Cloning And Functional Identification Of Circadian Clock Gene LHY In Hemerocallis Spp.

Hemerocallis is one of the most important edible and medicinal ornamental plants.Because the flowering period of a single flower is only about 24 hours,and there are naturally nocturnal and daytime flowering groups,Hemerocallis is a good model material for studying flower opening and biological clock phenomenon.In this study,we based on the polymorphic markers selected by our research group,155 Hemerocallis plants flowering time were analyzed by association analysis,and the molecular markers and their corresponding genes related to their characters were mined;the flowers of Hemerocallis citrina cv.‘Datong Huanghua’ and Hemerocallis fulva‘Suqian 3’at the same time point were selected for transcriptome sequencing,and the candidate genes related to flowering time were further mined in the differential genes;Candidate genes of flowering time in Hemerocallis was cloned,then the bioinformatics characteristics of the gene were analyzed;Based on this,subcellular localization and spatiotemporal expression of candidate genes were also studied;Its biological function was identified by using the technology of VIGS,in order to lay a foundation for analyzing the molecular mechanism of flowering time of Hemerocallis,and It provides a theoretical basis for further regulation of flowering time by heredity and exogenous substances.The main conclusions are as follows:Based on 43 EST-SSR polymorphic markers developed by the research group from the transcription group of Hemerocallis citrina cv.‘Datong Huanghua’,155 Hemerocallis germplasms were analyzed for the correlation of flowering time.12 markers were detected in GLM model,8 markers were associated with flowering time in MLM model,and 10 markers were significantly associated with flowering time in GLM and MLM model.Taking the flowers of Hemerocallis citrina cv.‘Datong Huanghua’and Hemerocallis fulva‘Suqian 3’at the same time point as materials,RNA was extracted to construct c DNA sequencing library,and transcriptome sequencing was carried out based on Illumina platform.A total of 50 GB of data was obtained.After filtering,335 897 532 of reads was obtained,and 72 828 unigenes were assembled,thus the transcriptome database of Hemerocallis was constructed.There were 41 594 differential genes,22 289up-regulated and 19 305 down regulated.There are 46 differential genes in the pathway of circadian rhythms-plant metabolism.Association analysis and transcriptome analysis showed that Unigene0049603 and c33464.graph_c0were the same gene,which was annotated as LHY（LATE ELONGATED HYPOCOTYL）.It was one of the key genes of circadian clock core oscillator.The clock gene Hc LHY of cauliflower was cloned by PCR.The total length of its c DNA is 1929 BP,encoding 642 amino acids,the relative molecular weight of the protein is 69470.67 Da,and the theoretical isoelectric point（PI）is 5.82.At the same time,the clock gene Hf LHY was cloned from Hemerocallis fulva‘Suqian 3’ with 1833 bp fragment length,610 amino acids,65943.38 Da relative molecular weight and theoretical isoelectric point（PI）is 5.81.The similarity of Hc LHY and Hf LHY was 94.42%.Bioinformatics predicted that LHY is atypical Myb transcription factor,located in the nucleus;LHY is a hydrophilic protein without transmembrane domain;,without signal peptide;The main structure of LHY gene coding protein is irregular curl,followed by α-helix;LHY protein contains a large number of phosphorylation sites,suggesting that the realization of LHY protein function is related to reversible phosphorylation regulation.Phylogenetic analysis showed that LHY of Hemerocallis was closely related to Ao LHY（XP₀20245416.1）of Asparagus.qRT-PCR was used to analyze the temporal and spatial expression characteristics of LHY gene.The results showed that the expression of LHY had obvious rhythmic phenomenon,and the highest expression in petals,which was significantly higher than that in leaves,pistils,stamens,calyx and roots.The full length of ORF of Hc LHY and Hf LHY was constructed into p CAMBIA1300-super1300-GFP（C）vector by DNA recombination technology,and subcellular localization vector was successfully constructed.The LHY protein was located in the nucleus of tobacco mesophyll cells infected by Agrobacterium.In this study,PDS was used as the reporter gene to infect the 4-week-old seedlings of Hemerocallis citrina cv.‘Datong Huanghua’ by constructing TRV-PDS recombinant vector,and induce gene silencing,which proved that TRV-VIGS system can be applied in the functional verification of Hemerocallis.The function of Hf LHY on the common variety of Hemerocallis fulva‘Stella de Oro 2’ was further verified by this system.The results showed that after LHY was silenced in the flowers of Hemerocallis,the flowering time was 4 hours earlier than that of the control plants,and the expression of endogenous LHY gene in TRV2-Hf LHY was significantly lower than that in TRV2.