Cloning And Expression Analysis Of Genes HfD14 And HfD27 Related Strigolactone In Hemerocallis Spp

Hemerocallis spp.is one of the ornamental flowers with a long history of cultivation in China,which belongs to the genus Hemerocallis.The monocotyledonous Hemerocallis spp.has a tillering mode similar to that of Gramineae,and tillering is an important character about the yield and ornamental of Hemerocallis spp..Tillering of Gramineae plants is a complex biological phenomenon,which is regulated by many hormones.It has been found that strigolactones（SLs）plays an important role in plant tillering,and its effect is the inhibition of buds.In rice,the growth regulation of tiller buds is regulated by strigolactone Max/RMS/D pathway,which may be conservative in higher plants.DWARF14（D14）is a key gene in strigolactone signal transduction pathway,which plays a role in hydrolyzing SLs and sensing SLs active molecules,and plays an important role in plant tillering.In the strigolactone synthesis pathway,DWARF27（D27）encodes the firstβ-carotene cis/trans isomerase,which appears in the first step of strigolactone biosynthesis.In this study,Hemerocallis fulva low tillering variety’Autumn Red’and multi-tillering varieties’M-1’,’M-2’,’M-3’and’golden doll’were used as research objects to explore the function of strigolactone synthesis gene HfD27 and receptor hydrolase gene HfD14,so as to provide new ideas for tillering and plant type regulation of Hemerocallis spp..The main contents are as follows:（1）D14 candidate gene HfD14 in Hemerocallis spp.was selected from the transcriptomic data of Hemerocallis spp.’Autumn Red’in our laboratory and verified by cloning.Bioinformatics analysis showed that HfD14 protein was hydrophobic and belonged toα/βhydrolase superfamily.According to the structure of rice Os D14protein,the key regions of HfD14 protein receptor pocket and hydrolase triad,α4-α7helix region(R143^HfD14-T155^HfD14,Y157^HfD14-V169^HfD14,P174^HfD14-N186^HfD14,P189^HfD14-F200^HfD14)were predicted.The genes of three varieties of Hemerocallis spp.with high tillering ability were screened by cloning in our laboratory,and the genes of’Autumn Red’D14 of Hemerocallis fulva.with low tillering ability were compared and analyzed by bioinformatics.There are differences in hydrophobicity among the four kinds of Hemerocallis spp.D14,but they all belong to hydrophobic proteins.The prediction of basic physical and chemical properties showed that there were also differences in isoelectric points of the four D14 proteins,which may lead to differences in the structure of proteins.Sequence comparison showed that compared with HfD14,the difference of amino acid 199(corresponding to V194^{At D14})of M-3was located in theα4-α7 helix region ofD14 protein,which may affect the binding ofD14 to strigolactones molecule.D52^{At D14}(corresponding to D57^HfD14)is the key gene in the interaction between At D14 and D3 protein,and the amino acid at position 57 of M-2 may affect the interaction between D14 and D3 protein.（2）D27 candidate gene HfD27 in Hemerocallis spp.was selected from the transcriptomic data of Hemerocallis fulva’Autumn Red’in our laboratory.The conservative domain analysis of HfD27 protein showed that the amino acids at position 155 to 23 belonged to the DUF4033 supergene family.The full-length CDS of HfD27 gene was cloned for the first time in Hemerocallis fulva.’Autumn Red’and multi-tillering variety’Golden Doll’by PCR technique.Sequence comparison shows that they have the same conservative domain,which may indicate that there is little functional difference between them.（3）In order to explore the expression pattern of HfD14 and HfD27 in Hemerocallis spp.,the subcellular localization of the these two genes was observed.The results showed that HfD14 was located in cytoplasm and nucleus,and HfD27 was located in chloroplast.Using different tissue parts of Hemerocallis fulva’Autumn Red’as materials,the tissue-specific expressions of HfD14 and HfD27 were analyzed by fluorescence quantitative PCR（Q-PCR）.The results showed that the expressions of HfD14 and HfD27 in axillary buds of Hemerocallis spp.were the highest,indicating that these two genes may be closely related to the tillering of Hemerocallis spp..（4）In order to further verify the function of HfD14 and HfD27,plant expression vectors 1301-ubq10-HfD14-Flag and 1301-ubq10-HfD27-Flag were constructed.The wild type Arabidopsis thaliana and Arabidopsis thaliana d14 mutants were transformed into the expression vector 1301-ubq10-HfD14-Flag for HfD14overexpression and functional complementarity experiments,and HfD27 was over-expressed in wild type Arabidopsis thaliana.The wild type Arabidopsis thaliana was infected by Agrobacterium tumefaciens,and the transgenic plants of T₀generation were obtained by resistance and PCR screening.Further studies are needed for subsequent molecular and phenotypic identification.