Transcriptional Regulation Mechanism Of PtFLC And CRISPR Technology System Establishment In Citrus

In China,Citrus is a very important economic crop,the planting area is very wide,but It has a juvenile phase of more than eight years,which seriously restricted its breeding process.So in the molecular biology of fruit trees,how to shorten its juvenile phase is a hot point and a difficult point.However,FLOWERING LOCUS C(FLC)is a key gene in the flowering regulation of Citrus,which inhibits flower formation.Citrus flowering is generally promoted by inhibiting PtFLC transcription and protein expression levels.CRISPR/Cas9 has shown its superiority in gene editing technology and has been rapidly applied to the research of a variety of organisms.It is more efficient and accurate than traditional transgenic technology;Compared with TALENs\ZFNs genome editing technology,it is more convenient,more efficient and more accurate,greatly reducing the probability of off-target.CRISPR/Cas9 plant gene knockout has been successfully applied to the study of gene modification in almost all plant cells.In citrus,there are few reports on the use of any current genome-editing methods.The technical system of CRISPR is still in the exploratory stage,not only the low success rate of transformation of citrus,but also the conditions of CRISPER in this species of citrus are still not clear,which both lead to the unsatisfactory editing efficiency in citrus.In this study,we use the Citrus,Arabidopsis and tobacco(Nicotiana tabacum)as materials,research the transcriptional regulation of PtFLC and the establishment of CRISPR technology in Citrus.The main results are as follows:1.This study identified and confirmed the transcriptional regulation effect of transcription factor PtHB-13 on FLC promoter.PtHB-13 interacts with PtFLC promoters and activates PtFLC transcription by double hybridization and double luciferase analysis of yeast monohybrid yeast.PtHB-13 is a gene in the HD-ZIP transcription factor family.The subcellular localization of PtHB-13 gene fused with EGFP was found to be located in the nucleus.2.In Arabidopsis thaliana and Tobacco,it was found that transgenic lines(T2generation)with overexpression of PtHB-13 showed late flower phenotype,which was consistent with the overexpression of PtFLC.3.Quantitative PCR analysis of different tissues of Citrus cultivars showed that PtHB-13 was the least expressed in roots and the highest in stems and leaves.After drought,low temperature and salt treatment,it was found that the expression of PtHB-13 in drought and salt treatment was different,while the expression of PtHB-13 in low temperature treatment was not changed,indicating that PtHB-13 gene may be responsive to environmental stress.4.CRISPR vector and technical system were successfully edited in cotton,soybean and other species,and CRISPR vector of PtFLC was constructed.After agrobacterium-induced genetic transformation of citrus and summer callus,there was no Indel in the sequence,indicating that this might not be an applicable system in Citrus.In summary,PtHB-13 binds to the PtFLC promoter and activates the expression of PtFLC.and PtHB-13 gene is located in the nucleus and has transcriptional activation activity,which is mainly expressed in the stems and leaves of plants.PtHB-13 overexpressed lines were late flowering phenotypes,and the episperm color was lighter than that of wild type.The expression patterns of PtHB-13 and PtFLC were the same under drought conditions.system of CRISPR on citrus has yet to be established,and efforts are needed to improve editorial efficiency.