Preliminary Study On The Biological Characteristics Of Red Walnut And The MYB Transcription Factor Related To Anthocyanin Expression Analysis

Walnut is the first of the four major nuts,common walnut pericarp is green,seed coat is yellow brown.According to the red walnut ’RW-1’pericarp,leaves and seed coat,the research group conducted transcriptome sequencing on the pericarp and leaves at different development stages.In this study,red walnuts were used as materials to investigate and analyze the appearance traits of branches and leaves,male and female inflorescence,fruit quality,and the traits separation of natural hybrid offspring.And based on MYBs transcriptome sequences that were significantly differentially expressed in transcriptome data and related to anthocyanin synthesis.The CDS sequences of three MYB transcription factor genes were cloned.The expression differences of red walnut with different phenotypes were verified in red and green leaves of natural hybrid progenies.Finally,bioinformatics analysis and subcellular localization analysis of MYBs were carried out.The main results are as follows:1.Investigation on the segregation of red walnut and quality traits of its natural hybrid offspring1)Comparison of red walnut maternal plant and green walnut,the color of the tree crown of red walnut is darker and red,and its leaves and branches are crimson.2)The average length of the male inflorescences of the red walnut is slightly shorter than that of the ordinary walnut,and the male inflorescence of the red walnut is darker.The female inflorescence of the red walnut is pink,while the male and female inflorescences of the ordinary walnut are both yellow-green.The germination rate of red walnut was about 30%,the germination rate of ordinary walnut pollen is 80%.The fruit of the red walnut is small,and peel,inner diaphragm and seed coat are dark red.The seed kernel is white in the same way as ordinary walnuts.The nutrients of red walnuts and ordinary walnuts kernels were quantitative analysised.It was found that unsaturated fatty acids were the main components of both.In addition,compared with ordinary walnuts,the crude fat content and the sucrose content of the red walnuts are higher.3)The progeny of natural hybrids of red walnut appear to have segregation of traits.The proportions of red walnut and green walnut in all progeny lines are about 5%and 95%,respectively.The tree vigor and root system of the red walnut are weaker than the green walnut,and the trunk of the red walnut is darker than that of the green walnut.4)Through the qualitative and quantitative analysis of the anthocyanin components in the leaves of the red and green walnuts the offspring from the red walnut.This indicates that the main components of anthocyanins in the offspring of walnut leaves are the derivatives of delphinium and cyanidin.2.Cloning and functional analysis of MYB transcription factor related to walnut anthocyanin metabolism1)The CDS sequences of JrMYB4,JrMYB12 and JrMYB306 were 846 bp,939 bp and 1,047 bp.That encoding 281,312 and 348 amino acids with relative molecular values of 31.70 kD,35.39 kD and 38.25 kD,respectively.From intron-exon structure analysis,it was found that both JrMYB4 and JrMYB12 contained two introns,and JrMYB306 contained an intron.Chromosome mapping results showed that JrMYB4 was located on Chrl of walnut,and both JrMYB 12 and JrMYB306 were located on Chr3 of walnut.2)Sequence analysis of three JrMYBs proteins in red and green walnuts and MYB proteins in other species showed that,The amino acid sequence of JrMYB4 transcription factor was not different between red walnut and green walnut.The amino acid residues at the 87-94 the amino acid sequence of JrMYB 12 of green walnut which is an insertion mutation of JrMYB 12 of relative red pulp walnut.The JrMYB306 protein sequence was compared with MYB transcription factor biosynthesis of anthocyanin from other species.The R2-MYB conserved domain of the N-terminal of JrMYB306 protein sequence was mutated with deletion of 52 amino acid residues.Phylogenetic tree results of MYB proteins related to anthocyanin synthesis in other species indicate that,JrMYB4 and JrMYB306 transcription factors in walnut belong to the same group as MnMYB4 in mulberry,NtMYB2 in daffodil and DcMYB1 in carrot.JrMYB12 is homologous to AtTT2 protein in Arabidopsis that is involved in proanthocyanidin synthesis,which is grouped with ZMC1 and ZMP1,which are related to anthocyanin synthesis in maize.3)Bioinformatics analysis of JrMYB4,JrMYB 12 and JrMYB306 protein revealed that the theoretical isoelectric point is 5.11,8.39,5.62,the protein stability index is 35.18,46.73,48.40,the total average hydrophobic index is-0.69,-0.78,-0.71,respectively,the values are less than zero,so it is hydrophilic protein,the most abundant amino acid is Ser.The prediction results of the transmembrane region of the three JrMYBs protein sequences showed that the JrMYB4,JrMYB 12 and JrMYB306 proteins had no transmembrane structure,and it was speculated that these three proteins were not membrane proteins.4)The specific expression analysis of the three JrMYBs genes in different developmental stages and different tissues of walnut showed that:the expression levels of the three JrMYBs genes in the red walnut seed coat increased gradually at three different development stages,while the expression levels in the green walnut seed coat decreased gradually at three different development stages.The expression of JrMYB4 and JrMYB12 in red walnut seed coat were higher than that in green walnut seed coat at each stage.In addition to the first stage,the expression level of JrMYB306 in red walnut seed coat was higher than that in green walnut seed coat at other two stages.The expression of JrMYB4,JrMYB12 and JrMYB306 in the progeny of red walnut in the red leaves and green leaves of the natural hybrid progeny of red walnut in the first and third stages were significantly higher than those in the progeny of green walnut.In the second period,the expression of JrMYB12 were similar in the leaves of progeny red walnut and green walnut,and the expression of JrMYB4 and JrMYB306 were lower in progeny red walnut than in progeny green walnut.5)The results of subcellular localization showed that JrMYB4,JrMYB12 and JrMYB306 were localized in the nucleus.The detection results of yeast double-heterozygous transcriptional activation activity showed that both JrMYB4 and JrMYB 12 had indicating transcription self-activation.JrMYB306 also is less transcriptional.To sum up,the growth of red walnut is weaker than that of ordinary walnut,and the character seperantion phenomenon appears in the natural hybrid offspring.In the red walnut leaves the main components of anthocyanins are delphinium and cyanidin derivatives,and the seed skernel contain a large amount of unsaturated fatty acids.The transcription factors of JrMYB4,JrMYB12 and JrMYB306 in red walnut have the function of regulating anthocyanin synthesis,which provides a theoretical research foundation for the regulation mechanism of walnut anthocyanin synthesis and the cultivation of new varieties.