Study On The Function Of EgMIXTA1 Transcription Factor In Eustoma Grandiflorum

In this study,choosing the EgMIXTA1 transgenic lines and wild type of E.grandiflorum which were cultivated in College of Life Science of Northeast Forestry University as the experimental materials.Using the molecular biotechniques,bioinformatics methods and RNAseq to predict and analyze the function of EgMIXTA1 transcription factor and its expression pattern combining with the results that have reported about the function of MIXTA and MIXTA-like in model and non-model plants.To research the function of EgMIXTA1 not only make the deeper understand on stress-tolerance in E.grandiflorum come true,improved the the quality of fresh cut flowers and got ornamental stronger plants,but also could offer a theoretical basis for studying molecular mechanism of R2R3 MYB Sub 9 gene family.The overall results were summarized as follows:1.To confirm the expression level of the EgMIXTA1 and the phenotype of E.grandiflorum by the methods about Real-Time PCR,Scannig electron microscope,Western blot,Water loss rate and Subcellular localization.The results indicated that EgMIXTA1 was more strongly expressed in over-expression transgenic lines than in the wild type.Comparing with the wild type,the SEM observation showed that cuticular waxes of over-expression transgenic lines obviously had a greater increase.Western blot analysis showed that the expression of EgMIXTA1 protein in the over-expression transgenic plants was higher than that in the wild type.Water loss rate of the leaves in the over-expression transgenic lines was significantly lower than that of the wild type,it meant that the drought stress tolerance had a greater increase in EgMIXTA1 over-expression transgenic lines.It was presumed that the cuticular waxes had the water retention function on the E.grandiflorum.At the same time,relying on the onion epidermal cell subcellular localization method,the result showed that EgMIXTA1 was located on the cell membrane and nucleus by using GFP on pK7WGF2-EgMIXTA1 overexpression vector.2.The phylogenetic tree of EgMIXTA1 was based on using bioinformatics technology.The results showed that EgMIXTA1 was similar to that the MYB gene of Petunia hybrida,and also had the homologous relation to the Anarrhinum bellidifolium(AbMIXTA-like3),Antirrhinum majus(AmMYBML3),etc.And a series of genes,such as Eg1g77670,Eg3g50800,Eg4g20960,Eg2g26250-KCS10,Eg5g52510-SCL8,KCS1,KCS2 and so on,were found in comparison with the genome database of the E.grandiflorum in the laboratory.The genes were analyzed quantitatively in the EgMIXTA1 transgenic lines and wild type plants.The result showed that the expression of these genes were up-regulated except for Eg5g52510-SCL8 in EgMIXTA1 overexpressing transgenic plants.3.The transcriptions of leaves from wild-type and Eg MIXTA1 overexpression transgentic lines were analyzed by RNA-seq.Furthermore,by comparing EgMIXTA1 transgenic plants and wild type plant transcript sequences,differentially expressed genes were obtained.In the EgMIXTA1 overexpression transgenic plants,there were 504 differentially expressed genes,of which 232 were up-regulated and 272 sequences were down-regulated.Combining with the correlation between the sequencing results and the annotation of differentially expressed genes in each database,20 genes related to EgMIXTA1 transcription factor function from EgMIXTA1 overexpression transgenic plants and wild type plants were selected and quantitatively analyzed.The gene ID were named by c87990,c80418,c72766,c83250,c65763,c27131,c54864,c80865,c32894,c71819,c35983,c80696,c90308,c82147,c66358,c80916,c29911,c72094,c79034,c89150.And the quantitative results was consistent with the RNA-seq in addition to c80696.In this study,30 target genes that were regulated by EgMIXTA1 transcription factor were obtained by bioinformatics and RNA-seq.These genes were involved in the regulation of plant epidermal cell morphology including Eg1g77670,Eg3g50800,Eg4g20960,Eg2g26250-KCS10,Eg5g52510-SCL8,the response to the hormone signal and the optical signal including c87990(ABR1-like),c80418(PRE6-like),c54864(PINOID 2),c80265(PID2),c82147(GH3.1),c66358,c80916(SAUR71),c72094(OPR7),the response to abiotic and biotic stress including c35983(HSC80),c89150(MDH),participating into the growth and apoptosis of plant cells including c65763(fabB),c32894,c71819,c80696(UPTG2),c90308(Clp1),c29911(ST3),c79034(XTH),the synthesis of long-chain fatty acids that the main components of the plant epicuticular wax and the lipid,including KCS1,KCS2(c83250),KCS9,CER10,KCS20,c72766(CER3),c27131(SBH1).