Functional Research Of FT-like Gene OsFTL4 In Rice

As an important cereal crop and economic crop,the appropriate growth period of rice has great influence on the yield.Flowering,as an important part,determines the transition from vegetative growth to reproductive growth.The FT-Like genes of PEBP family playing an important role in the regulation of flowering,and playing an antagonistic role with the TFL1-Like genes.At present,there have been some studies on the molecular biological mechanism of rice flowering,and many key genes and important regulatory pathways have been discovered.But there are still many genes whose functions and regulatory pathways need further exploration.Based on the research of FT gene in arabidopsis and FT-like gene in rice,OsFTL4 was selected to investigate it role in rice growth and development by constructing the OsFTL4 CRISPR/Cas9 knock-out mutant in the background of Guangluai4.the main results are as follows:1.Two different transcripts were obtained when the OsFTL4 gene was amplified,one of which was named OsFTL4.1,as published by the National Rice Data Center,the other,named OsFTL4.2,appears variable splicing at second intron and causes early termination of transcript.Subcellular localization showed that OsFTL4.1 had fluorescence signal in both nucleus and cytoplasm,while OsFTL4.2 had fluorescence signal only in nucleus.Different transcripts had different localization results in cells.Quantitative real-time PCR(qRT-PCR)analysis indicated that OsFTL4.1 and OsFTL4.2 constitutively expressed in all examined tissues,including root,stem,young leaf,old leaf,sheath,node and panicle.The highest transcript level was detected in node.2.A phylogenetic tree results showed that OsFTL4 had high homology with RCN.3.Using the CRISPR/Cas9 system to knock out the gRNA site at the first exon of OsFTL4,the successful vector was introduced into the Callus of Guangluai4 by agrobacterium-mediated method,and the mutant plants were obtained,two types of homozygous mutations were obtained after sequence analysis:Single base Mutation with G insertion and double base mutation without GT.Phenotypic identification of osftl4 mutants and wild-type plants showed that the flowering time of rice was significantly changed earlier after osftl4 deficiency,23 days in advanced in Hainan and 16 days in Yangzhou.4.The experimental results of seed germination showed that OsFTL4 was involved in the regulation of seed germination and was regulated by GA and ABA.5.Wild type and mutant seedlings were treated with different concentrations of hormone solution.The results showed that the seedling height of osftl4 mutant was lower than that of wild type without hormone treatment,However,there was no significant difference in seedling height between the wild type and the mutant treated with 10-12mol/LGA solution and 10-12mol/LABA solution.Indicating that osftl4 affected the morphogenesis of rice seedlings and was regulated by GA and ABA.Expression analysis showed that OsFTL4 was involved in GA and ABA Pathways,and had negative regulation on GA and ABA related Synthetase.6.The experiment of expression analysis after photoperiod treatment showed that OsFTL4 was involved in the photoperiod regulation pathway,located in the downstream of OsG1 and OsHd1,and regulated by OsGI and OsHd1.Under SD conditions,OsFTL4 and OsHd3? had a competitive relationship.7.The results of drought and salt treatments showed that the wild type was more tolerant to drought and salt than osftl4,indicating that OsFTL4 was involved in the regulation of drought and salt tolerance in rice8.The results of yeast two hybrid showed that:OsGF 14e interacted with OsFTL4.1,OsFTL4.2 and OsFD 1 simultaneously,indicating that a FAC-like complex could be formed between them;Neither OsGF14g nor OsGF14h interacted with OsFD1 and OsFTL4.1,only interacted with OsFTL4.2,indicating the functional differentiation between different transcripts of OsFTL4.