| MYB transcription factor is one of the largest transcription factor families in plants.It is involved in plant growth and development,primary and secondary metabolism,cell morphogenesis,hormone signal transduction,development regulation and response to biotic and abiotic stresses.Seven differentially expressed MYB genes were found in the transcriptome study of mulberry vein belt associated virus infection in mulberry leaf tissue.In order to further study the function of these genes,fluorescence quantitative PCR was used to detect the expression of MYB genes in mulberry vein belt virus infected plants.The genes were cloned and their amino acid sequences were analyzed by bioinformatics methods,and their amino acid sequences were verified by yeast one hybrid The expression patterns of transgenic tobacco under different tissue and abiotic stresses were detected,and the homologous genes were transformed into tobacco to analyze the performance of transgenic tobacco under drought and virus stress.The results are as follows:1.The expression levels of 7 candidate MYB genes were detected by fluorescence quantitative PCR.The results showed that the expression levels of Ma MYB4,Ma MYB30 and Ma MYB308 in diseased leaves were significantly up-regulated,which were 2.41,3.96 and 6.4 times of those in healthy leaves.Ma MYB16 and Ma MYB124 were down regulated,and 0.5 times of that in healthy leaves.There was no significant difference in transcription levels of Ma MYB73 and Ma MYB1R1 in diseased and healthy leaves,the results were consistent with the results of transcriptome sequencing.2.The ORF fragments of 7 candidate MYB genes were cloned and the recombinant plasmid p GBKT7 was constructed and transferred into yeast Y2 H gold.The results showed that Ma MYB4,Ma MYB16,Ma MYB30 and Ma MYB308 had self activation activity,while the other three genes had weak or no self activation ability.3.The c DNA sequences of Ma MYB4,Ma MYB30 and Ma MYB308 were cloned and analyzed by bioinformatics.The full length of Ma MYB308 was1266 bp,and the coding region was 1026 bp.It contained a 25 bp untranslated region(UTR)and a 215 bp 3′untranslated region,which could encode 341 amino acids.The encoding region of Ma MYB30 was 864 bp,encoding 288 amino acids.Ma MYB4 contains an 825 bp translation region,encoding 275 amino acids.The three genes contain two DNA binding domains,which are typical R2R3 MYB transcription factors.4.The expression patterns of Ma MYB4,Ma MYB30 and Ma MYB308 were analyzed by fluorescence quantitative PCR.It was found that the three genes were tissue-specific,and their expression levels in roots were more than20 times of those in stems and leaves.Under abiotic stresses,the three genes showed different degrees of stress,among which the induction of drought,low temperature,SA and ABA was the most significant,and the highest expression level reached 80% of the control Times.5.The homologous gene of Ma MYB4 was transformed into tobacco Nt MYB4,and 13 tobacco strains with overexpression of Nt MYB4 were obtained.Fluorescence quantitative PCR was used to detect the expression of phenylpropanoid metabolism related genes in transgenic and wild-type tobacco.It was found that dihydroflavonol 4-reduetase(DFR)and cinnamate-4-hydroxy-lase(C4H)were down regulated to 0.003 and 0.48 times of wild type respectively in transgenic plants.The results showed that the tolerance of transgenic tobacco to drought was lower than that of wild-type tobacco,indicating that Nt MYB4 may be a negative regulator in tobacco regulation of drought stress;after TMV infection,the onset time of wild-type tobacco was earlier than that of transgenic plants,and the degree of disease was greater than that of transgenic plants.It was speculated that Nt MYB4 might be involved in the regulation of tobacco to biological stress. |
