| Vernicia fordii is one of the four woody oil species in China.The oil extracted from its seeds is called tung oil,which is a kind of high quality drying oil.Vernicia fordii is monoecious plant unisexual flowers.The female flowers of Vernicia fordii have male structure in the early stage,and the timely degradation of the male structure can ensure the normal formation of female flowers.However,the female flowers of tung tree per plant are few and often have abnormal development,resulting in the lower seed setting rate.In order to clarify the molecular mechanism of female flower development,the research group screened the key candidate gene VfMYB35-1(VF05224)for early male structural degeneration of female flowers based on genome and transcriptome data.The biological function of VfMYB35-1 gene was studied by using gene cloning technology,bioinformatics technology,real-time fluorescence quantitative technology,yeast two-hybrid technology and transgenic technology,aiming to reveal the molecular regulation mechanism of VfMYB35-1 in the process of flower development and provide scientific basis for the formulation of high-yield cultivation technology of Vernicia fordii.Finally,increase the yield per unit area.The main findings are as follows:1.A member of VfMYB35 gene was obtained by cloning.In this study,a member of VfMYB35 gene was isolated and named VfMYB35-1 by transcriptomic data analysis and RTPCR cloning technology.The full length CDS sequence of this member was 1092bp,encoding 363 amino acids,belonging to the R2R3-MYB type of MYB gene family,and was most closely related to Jatropha curcas.Subcellular localization showed that the protein was localized in the nucleus and had the nuclear localization characteristics of a transcription factor,playing an important regulatory role in the nucleus.2.The interaction protein of VfMYB35-1 gene was screened.In this study,five candidate proteins interacting with VfMYB35-1 transcription factors were screened by using the yeast two-hybrid library in the nuclear system,including anthocyanin protein GLABRA2,flower homolog protein GLOBOSA,disease response protein 206.Cytochrome and HDT.GLABRA2 and Cytochrome are related to pigment.GLOBOSA protein,a member of MADS-Box gene family,affects floral organ formation.HDT protein is related to the development of female gamete,filament and anther.Resistance response protein 206 is associated with abiotic stress.3.The molecular mechanism of VfMYB35-1 gene in flower development was elucidated.Using Arabidopsis thaliana and Nicotiana tabacum as materials,we found that the transgenic Arabidopsis thaliana VfMYB35-1 had 5 stamens,and a few of the filaments and anthers were dry and transparent,with obvious abortion,by means of transgenic technology,paraffin section technology and microscopic observation technology.Fruit pods spacing and pods length decreased significantly,and whole branch abortion occurred in some main branches of transgenic Arabidopsis thaliana.In transgenic Nicotiana tabacum VfMYB35-1,elongated styles,depressed anthers,late dehiscence,and petalized anthers were observed.The number of pollen and pollen vigor in a single anther were significantly lower than those in the wild type.The fruit pods of transgenic Nicotiana tabacum VfMYB35-1 were smaller,and the number of seeds in a single pod was significantly reduced,but the seeds were significantly larger than those in the wild-type.QRT-PCR analysis showed that,NtDYT1,an upstream gene of NtMYB35 associated with tapetal development,plays an inhibitory role in anther’s development of transgenic tobacco.Overexpression of exogenous gene VfMYB351 in tobacco plays a regulatory role in the development of anther,and can affect the B genes(NtAP3,NtPI)and interaction genes(NtGLOBOSA,NtHDT,NtGLABRA)to promote anther flapification.4.A new function of VfMYB35-1 gene regulating flower color was found.Not only did the petals of the transgenic tobacco become elongated,they became more brightly colored.The fluorescence quantitative results of NtMYB35,NtGLABRA,NtCytochrome,NtGLOBOSA and NtHDT in transgenic tobacco suggested that they might be involved in the regulation of flower color,among which NtGLABRA was significantly up-regulated in transgenic tobacco petals.It is speculated that the overexpression of VfMYB35-1 gene can regulate the synthesis and accumulation of anthocyanidins.Eventually,the petals become more colorful. |
