Preliminary Investigation On Molecular Mechanism Controlling Floral Symmetry In Soybean

Soybean (Glycine max (L.) Merr) belongs to Glycine genus, Faboideaesubfamily, Leguminosae family. It is one of most important food, oil and feed crops inthe world. Soybean is an ancient polyploidy, its genome duplications occurred twice,resulting in a highly duplicated genome with nearly75%of the genes present inmultiple copies. The molecular mechanism from model plant may vary with moregene copies emerging from soybean. In this study, we investigated the molecularmechanism controlling floral symmetry in soybean, analyzed the function divergencyof genes in the network to pave the path for soybean genetic modification.Previous studies on the model plant, Antirrhinum majus, demonstrated that therewere mainly four cardinal genes in the development of dorsoventral asymmetry:CYCLOIDEA(CYC), DICHOTOMA(DICH), RADIALIS(RAD)and DIVARICATA(DIV).CYC and DICH genes which belonged to the TCP transcription factor family wereinvolved to the dorsal identity of flower development. Both RAD and DIV genesbelonged to the MYB transcription factor family, and RAD gene promoted dorsalindentity while DIV to the ventral identity. CYC and DICH genes were expressed indorsal floral primordia, and then activated the expression of RAD gene. DIV gene wasinhibited by the RAD gene expression in the dorsal and lateral petals. Therefore thedorsal identity was expressed in adaxial domain of dorsal and lateral petals indifferent degree, forming three different morphology petals.Based on genome database and bioinformatics method, homologous genes ofCYC/DICH, RAD and DIV were characterized from soybean in this study. Three TCPgenes, GmTCP1, GmTCP2and GmTCP4which were closer to the CYC gene wereisolated from soybean. By transgenic technology, the overexpression and gene silence of these three genes experiments illustrated that GmTCPs genes, GmRADs genes andGmDIVs genes were involved in the control of flower bilaterally symmetry in soybean.More factors were involved in soybean floral symmetry regulation comparing withmodel plant. Altering expression of these genes in Arabidopsis gave rise to dissimilarphenotypes of transformants, which suggested that their funtions were divergent. Wealso set up and optimized the system of Agrobacterium-mediated soybeantransformation using the cotyledonary node and the system of biolisticstransformation using the embryonic tips of soybean, which provided a good platformfor soybean function analysis. The results were as follows:1. Establish the system of Agrobacterium-mediated soybean transformation usingthe cotyledonary node and the system of biolistics transformation using the embryonictips of soybeanThe effects of13cultivars and two selection agents were studied during theprocess of shoot regeneration and transformation. The results showed that the cultivar‘Shanning14’ induced highest number of multiple shoots among tested13cultivars,and hygromycin B was better than glufosinate during transformant selection ofcultivar ‘Shanning14’. The optimal transformant selection scheme was a gradientconcentration of hygromycin B from3mg L-1to8mg L-1. Transformation efficiencywas3.2%based on the number of transformed events. By biolistics transformation ofsoybean embryonic tips, we acquired a number of transgenic plants, and thetransformation efficiency of this method was9.3%based on the number oftransformed events.2. Cloning of the TCP homologous genesFifty-eight CYC homologous genes, five RAD homologous genes and five DIVhomologous genes were characterized from soybean phytozome v7.0database. Thephylogenetic analyisof TCP, RAD and DIV homologous genes were constructed bythe MEGA5software from Antirrhinum majus, Zea mays, Oryza sativa, Arabidopsisthaliana, Medicago truncatula and Glycine max. Three TCP genes, GmTCP1, GmTCP2and GmTCP4, closer to the CYC gene were cloned from soybean, and theirsense expression vectors, antisense expression vectors and VIGS vectors wereconstructed in this study.3. Function analysis of GmTCP1, GmTCP2and GmTCP41) Phenotyping of transgenic soybean of GmTCP1, GmTCP2and GmTCP4: ByAgrobacterium-mediated genetic transformation and biolistics transformation, thesense expression vectors of GmTCP1, GmTCP2and GmTCP4were transformed intosoybean cultivar ‘Shanning14’. Meanwhile, a series of VIGS transgenic lines ofGmTCP1, GmTCP2and GmTCP4were also generated in this study. Theoverexpression of GmTCP2and GmTCP4conferred to ectopic dorsal indentityshifting in other petals, concurring with morphological phenotypes of epidermal cells.No obvious phenotype was oberserved in GmTCP1overexpression transgenic lines.The VIGS transgenic lines of GmTCP1, GmTCP2and GmTCP4were all withabnormal tubular cup dorsal petals to various extent. The phenotypes of scanningelectron microscope on epidermal cells were coincident with their morphologicalvariations.2) Phenotyping of transgenic Arabidopsis of GmTCP1, GmTCP2and GmTCP4:To further study the function of GmTCPs genes, the sense and antisense vectors ofGmTCP1, GmTCP2and GmTCP4were transformed into Arabidopsis thaliana. TheGmTCP2overexpression transgenic lines grew quickly with larger leaves and floralorgans, while the antisense transgenic lines showed opposite phenotype. TheGmTCP4overexpression transgenic lines induced more round leaves, while theantisense transgenic lines had no difference with wild type. There was no obviousphenotype of GmTCP1transgenic lines with both overexpression and antisensevectors.4Interactions of GmTCPs, GmRADs and GmDIVsReal time-PCR analysis result suggested that GmTCPs and GmRADs genes wereexpressed in the dorsal petal, while the GmDIVs genes were expressed in the ventral petal. The transcription levels of GmTCPs genes and GmRADs genes wereconstitutively up-regulated in the GmTCP2and GmTCP4overexpression transgenicplants, but the transcription levels of GmDIVs genes were reduced in the sametransgenic plants. On the contrary, the VIGS lines gave rise to the opposite result. Theabove results suggested that GmTCPs genes, GmRADs genes and GmDIVs genesinvolved in the control of flower bilaterally symmetry in soybean. GmRADs genesmay be downstream of GmTCPs genes. GmRADs may antagonise GmDIVs genes inthe control of flower symmetry.The innovations of this thesis:1. It was the first research to illustrate that alteration expression of TCP genecould cause the tubular cup dorsal petal. This provided the clues for studying thefunction divergency of TCP gene and indentifying their new interaction factors.2. It was proved that more factors were involved in soybean floral symmetryregulation comparing with model plant. GmTCPs, GmRADs and GmDIVs genesinvolved in the control of flower bilaterally symmetry in soybean, which is morecomplicated than Antirrhinum majus as more factors imported.3. The divergent phenotypes were observed from transgenic Arabidopsis ofGmTCP1, GmTCP2and GmTCP4genes, which suggested that their specifities aredifferent. GmTCP2could function in both soybean and Arabidopsis via regulting cellproliferation and differentiation.4. We established and optimized Agrobacterium-mediated transformation andbiolistics transformation platforms of soybean, which availed to soybean genefunction indentification in the future.