Cloning And Primary Characterization Of CiMYB61 In Chrysanthemum Indicum Var.Aromaticum

Chrysanthemum indicum var.aromaticum is a kind of wild plant that has been widely concerned in recent years.It has many excellent characters.MYB is a large family of transcription factors,which plays an important role in regulation of secondary metabolism.In order to study the role of MYB in C.indicum var.Aromaticum,based on the transcriptome data obtained in the early stage,the MYB transcription factors family of C.indicum var.Aromaticum was analysed and a MYB gene,which was named as CiMYB61,was chosen for further study.The main results are as follows:1.In the transcriptome data of C.indicum var.Aromaticum,62 MYB transcription factors were identified,including 26 1R-MYB transcription factors,35 R2R3-MYB transcription factors,and one 4R-MYB transcription factor.Of all the MYB transcription factors,31 sequences with complete ORF were predicted.Under the treatment of methyl jasmonate,29 genes were up-regulated and 33 genes were down regulated,of which 24 genes were significantly different.4 of 62 MYB transcription factors were not annotated by the GO biological process.According to the analysis of transcriptome data,the target gene was screened and named as CiMYB61.2.The target fragment was amplified by gradient PCR using the cDNA of C.indicum var.Aromaticum as a template,and CiMYB61 was successfully cloned.The length of the ORF was 1008bp and encoded 335 amino acids.Domain analysis showed it to be a R2R3-MYB transcription factor.q-PCR results showed that CiMYB61 gene had the lowest expression level in roots and the highest expression level in stems,and similar expression levels in leaves and flowers.Under the treatment of 100umol/L methyl jasmonate,the expression level of CiMYB61 gene increased at first and then decreased with the prolonging of treatment time.The expression level of CilMYB61 gene reached the maximum value when treated for 4 hours.3.The pBI121-CiMYB61-GFP plant expression vector was constructed by double enzyme digestion and transferred into tobacco by Agrobacterium-mediated method.The transgenic tobacco was detected by PCR.The results showed that the exogenous CiMYB61 had been integrated into the tobacco genome.The subcellular localization analysis of CiMYB61 gene in onion epidermal cells was performed by particle bombardment.The results showed that CiMYB61 protein acts on the nucleus.Three transgenic CiMYB61 lines,CiMYB61-S7,CiMYB61-S2 and CiMYB61-S1,were selected to obtain transgenic T1 plants.4.Compared with control WT and EV,transgenic tobacco showed smaller leaf area,shorter leaf length,and smaller leaf width.Leaf edges of transgenic tobacco were neat,and the hardness of leaf and stem increased.Among the three transgenic lines,CiMYB61-S7 showed more distinct traits,followed by CiMYB61-S2 and CiMYB61-S1.Scanning electron microscopy(SEM)was used to compare the cell structure of the same position in the fourth quarter of stems of transgenic tobacco and wild type tobacco.The xylem cells of CiMYB61 transgenic tobacco were arranged more closely and the woody cloth range was wider.The Wiesner experiments show the same result.Ultraviolet spectrophotometry was used to determine the lignin content in transgenic tobacco and non-transgenic tobacco.The results showed that the content of lignin in transgenic tobacco was significantly increased.Further qPCR validation showed that the exogenous gene CiMYB61 can regulate the expression of key enzyme genes in tobacco lignin synthesis.