Construction Of RNAi Vector Of CiMYB61 Gene In Chrysanthemum Indicum Var.aromaticum And Preliminary Study On Its Effect On Leaf Trichomes Of Chrysanthemum Indicum

Chrysanthemum indicum var.aromaticum is a rare perennial herb,which has a high value in medicinal and ornamental properties,so it has broad development prospects in garden applications.MYB is a large and feature-rich family of transcription factors that play an important role in many biological processes.In this study,the CiMYB61 gene of Chrysanthemum indicum var.aromaticum,which was successfully cloned in the early stage of the research group,was taken as the research object.In order to comprehensively study and enrich the function of CiMYB61 gene of Chrysanthemum indicum var.aromaticum,this study constructed its RNAi vector,and genetically transformed the Chrysanthemum indicum.Over-expressed CiMYB61 transgenic Chrysanthemum indicum and inhibited expression of CiMYB61 transgenic Chrysanthemum indicum were obtained respectively,and the preliminary functional analysis of CiMYB61 transgenic Chrysanthemum indicum,the main research results are as follows:1.Constructed the RNAi vector of CiMYB61 gene of Chrysanthemum indicum var.aromaticum,and selected 308 bp as the RNAi interference fragment from the sequence of CiMYB61 gene ORF region obtained in the laboratory.The sense and antisense fragments were respectively connected to the corresponding positions on the p HANNIBAL intermediate expression vector,and the PDK-intron interval was added in the middle to achieve the silencing effect.The constructed CiMYB61(+)/p HANNIBAL/CiMYB61(-)containing the hairpin structure was inserted into the plant expression vector p ART27,and the RNAi vector p RNAi-CiMYB61 of the CiMYB61 gene of Chrysanthemum indicum var.aromaticum was successfully constructed.Then,the successfully constructed p RNAi-CiMYB61 recombinant plasmid was successfully introduced into Agrobacterium tumefaciens by electrotransformation,and finally the Agrobacterium bacterium liquid containing p RNAi-CiMYB61 recombinant plasmid was used.2.PBI121-CiMYB61 recombinant plasmid was transformed into Chrysanthemum indicum by agrobacterium-mediated method,and the over-expressing CiMYB61 transgenic positive Chrysanthemum indicum was initially obtained through resistance screening.It was successfully reverse-transcribed into RNA on the basis of better quality of RNA extraction c DNA,the method of detecting the relative expression of CiMYB61 gene in resistant plants by PCR detection of resistant plants and qRT-PCR confirmed that two overexpressing CiMYB61 transgenic positive Chrysanthemum indicum strains had been successfully obtained.3.The optimal concentration of Spec for leaf disc screening during RNAi genetic transformation of Chrysanthemum indicum is 10mg/L,the optimal critical tolerance concentration of Spec for rooting screening resistant seedlings is 12mg/L,and the antibacterial concentration of Cef is 200mg/L.Then the agrobacterium-mediated method was used to transfer the p RNAi-CiMYB61 recombinant plasmid into the Chrysanthemum indicum,and the resistance screening CiMYB61 transgenic positive Chrysanthemum indicum was initially obtained through resistance screening.The RNA was successfully reverse transcribed on the basis of ensuring high RNA extraction quality.The method of detecting c DNA and detecting the relative expression of CiMYB61 gene in resistant plants by PCR detection of resistant plants and qRT-PCR confirmed that one plant that inhibited the expression of CiMYB61 transgenic positive Chrysanthemum indicum was successfully obtained.4.Observed the morphology and density of trichomes of wild-type Chrysanthemum indicum(WT),over-expressed CiMYB61 transgenic Chrysanthemum indicum,inhibited expression of CiMYB61 transgenic Chrysanthemum indicum,and found that there were differences in morphology of the three T-shaped non-glandular hairs;The T-shaped non-glandular hair density was the highest,followed by the wild-type Chrysanthemum indicum,and finally the inhibition of the expression of CiMYB61 transgenic Chrysanthemum indicum lines;the length and width of the glandular hairs of the over-expressed CiMYB61 transgenic Chrysanthemum indicum lines were significantly higher than those of the other two lines.The density of glandular hairs that inhibited the expression of CiMYB61 transgenic Chrysanthemum indicum was the most sparse.