Study On The Regulation Mechanism Of MYC Transcription Factors Participating In Latex Production In Hevea Brasiliensis

Natural rubber is mainly from the Hevea brasiliensis,which is one of the main economic crops in Hainan Province.The latex is synthesized and stored in the laticifer,and the number of secondary laticifer affects the latex yield.laticifer differentiation has been shown to be associated with the jasmonic acid signaling pathway,in which JAZ proteins inhibit the expression of downstream genes by binding to MYC transcription factors,myc is a b HLH-like transcription factor that can interact with G-box cis-acting elements.Our group has demonstrated the interaction between HbJAZ1 and HbMYC3 in the previous study,and initially identified the interaction between MYCs and G-box cis-acting elements.In order to further screen and identify the downstream target genes that are regulated by HbMYCs transcription factors and related to rubber production in the JA signal pathway of rubber tree,Our group screened the target genes related to rubber production and those promoters including G-box cis-acting elements on the whole-genome of rubber tree by using bioinformatics software ind EPendently developed.Finally,40 eligible genes were screened.In this paper,we studied two of the genes,HbREF and HbHMGR1.The main research content and results are as follows:(1)To further verify that the promoter sequences of HbREF and HbHMGR1 used in the experiment contain G-box(CACGTG)cis-acting elements,we used the online promoter analysis software Plant CARE and PLACE.Bioinformatics analysis showed that the promoters of both genes contained G-box cis-acting elements.(2)Using the whole genomic DNA of rubber tree as a template,the full-length promoter sequences of the rubber-producing genes HbREF and HbHMGR1 were cloned,the sizes were: 805 bp and 589 bp.(3)In order to verify whether HbMYCs can interact with HbREF and HbHMGR1 containing G-box cis-acting elements,we used yeast one-hybrid assay to verify.The results showed that: HbMYC2,HbMYC3,and HbMYC4 can interact with HbREF and HbHMGR1,respectively.(4)In order to further verify the interaction between HbMYCs and HbREF,HbHMGR1,we used single Luciferase reporter gene assay to verify.The results showed that: HbMYC2,HbMYC3,and HbMYC4 can interact with HbREF and HbHMGR1,respectively,and with HbREF interaction is stronger.(5)In order to further verify the interaction between HbMYCs,HbREF,and HbHMGR1,we tested it using the double Luciferase reporter gene assay.The results showed that: HbMYC3 and HbMYC4 can interact with HbREF and HbHMGR1,respectively,and with HbREF Interaction is stronger.(6)In order to investigate the difference in HbMYCs expression in rubber leaves of rubber trees at four different periods,we performed differential expression detection using RT-PCR technology.The results showed that: the expression of MYCs from the stage1 to the stage3,showed a constant decline.In the fourth period,the expression of HbMYCs increased again.(7)We want to further explore the effect of Me JA treatment on the expression of MYCs after four-stage rubber leaves.The REFore,the RT-PCR technology was used to detect differential expression of four-stage leaves in different periods after Me JA treatment.The results showed that: The expression of MYCs increased first and then decreased in stage1,and decreased first and then increased in stage2 and stage3,the expression showed an irregu LAR trend in the fourth stage.(8)To further identify the function of the HbMYC gene,we performed the phenotypic observation experiment in Arabidopsis,the results showed that: The phenotype of the myc2/3/4 triple mutant was significantly reduced,the root length was shortened,the leaf area was reduced,and the plant size became smaller,both myc2 and myc3 mutants became darker in leaf color,increased trichome,reduced leaf area,early flowering and higher plant height,after complementing the myc2 mutant with HbMYC2,the corresponding phenotype disappeared,and the opposite phenotype appeared after HbMYC3 overexpression.(9)In order to further prove that the interaction between HbMYCs,and HbREF,HbHMGR1 is performed in the G-box region,we designed a short sequence containing the G-box element on the promoter,and a corresponding short mutation of the G-box element Sequences,and these short sequences were constructed on the Ab Ai vector for subsequent single-hybrid experiments.(10)In order to further prove that the HbMYCs transcription factor interacts with HbREF and HbHMGR1 through the C-ter minus,we constructed AD-MYCs C-ter minus and AD-MYCs D-ter minus vectors for subsequent single-hybrid experiments.(11)A 28a-MYCs prokaryotic expression vector was constructed to provide a basis for subsequent EMSA.In summary,we conclude that HbMYCs transcription factors can interact with the gel-producing genes HbREF and HbHMGR1;Me JA has an effect on MYCs expression;HbMYC2 can complement the phenotype of the Arabidopsis mutant,it has good homology with At MYC2.In addition,since early flowering and increased trichome belong to the plant stress tolerance phenotype,it is speculated that HbMYC2 and HbMYC3 are related to plant stress resistance.These conclusions provide more theoretical basis for the connection between the rubber tree production mechanism and the jasmonic acid signaling pathway.