Research On The MdTAS4-siR81(-) Targeted MdBHLH3 Roles In Regulation Of Anthocyanin Biosynthesis And Analysis Of Apple WD40 Protein Family

Anthocyanin content determines the color of apple peels.At present,studies on apple anthocyanin mainly focus on the transcription level,but there are relatively few studies on post-transcriptional levels.Small RNA mainly workson the post-transcriptional level and thus affects plant phenotypes.In this study,MdTAS4-siR81(-),the small RNA with differential expression was firstly found out from the high-throughput sequencing result of small RNA of the unbagged 'German apple' green peel and bag-removed ‘Granny Smith' red peel.Moreover,the function of MdTAS4-siR81(-)in fruit coloring was studied.WD40,bHLHand MYB compound can affect the formation of anthocyanin through regulating structural genes.At present,MYB and bHLH family of transcription factors have been fully identified in apples,but there is no related report about WD40 protein.Identification,classification,evolution and expression of WD40 gene in the new genome of apple were analyzed.The following results have been obtained:1.In the high-throughput sequencing results of small RNA,MdTAS4-siR81(-)in the green peel of the unbagged 'German apple' was significantly higher than that of the bag-removed ‘Granny Smith'.In the coloring process of “Granny Smith” and ‘Starkrimson',MdbHLH3 had an identical change trend with the anthocyanin content,while MdTAS4-siR81(-)had an opposite change trend with MdbHLH3.5'RACE demonstrated that MdTAS4-siR81(-)could realize target cleavage of MdbHLH3 both in “Granny Smith” and “Starkrimson”.The transient overexpression of MdbHLH3 in apple peels could promotes the expression of the structural genes MdDFR,MdANS and MdUFGT to promote the accumulation of anthocyanins,while transient overexpression of MdTAS4-siR81(-)inhibited accumulation of anthocyanin.Overexpression of MdbHLH3 could increase the proanthocyanidin content in the Arabidopsis seeds,while MdTAS4-siR81(-)could target MdbHLH3 and further inhibit the accumulation of proanthocyanidin in the arabidopsis seeds.At the same time,overexpression of MdbHLH3 could accumulate anthocyanins in the arabidopsis seedlings,while MdTAS4-siR81(-)could target MdbHLH3 and inhibit anthocyanin accumulation in the Arabidopsis seedlings.2.A comprehensive search and identification of new apple genes were carried out to find out 346 WD40 genes.They were named as MdWD40-1 to MdWD40-346 according to their positions on the chromosome.Based on the structural domain composition,the 346 apple WD40 genes were divided into 9 subfamilies(A-I).Meanwhile,phylogenetic analysis of WD40 genes in apples and Arabidopsis was made.The results showed that they were polymerizedto 14 different groups and the intron exon structure showed a similar structure in each group.In addition,the collinearity analysis showed that the large-scale amplification of WD40 in apples was caused by the modern genome-wide replication events of Apple.19 candidate genes related to drought and low temperature were selected through GO annotation and homologous comparison with Arabidopsis,and the quantitative results showed that there was one differential expression for the 19 candidate genes in different tissues and organs.Through ABA,drought and cold treatment,eight genes(MdWD40-17,24,70,74,219,256,283and307)were finally discovered,which responded to at least one of ABA,drought,and low temperature.