Molecular Cloning And Functional Characterication Of DiMYB1 In Davidia Involucrata Baill.

Dove tree(Davidia involucrata Baill.)is a deciduous tree belonged to the Nyssaceae family.It is a relic species of tertiary tropical flora,one of the first class national protect plants,and a endemic species of China.It is of high value for the aspects of appreciation and academic studies.According to the transcriptome sequencing data of the fruits and seeds of Davidia,,a gene involved in proanthocyanin biosynthesis was selected to investigate in the present study.The target gene was identified to encode a MYB1 transcription factor by cloning and sequence analysis,and named as DiMYB1 to indicate the first identified MYB gene in Daviaia.Finished works in this study are as follows:1.Cloning and identification of DiMYB1 from Davidia.Based the sequencing data of the ORF of DiMYB1,the full-length ORP sequence was cloned from the cDNA of the abortive seeds of Davidia by PCR using specific primers.We submitted the gene sequence to NCBI and got the GenBank number of KR996175.Furthermore,the bioinformatics analysis of the DiMYB1 sequence were performed using online software.2.The expression profiles of DiMYB1.?Expression profile in different Davidia tissues and organs.Expression levels were detected by qRT-PCR.The result showed that DiMYBl had the highest transcript abundance in purple young leaves,followed by purple stamens,while had very low transcript abundance in buds,stems,leaves,bracts,fruits,and seeds.? Expression profile in normal and abortive seeds of Davidia.Transcript abundance of DiMYB1 in abortive seeds was significantly higher than that in normal seeds,and reached the peak in the middle developmental stages in abortive seeds.? Expression profile in Davidia leaves in different colors.DiMYB1 had the highest transcript abundance in purple leaves,followed by intermediate color(in the middle of purple and yellow)leaves,and a low expression level in green leaves.3.Prokaryotic expression of DiMYB1 gene.Prokaryotic expression vector named pET-28a(+)-DiMYB1 was constructed successfully and transferred into E.coli,The DiMYB1 protein was induced by IPTG to express effectively.DiMYB1 protein was produced in the form of inclusion body.4.The preliminary functional verification of DiMYB1 in Arabidopsis thaliana.The plant over-expression vector pB1-121-DiMYB1 was successfully constructed and transferred into agrobacterium GV3101.Floral-dip method was used to transform Arabidopsis thaliana.After harvesting the seeds,transgenic plants were screened through growing on the medium with kanamycin added.T2 generation of transgenic plants were successfully obtained by inbred.Compared with the wild-type plants,the transgenic plants showed purple color in the leaves of young seedling;most of them appeared obvious changes in the shape and number of leaves,some of them appeared albino in the blade;stem slender;a few plants have mature pods in purple;Some pods are not full,and the seeds are shriveled;slim stems,drawf phenotype,and flowering in early period.Further research should be focused on homozygous transgenic plants screening,which is critical for function analysis of DiMYB1.