| Tung tree(Verniciafordii)is one of the four major woody oilseed species in China,with unisexual flowers,the female flowers of which have a hermaphroditic structure in early development.The stamen degeneration in female flowers is a guarantee for the normal formation of female flowers,otherwise it will lead to abnormal development of the ovary in female flowers and reduced fruit set rate.Our group screened the key candidate transcription factor VfMYB35-1 that caused the degeneration of male structure in female flowers based on the analysis of Tung tree genome and transcriptome data.Then,five candidate genes interact with VfMYB35-1 were screened namely VfGLABRA,VfGLOBOSA,VfQCR8,VfHDT,and VfDIR6 by yeast two-hybrid.According to these results,we investigated the interactions of VfGLOBOSA,VfQCR8,VfHDT,and VfDIR6 with VfMYB35-1,and the possible synergistic relationships between them by gene cloning,bioinformatics analysis,BiFC,GST Pull Down and double gene co-transformation.This study aimed to provide a theoretical basis for uncovering the molecular regulation mechanisms of female flower development in Tung tree.The main findings are as follows:1.The interactions of VfQCR8,VfHDT and VfDIR6 with VfMYB351 were further validated.The BiFC vectors pNC-BiFC-Ecn and pNC-BiFC-Enn and the protein expression vectors pGEX-4T-1 and pCold TF were constructed in this study.The results of BiFC showed that VJMYB35-1 did not fluoresce yellow with the four interacting candidate genes in Nicotiana tabacum,and the GST Pull Down results indicated that VfMYB35-1 did not bind to the four interacting candidate proteins in vitro.Based on the results of BiFC and GST Pull Down,VfMYB35-1 may not have direct interactions with VfGLOBOSA,VfQCR8,VfHDT and VfDIR6.2.Overexpression of the VfMYB35-1 gene affected the expression of floral developmental pathway genes in Nicotiana tabacum.Transcriptomic analysis of floral organs from VfMYB35-1 transgenic Nicotiana tabacum positive plants revealed that overexpression of VfMYB35-1 suppressed class A gene expression and promoted class B,C,D and E gene expression of floral organs.In addition,the expression of homologs of reciprocal candidate genes such as VfGLOBOSA was significantly altered in Nicotiana tabacum.Among them,the expression of NtDIR6 in the petalized anthers of transgenic positive plants was 9 times that of wild-type(WT)anthers,and the expression of NtGLOBOSA in the petals was 8.2 times that of wild-type(WT).The results indicate that VfMYB35-1 can regulate the expression of floral organ characteristic genes and NtGLOBOSA,NtQCR8,NtHDT,and NtDIR6.Since there is no direct interaction between VJMYB35-1 and candidate genes such as VJGLOBOSA,VfMYB35-1 may indirectly affect the expression of these genes by regulating other pathways,thereby regulating female flower development.3.VfMYB35-1 had a synergistic effect with three candidate genes.The three coexpression vectors pDT7-VJMBY35-1-VfGLOBOSA,pDT7-VfMBY35-1-VfDIR6,pDT7-VfMBY35-1-VfQCR8 and three single gene expression vectors pDT7VfGLOBOSA,pDT7-VfDIR6,pDT7-VfQCR8 were transferred into wild-type Arabidopsis thaliana,respectively.PCR and Western Blot results showed that all genes could express normally.Phenotypic analysis revealed that all plants transformed with the VfMYB35-1 gene showed pod abortion.Paraffin section results showed that abnormal pollen development was one of the causes of plant abortion.The cotransformed positive plants of pDT7-VfMBY35-1-VfGLOBOSA and pDT7-VfMBY351-VfQCR8 showed ectopic formation of secondary flowers in the axil of sepals compared with the positive plants of single-transformed VfMYB35-1,VfGLOBOSA and VfQCR8.The pDT7-VfMBY35-1-VfGLOBOSA co-transformed positive plants showed anther petalization.RT-qPCR showed that the expression level of VJMYB35-1 cotransformed with other genes to wild-type Arabidopsis thaliana was significantly higher than that of VfMYB35-1 single-transformed.These results illustrate VfMYB351 and VfGLOBOSA,VfDIR6,VfQCR8 genes regulated each other ’s expression in an indirect way. |
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