| Objective: Walnut(Juglans regia L.)belongs to the genus Walnut in the family Walnutaceae and is an important timber,ecological,economic and bioenergy tree species that is widely cultivated in many countries and regions around the world.Studies have shown that GI(GIGANTEA),CONSTANS-Like,NF-Y(NUCLEAR FACTOR Y)and PEBP(phosphatidyl ethanolamine-binding protein)genes play an important role in flower formation induction.In order to investigate their functions in walnut flower bud differentiation,the GI gene,COL and PEBP gene families in walnut were analyzed to further investigate the potential mechanism of the synergistic role of Jr CO proteins and JrNF-Ys proteins in regulating the transcription of Jr FT genes during walnut flower bud differentiation,which is important for walnut variety improvement,quality improvement and yield enhancement.Methods: Samples of female flower buds and leaf buds of Juglans regia cv.‘Xinxin No.2’ were selected at different stages of differentiation and at the same time as the beginning of morphological differentiation of female flower buds.Using bioinformatics methods,we screened and identified the number of genes in the COL and PEBP gene families,and analyzed their gene structures,conserved structural domains,physicochemical properties,evolutionary relationships,covariance analysis,expression in different tissues,and the differences in expression of target genes in female flower buds at different stages of differentiation by quantitative real-time fluorescence PCR(q RT-PCR).The c DNA obtained by reverse transcription of RNA extracted from ‘Xinxin No.2 ’leaf tissues was used as a template to clone the relevant genes and investigate the correlation between the expression patterns of CO and FT in female flower buds at different differentiation periods.Using yeast monohybrid and yeast two-hybrid assays,we systematically investigated the mechanism of role of walnut Jr CO and NF-Y family members in the synergistic regulation of Jr FT(Jr PEBP10)transcription process.Results: The main research and results of this thesis are as follows.(1)A total of 55 COL genes were identified in the whole genome of walnut,unevenly distributed on16 chromosomes and one Scaffold,named in order as Jr COL1-Jr COL55,which were divided into three subclades by evolutionary tree analysis and had similar conserved motifs and gene structural positions in the same branch of genes.The Jr COL family genes in ‘Xinxin No.2’ have tissue-specific expression.q RT-PCR analysis of the Jr COL genes at different times in ‘Xinxin No.2’ female flower buds showed that Jr COL1,Jr COL35,Jr COL50a-f and Jr COL52 had consistent expression trends in ‘Xinxin No.2’ female flower buds,with Jr COL50a-f playing an important role in the differentiation of ‘Xinxin No.2’ female flower buds.It is hypothesized that the Jr COL family genes play important functions in walnut leaf development.(2)A total of 53 genes possessing the PEBP structural domain were identified from five walnut families.The walnut PEBP gene family can be divided into four subgroups,including FT-like,TFL-like,MFT-like,and PEBP-like subgroups.The results of gene duplication and covariance analysis indicated that the evolution of PEBP genes was mainly subject to purifying selection and segmental duplication was the main driver of the evolution of the PEBP gene family in walnut.q RT-PCR results indicated that the walnut PEBP gene family plays an important role in female flower bud differentiation and the majority of Jr PEBP genes were highly expressed in leaf buds and female flower buds.(3)JrNF-YB4 and JrNF-YB6 directly activate the expression of Jr FT(Jr PEBP10).The binding of Jr CO1,JrNF-YB4 and JrNF-YB6 to the promoter of Jr FT gene was demonstrated using yeast single-hybrid assay and dual-luciferase transient expression assay.The results demonstrated that the Jr CO1 could not interact with the promoter of Jr FT,and JrNF-YB4 and JrNF-YB6 could directly interact with the promoter of Jr FT.(4)It was demonstrated by yeast two-hybrid assay and bimolecular fluorescence complementation assay that Jr CO1 protein can interact with JrNF-YB4 and JrNF-YB6 proteins,respectively.We found that JrNF-YB4 and JrNF-YB6 could directly bind the promoter of the Jr FT gene,while Jr CO1 could not directly bind the promoter of the Jr FT gene.In addition,Jr CO1 interacted with JrNF-YB4 and JrNF-YB6,respectively,and dual luciferase experiments showed that Jr CO1 interacted with JrNF-YB4 and JrNF-YB6,respectively,and then improved the Jr FT gene promoter binding ability of Jr FT gene promoter.(5)It was concluded from yeast three-hybrid experiments that Jr CO1 proteins can interact wi th JrNF-YB and JrNF-YC proteins to form trimeric complexes,i.e.,Jr CO1-JrNF-YB4-JrNF-YC1,Jr CO1-JrNF-YB4-JrNF-YC3,Jr CO1-JrNF-YB4-JrNF-YC7,and Jr CO1-JrNF-YB6-JrNF-YC7.The Jr CO1 protein may first form a dimeric complex by binding to the JrNF-YB protein(as an intermediat e)and then forming a trimer with JrNF-YC to positively regulate the transcriptional expression of the Jr FT gene by binding to the promoter,thereby affecting the differentiation of female flower bu ds in walnut.(6)The walnut Jr GI gene was cloned.The full length of Jr GI is 3516 bp,encoding 1171 amino acids,with a molecular weight of 128.60 k Da,and is a hydrophilic protein;tobacco subcellular localization showed that the Jr GI protein is located in the nucleus.RT-q PCR analysis of Jr GI,Jr CO and Jr FT genes at different time periods of ‘Xinxin No.2’ female flower buds showed the highest expression in female flower buds at the critical stage of morphological differentiation,and it was speculated that Jr GI plays an important role in the differentiation of female flower buds at this time.In addition,Jr GI genes were expressed in all tissues of ‘Xinxin No.2’,and the highest expression was found in the leaves,and it is speculated that Jr GI genes also play an important function in the development of walnut leaves. |
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