carried out,through Basta screening and PCR detection,transgenic lines with single base mutations and fragment deletions were detected,and then seeds were seeded to obtain for phenotypic observation E1 mutants.We found that PhWRIL1 knockout petunias showed dwarf phenomenon,smaller leaf area and delayed flowering time compared with control.The E1 generation mutant strains were used for in vitro culture of Petunia with 0.5 ?mol /L 6-BA,the number of regenerated shoots and culture quality of explants of PhWRIL1 fixed-point knockout vector E1 mutant strains were lower than those of control,indicating that the ability of phwril1 to regenerate adventitious buds was inhibited.