Cloning And Mechanism Of Gene OsPCL1 In Controlling Of Heading Date In Rice

PCL1(PHYTOCLOCK 1),is one of the most critical circadian clock genes,which belongs to the transcription factor family of MYB.A long basic vegetative phase mutant,named lbvp,was obtained from the progeny of radiant mutagenesis of a Japonica rice varieties nipponbare.Using bulked segregant sequencing analysis,we idetified as the candidate gene of LB VP.Based on these results,the present project further aims to confirm the function of OsPCL1,analyze its expression characteristics,and screen and verify its interaction protein.The main results were as follows:(1)Identification of the candidate genes.The wild type DNA pool and mutant DNA pool was constructed.The candidate gene of LB VP was identified as OsPCL1 through bulked segregant sequencing analysis and by sequencing verification.A single nucleotide substitution of C to T occurs in the mutant,causing Arginine to Tryptophan.(2)Functional verification of the OsPCL1.A complementary vector containing OsPCL1 was constructed and transformed to lbvp mutant.Taltaly 25 transgenic lines were obtained,and the character of the mutant compeletly restored to the same with wild type rice.The knockout vector of OsPCL1 was constructed through CRISPR/Cas9 gene editing system,and transformed into Nipponbare.Taltaly 11 heterozygous transgenic lines were obtained,and homozygous mutant plants were identified from the T1 generation.The phenotype of homozygous mutant is consistent with that of lbvp plant.The results of these two experiments indicate that OsPCL1 is a key gene to control heading date in rice.(3)The expression characteristics of OsPCL1.qRT-PCR analysis showed that OsPCL1 was expressed in roots,stems,leaves,spikes and shoot in rice,and the highest expression was detected in panicles.The vector combined the promoter of OsPCL1 with the GUS gene was constructed and transformed into Nipponbare.Totally 23 transgenic lines were obtained.The results of GUS detection showed that the OsPCL1 was expressed in all of the detected tissues,including root,stem,leaf,leaf sheath,pulvinus,shoot,spike and endosperm in rice.(4)Subcellular localization of OsPCL1.The ORF sequence of OsPCL1 was connected to GFP and constructed a fusion expression vector,which was transformed into onion epidermal cells and tobacco leaves by Agrobacterium.The resuls showed that OsPCL1 was expressed in nucleus.(5)The relationship between OsPCLl and other flowering related genes in rice.The expression levels of ten flowering related genes,including OsPCL1,OsGI,OsLHY,OsELF3-2,OsPRR37,OsPRR95,Hdl,Ghd7,Ehd1,Hd3a and OsELF3-1,were detected from Nipponbare and lbvp mutants under short-day conditions by qRT-PCR.The results showed that the expression of the mutant were significantly increased in OsG1 Ghd7,and OsPRR37,wheras were obviously decreased in other 7 genes.Interestingly,almost no expression was detected in Hd3a gene in lbvp mutant under short-day treatment of 30 days.(6)The interaction protein of OsPCL1.Bioinformatics analysis showed that OsPCL1 probably interact with OsELF3-1 and OsELF3-2.The double molecule fluorescent fusion vector was constructed and transformed into the onion epidermal cells and tobacco mesophyll cells by Agrobacterium tumefaciens.The results showed that OsPCL1 was interacted with both OsELF3-1 and OsELF3-2.