Molecular Mechanism Of MdMYB111 And MdWRKY40 Involved In Anthocyanin Biosynthesis In Red?fleshed Apple

Anthocyanins are important secondary metabolites in plants and belong to flavonoids.It is a natural plant pigment,which is distributed in many tissues and organs of plants.It plays an important role in plant growth and development.It is also the main pigment that determines fruit coloring.It has the effects of preventing cardiovascular disease,anti-oxidation,and anti-aging.Good for human health.Its biosynthetic pathway is affected by many external factors,such as temperature,endogenous hormones,and light.MYB,bHLH,WD40 and DELLA proteins are transcription factors that are mainly involved in regulating anthocyanin synthesis.As an important family of transcription factors in plants,WRKY plays a key role in plant growth and abiotic stress response.It has been studied in many directions such as disease resistance and salt stress.It is clear that the excellent flavonoid germplasm ‘Zihong No.1’ selected by the research group is used to study the mechanism of its participation in anthocyanin synthesis and provide theoretical support for subsequent breeding work.In this study,the red meat callus induced by the young leaves of ‘Hong Cui No.1’apple and ‘Zihong No.1’ were used as experimental materials and relevant experiments were carried out.The main results are as follows:1.The MdWRKY40 sequence has an open reading frame of 966 bp and is capable of encoding 321 amino acids.The phylogenetic tree analysis showed that MdWRKY40 is on the same evolutionary branch as Arabidopsis AtWRKY18 and AtWRKY40.Through amino acid sequence analysis,we found that MdWRKY40 protein contains a leucine zipper motif and WRKY domain.2.MdMYB111 was overexpressed in apple callus,and red meat callus changed from purple red to dark yellow,indicating that it had an inhibitory effect on anthocyanin synthesis,while overexpression of MdWRKY40 showed almost no change,but in the overexpressed MdMYB111 On the basis of over-expression of MdWRKY40,we found that the callus that faded to dark yellow gradually turned pink again,indicating that the two participated in the regulation of anthocyanins under a certain mechanism.3.Yeast two-hybrid experiments and two-molecule fluorescence complementary experiments showed that MdWRKY40 can interact with itself to form homodimers,while MdMYB111 does not interact with MdWRKY40;gel retardation experiments(EMSA)show that MdMYB111 can interact with The MRE element on the MdANS promoter binds,thereby inhibiting the expression of MdANS,and MdWRKY40 can be bound to the W-box of the MdANS promoter.When they work together,they can reduce the inhibitory effect of MdMYB111 on MdANS.4.The fragment obtained by knocking out the Leu zipper motif in MdWRKY40 by overlap PCR technology was named LLSMdWRKY40,and it was found that it could no longer form homodimers through its own interaction.In the presence of the two,the inhibitory effect of MdMYB111 on anthocyanins could not be weakened;the fragment obtained by knocking out the C-X5-C sequence was named LCSMdWRKY40,making it unable to bind the W-box of the MdANS promoter,and the above steps were repeated After that,the results were the same.