The Mechanism Of Protein Kinase Hd16 Regulating Rice Heading Date In Short Day

Rice(Oryza sativa L.)is a widely cultivated facultative short-day plant.Flowering time also means heading date in gramineous plants,as a crucial agronomic trait,it can directly affect yield and regional adaptability of plant.Therefore,it is of great significance to study the influencing factors of this trait and make the plant flower at appropriate plantation and time.Heading date,as a complex quantitative character,is jointly controlled by the internal gene network and external conditions such as light and temperature.A number of key genes that control heading date have been identified and cloned,and protein kinases have been found to play an important role in the molecular mechanism of rice heading date.In rice,Hd16(Heading Date16)/EL1(Early flowering 1)codes casein kinase CKI,a negative regulator of heading date under long daylight conditions.It is known that Hd16 can inhibit the expression of Ehd1,Hd3 a and RFT1 by phosphorylation of Ghd7/Hd4,suppressing rice flowering in long day conditions.hd16 mutant suppresses rice flowering in long day conditions but no significant change was observed in short day conditions.Hd16 also interacts with Os PRR37/Hd2 and phosphorylates the middle and C-terminus of Os PRR37/Hd2,but the exact phosphorylation site remains to be identified.Due to the broad spectrum of substrates,in addition to the known phosphorylation substrates of Hd16,it is not clear whether there are new substrates phosphorylated by Hd16 that can interact with Hd16 and form a new pathway to regulate heading date of rice.Therefore,this study used EMS-induced Hd16 point mutation(SNP)material and found that the mutant flowered later in short day conditions instead.Also,homozygous stable mutants were obtained by CRISPR-Cas9 to further determine the function of Hd16 under short-day conditions.At the same time,yeast screening bank and Co-IP mass spectrometry were used to try to find a new interaction substrate of Hd16,so as to explore the regulation process of the tyrosine kinase gene Hd16 on rice heading date in short day.The results are as follows:1.hd16 mutants showed a long-day early-flowering and short-day late-flowering phenotypeIn the EMS mutant population,two single non-synonymous single nucleotide polymorphism(SNP)mutants were found to flower early under long day conditions.Cap and d Caps identification showed that SNP-1 of Hd16 was located in the ninth exon kinase domain,transforming from alanine(Ala)to valine(Val).SNP-2 of Hd16 was located in the 15 th exon and changed from threonine(Thr)to isoleucine(Ile).The protein sequence alignment revealed that the loci were very conserved,and the late flowering phenotype was also found during the planting process under short day conditions.After years of breeding,a homozygous mutant Hd16 was obtained by knockout of Hd16 gene using CRISPR-Cas9 technology.Phenotypic identification showed that hd16 mutant flowered early under long day conditions,but late under short day conditions.2.The transgenic plants with overexpression of Hd16 headed earlier under long day conditions but later under short day conditionsThe overexpression vector of Hd16(p GWB12-Hd16)fused with Flag label was constructed and infected with Nipponbare callus by agrobacterium-mediated genetic transformation.Realtime PCR and Western Blot were used to test the transcription and protein levels of four stable lines,and Hd16 overexpressed transgenic plants were obtained.Phenotypic analysis showed that Hd16 overexpressed plants advanced heading in long day conditions but delayed heading in short day conditions.3.Hd16 is located in the nucleus and the proteins which interacted with Hd16,identified using yeast screening bankThe complete coding region(CDS)sequence of Hd16 was linked to the Gateway system vector p H7WGF2.0,which was fused with green fluorescent protein GFP.The subcellular localization of Hd16 was detected by transient expression system of tobacco.The results showed that Hd16 was located in the nucleus.Four candidate proteins were found using yeast screening bank.In addition to Hd2 whose interaction with Hd16 had already been found in previous studies,only candidate protein OSPP2A-1 showed weak interaction with Hd16 in yeast double-hybrid point-to-point verification.However,no interaction was found in tobacco LCI assay,so further study was not carried out.4.Hd16 regulates rice heading date by binding to Hd2 under short-day conditionsSince Hd2,which had been discovered to interact with Hd16 in previous research,was found using yeast screening bank,yeast point-to-point experiments were carried out to verify the interaction between Hd16 and Hd2.To learn the genetic relationship between Hd16 and Hd2,based on the phenotypic analysis of single and double mutants of hd16 and hd2,it was inferred that the promotion of heading in short day because of Hd2 depended on the functional Hd16 gene.As for the unknown phosphorylation site of Hd16 and its post-phosphorylation function,the most likely phosphorylation site was predicted by GPS 5.0-kinase-specific Phosphorylation Site Prediction.In the future,the site will be further verified by vivo and in vitro protein phosphorylation experiments,and site-directed transgenic mutation will be used for final confirmation.5.Candidate proteins interacting with Hd16 were identified by immunoprecipitation and mass spectrometry(IP-MS)Immunoprecipitation(Co-IP)experiment was carried out with the fusion Flag-tagged protein extracted from the overexpressed transgenic plants of Hd16,and the SDS-PAGE strips dyed by Coomassie bright blue were sent to the company for Mass Spectrometry(MS).Fifty-six proteins that might interact with Hd16 were identified by IP-MS,and four candidate proteins were identified for further verification by comprehensive analysis of the number of peptides and protein functions.This study identified the effect of Hd16 on rice heading date under short day conditions and attempted to analyze its functional mechanism,providing theoretical guidance and technical support for improving the regulation network of heading date and adjusting the regional adaptability of rice varieties.