Identification Of MYB Transcription Factors In Lycium Ruthenicum Murr.and Function Reseaching Of LrMYB1

MYB is one of the most common transcription factor families in plants.It is widely involved in the growth and metabolic regulations of plants,including the differentiation of epidermal cells,stomatal development,flavonoid biosynthesis and so on.At present,there are few researches on the MYB transcription factor of Lycium ruthenicum Murr.So 3 diffirent period fruit of L.ruthenicum Murr.were used as material to conduct transcriptome sequencing to identify the MYB genes related to fruit color.The main results are as follows:1.A total of 194385 transcript were obtained using Illumina HiSeqTM2000,a second-generation sequencing platform.83 transcription factors of MYB family were annotated.From analysis of differential expression of MYB genes of transcriptome sequencing(RNA-Seq)and differential expression data based on fluorescence quantitative PCR confirmed that the expression of LrMYBl,LrMYB3,LrAN2 and was significantly different with the change of fruit color.in L.ruthenicum Murr..2.Based on the sequence of MYB1 from transcriptome sequencing,we have cloned a MYB gene that related to fruit color,named LrMYB1 The gene is 1016bp in length and 771 bp in open reading frame,encoding a protein of 256 amino acids.Through the transient expression system of tobacco,the result of laser confocal microscopy showed that LMYB1 located in nucleus.3.we optimized the regeneration system of leaves in Lycium barbarum Murr.,At the same time,we have build 3 expression vector,such as LrMYB1-pMDC32,LMYBl-pMDC43 and LrMYB1-pMDC85.The method of Agrobacterium-mediated in leaf were used to transform,and the transformation system was optimized.10 transgenic plants were obtained and there are 6 transgenic plants of the L.barbarum Murr.that are identified to positive plants after PCR.4.By detecting the content of flavonoids in positive plants and wild-type plants,we found that the content of flavonoids in the positive plants was significantly higher than that of wild-type plants,indicating that the gene can positively regulate the accumulation of flavonoids.We screened the genes related to biosynthesis of flavonoids and anthocyanin in L.ruthenicum based on its RNA-Seq sequencing.And the sequence of these genes were used as templates to design primers.The wild-type plants were used as control,the relative expression of flavonoid-related genes in positive plants and wild-type plants was detected by fluorescence quantitative PCR and found that the expression of CHS,F3H,FLS,F3'H,F3'5'H,DFR and ANS in positive plants was significantly higher than that in wild-type plants,which indicating that LrMYB1 may regulate the content of flavonoids by up-regulating the expression of these genes.