| Chrysanthemum indicum var.aromaticum is a variety of Chrysanthemum indicum discovered in recent years.Its flowers,leaves,and roots are full of aromas.It can be used for extracting essential oil and processing medicinal materials.It is a kind of plant with high value of medicinal exploitation and garden application.Anthocyanin biosynthetic pathway is an important branch of flavonoid metabolic pathway,so it is important to study the metabolic pathway of flavonoids to improve the flower color of garden plants.MYB transcription factors are involved in the regulation of flavonoid synthesis in plants.In this study,a MYB gene was cloned from Chrysanthemum indicum var.aromaticum.Through transgenic method,CiMYB4 was overexpressed in Nicotiana tabacum,and the function of CiMYB4 gene was preliminarily explored.Furthermore,genetic transformation of chrysanthemum was carried out in order to provide a reference for the regulatory network of flavonoid metabolism pathways in Chrysanthemum indicum,and also to provide a theoretical basis for chrysanthemum flower color innovation.Based on the transcriptome data of Chrysanthemum indicum var.aromaticum,CiMYB4 gene was screened and cloned in this study,and the CiMYB4 gene was analyzed by bioinformatics method.Double enzyme digestion method was used to construct plant over-expression vector to carry out genetic transformation of Nicotiana tabacum and preliminarily explore the regulatory effect of CiMYB4 gene.Then the CiMYB4 gene was transferred into Chrysanthemum indicum,which laid a foundation for the later study on the metabolic pathway of CiMYB4 gene on flavonoids and color changes of Chrysanthemum indicum.The experimental results are as follows:1.An open reading frame(ORF)of MYB gene was cloned by RT-PCR,with a length of 846bp and 281 amino acids encoded.It was named CiMYB4 gene.The relative molecular weight of the protein encoded by this gene was 31720.79,and the theoretical isoelectric point(pI)was 8.77.It was an unstable protein with strong.Hydrophilicity multiple sequence alignment and phylogenetic analysis showed that the protein CiMYB4 was a typical R2R3 type of MYB transcription factors with two R domains in the N-terminus.It is predicted that this gene may have inhibitory factor.CiMYB4 is more homologous to Helianthus annuus,Artemisia annua,Lactuca sativa and Cynara cardunculus,and the similarity with MYB similarity in Artemisia annua reached 92%.2.The pBI121-CiMYB4 plant overexpression vector was successfully constructed by double enzyme digestion of CiMYB4 gene.The subcellular localization analysis of CiMYB4 gene in onion epidermal cells was performed by particle bombardment.The results showed CiMYB4 protein can locate on the nucleus.The recombinant plasmid pBI121-CiMYB4 and pBI121 plasmid were introduced into Agrobacterium by electrotrans formation and transformed into Nicotiana tabacum by agrobacterium-mediated method.Positive plants were preliminarily obtained through two resistant rooting screening,and false positive was excluded by RNA extraction and PCR detection,proving that positive tobacco lines were successfully obtained with CiMYB4 gene transfection and pBI121 empty vector transfection.Three CiMYB4 overexpressed lines were selected and sown for resistance screening to obtain transgenic T1 generation plants.qRT-PCR results showed that CiMYB4 gene inhibited the expression of flavonoid metabolism-related genes.3.CiMYB4 gene was transferred into wild-type Chrysanthemum indicum by agrobacterium-mediated method,and the positive plants were preliminarily obtained by rooting screening.By using geNorm and NormFinder software,the expression stability of 5 candidate reference genes in Chrysanthemum indicum root,stem,leaf and flower was analyzed.The results showed that the expression of CmEF1a was the most stable in different tissues.CmEF1a was used as internal reference primer in qRT-PCR,and the results showed that CiMYB4 gene overexpressed plants had been successfully obtained. |
