Functional Analysis Of Flowering-Related Genes In Hemerocallis And CRISPR/Cas9 System Mediated Function Knockout Of Flowering Genes

Hemerocallis fulva is a perennial root flower native to China.Continuous flowering is both an excellent ornamental trait of Hemerocallis and an important goal of breeding,but relies on traditional hybrid breeding cycles to be slow and large workload.It is particularly important to use molecular biotechnology to solve the molecular mechanism of continuous flowering of Hemerocallis and promote the breeding process.Studies have shown that TERMINAL FLOWER1(TFL1),SHORT VEGETATIVE PHASE(SVP)and APETALA1(AP1)genes play an important role in the process of plant flower transformation.With reference to the early transcriptome data of the research group,isolate the flowering related genes TFL1,SVP and AP1 of the continuous flowering Hemerocallis'H2006001',and preliminarily identify their expression patterns and biological functions to explore the function of the flowering related genes;using Csy4-mediated CRISPR/Cas9 technology to knock out AtTFL1,AtSVP and AtAP1 genes in Arabidopsis to further elucidate the molecular mechanism of flower-related genes;the first construction of Hemerocallis PDS and TFL1 gene CRISPR/Cas9 knockout vectors,laying a foundation for the realization of Hemerocallis gene editing basis.The main results are as follows1.Using RT-PCR technology to isolate three flowering related genes HkTFLl,HkSVP and HkAP1 of continuous flowering Hemerocallis 'H2006001',the total length of the sequences were 522bp,684 bp and 774 bp,encoding 173,227 and 257 amino acids respectively,BLASTX alignment shows that HkTFLl belongs to the gene of PEBP family,HkSVP and HkAP1 belong to the gene of MIKC type MADS-box family.qRT-PCR detected that HkTFLl and HkSVP were mainly expressed in vegetative organs,but lower in reproductive organs;the expression pattern during flower bud differentiation showed a gradual downward trend;HkAP1 gene was mainly expressed in reproductive organs,compared with vegetative organs low;the expression level during the flower bud differentiation of Hemerocallis gradually increased until the highest expression level in the ovule and anther formation stage.Subcellular localization showed that HkTFL1 protein was localized in the cytoplasm;HkSVP and HkAPl proteins were localized in the nucleus.The overexpression of HkTFLl and HkSVP genes in Arabidopsis inhibits the floral transition,promotes the number of inflorescence branches and affects the morphogenesis of floral organs;the overexpression of HkAP1 gene in Arabidopsis promotes the floral transformation and affects inflorescence structure and floral organ development2.The yeast two-hybrid technology was used to analyze the interaction mode of HkTFL1,HkSVP and HkAP1 proteins,and different yeast transformants composed of bait carrier and prey carrier were moved to three-deficient medium for growth,showing that the transformants can grow normally on the plate.The colonies developed by X-Gal turned blue,and the results showed that the two-way hybridization between HkAPl,HkSVP,and HkTFL1 proteins can directly interact to jointly regulate the growth and development of plants3.Construction of CRISPR/Cas9 knockout vectors for AtAP1,AtSVP and AtTFL1 key genes for Arabidopsis flower formation,as well as three genes knockout vectors.In the 23 mutant systems produced by pDIR21-AP1,8 strains were detected with target sequence deletion,sequencing revealed 4 types of deletions.In the 17 mutant systems produced by pDIR21-SVP,5 strains were detected with target sequence deletion,sequencing revealed 3 types of deletions.In the 30 mutant systems produced by pDIR21-TFL1,10 strains were detected with target sequence deletion,sequencing revealed 4 types of deletions.Mutants of each gene include deletion heterozygosity,deletion homozygous,and chimera mutant types.In the mutation system generated by the three gene knockout vector pDIR21-Triple,2 strains had a three-gene target sequence deletion,8 strains had a two-gene target sequence deletion,and 15 strains had a single-gene target sequence deletion.In addition,the development of AP1,SVP,TFL1 and three gene knockout mutants inflorescence meristems and floral meristems showed different degrees of abnormal structures.The inflorescence branches of AP1 and SVP mutant plants increased;the inflorescence of TFL1 mutant plants changed from infinite inflorescence to limited inflorescence;the inflorescence of three-knockout mutants had limited growth and the plants quickly entered senescence and could not form flowers4.Select Hemerocallis PDS and HkTFL1 genes,and construct a CRISPR/Cas9 vector.In the transgenic 'hongsanjiao' that obtained the pROKII-Cas9-PDS-1 target,three strains were detected with base mutations T to G,but they were located upstream of the sgRNA-PDS-1 region,which did not meet the expected cleavage site.Among them,2#pROKII-Cas9-PDS-1 resistant seedlings had obvious leaf albinism,but part of the leaf color recovered in the later period of cultivation,and the plant height grew slowly and remained dwarf.No signs of editing were detected in the other vectorsTo sum up,this study is based on exploring the expression characteristics of HkTFL1,HkSVP and HkAP1 genes of the Hemerocallis 'H2006001',by observing the morphological changes of transgenic plants and the study of protein interaction,the function of flowering genes was clarified Use of CRISPR/Cas9 technology to edit the Arabidopsis AtAP1,AtSVP and AtTFL1 genes deeply elucidated the molecular mechanism of multiple regulatory genes during flower development,which laid the foundation for the use of gene editing technology to improve the traits of flower plants.Due to the lack of genomic reference,the design of highly efficient sgRNA is limited,resulting in low efficiency of CRISPR/Cas9 mutations in Hemerocallis,resulting in off-target.