The Study On The Screening Of MYB Genes Involved In Drought Response And Its Regulatory Mechanism In Vaccinium Corymbosum L.

Blueberry(Vaccinium corymbosum L.)is an important economic perennial shrub,which has high economic and health value due to high content of anthocyanins in fruits.As a newly developed economic industry in China,the blueberry industry has a huge development and economic power.As a perennial shrub,blueberry is characterized by shallow-rooted property,with thin and undeveloped root hair,and it highly susceptible to drought stress.Due to global warming and frequent drought,in recent years,the yield and quality of blueberry production has significantly lowers,while the response mechanism of blueberry in response to drought stress still remains elusive.RNA-seq technology is a primary tool for studying gene expression,which is also a powerful tool for exploring key pathways and genes in the stress response.In this study,we used RNA-seq technology,weighted gene co-expression network(WGCNA)and protein–protein interaction networks(PPI)analysis to explore the potential drought-tolerance regulatory network based on physiological and biochemical changes for blueberry to respond to drought,and to verify key regulate pathways.Meantime,we analyzed the important MYB TFs family were screened for identification and identified the key Vc MYBs using differential gene expression(DEGs)analysis and expression correlation network analysis.Furthermore,we elucidate the characteristics and biological functions of the key Vc MYBs were identified by heterologous expression in Arabidopsis,so as to provide a new understanding of the mechanism of blueberry response to drought stress.The main achievements of this study are as follows:?The physiological and biochemical experiments showed that the antioxidant enzyme activity,reactive oxygen species(ROS)accumulates,stomatal density,anthocyanin concentration,abscisic acid(ABA)content and zeatin(ZT)content could be enhanced remarkably under drought stress;the stomata of leaf,gibberellic acid(GA₃)content,indoleacetic acid(IAA)content,chlorophyll and corresponding fluorescence parameters could be decreased significantly,and the growth of blueberries be inhibited.?Transcriptome analysis showed that 4670 genes were differentially expressed in the leaves,and2961 genes were differentially expressed in the roots,which the DEGs were defined with fold change?5.DEGs functional analysis showed that MAPK signaling transduction,plant hormone signal transduction,reactive oxygen species metabolic and biosynthetic process,AP2/ERF TFs family,MYB TFs family,b ZIP TFs family,flavonoid biosynthesis and monoterpenoid biosynthesis could be GO and KEGG significantly enriched,and eight important genes in response to drought stress were screened out.?MYB TFs family analysis showed that 229 non-redundant MYB sequences were identified in the blueberry genome,and divided into 23 subgroups.A total of 102 Vc MYB DEGs with a significant response to drought stress were identified,of which 72 in leaves and 69 in roots,and 8 differential expression genes with a??20-fold change in the level of expression.17 DEGs had a higher expression correlation with other MYB members.?The results show that Vc MYB14 and Vc MYB108 are mainly localized in the nucleus by transient expression in N.benthamiana.?Hormone treatment experiments showed that Vc MYB14 was induced by GA₃,salicylic acid(SA)and IAA,but it had no remarkable response to ABA and methyl jasmonate(Me JA)treatments.Vc MYB108responded significantly to GA₃,SA,ABA and Me JA,but not to IAA.?Using a quantitative fluorescent q RT-PCR assay,we tested Vc MYB14 and Vc MYB108 expression was highest in the leaves,followed by the roots.?The yeast assay results showed that Vc MYB14 and Vc MYB108 transcription factors have transcriptional activity of full-length.Meanwhile,Vc DREB1A and Vc KUA1,two interaction proteins of Vc MYB14,were verified via yeast two hybrid assay;and Vc MYB108 interacts with Vc ABF4.?By overexpression of Vc MYB14 in Arabidopsis,we find that overexpression of Vc MYB14significantly represses mature plants'and seedlings'tolerance to drought stress,and overexpression of Vc MYB14 not affect the plant growth or development.It is further found that the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)are significantly decreased in transgenic plants under drought stress,and the content of malondialdehyde(MDA)was significantly increased.Meanwhile,the expression of stress-responsive genes is obviously inhibited in Vc MYB14 overexpression lines.?By overexpression of Vc MYB108 in Arabidopsis,we find that overexpression of Vc MYB108significantly increase mature plants'and seedlings'tolerance to drought stress,and overexpression of Vc MYB108 not affect the plant growth or development.It is further found that the activities of SOD,POD,CAT and the anthocyanin concentration are significantly increased in transgenic plants under drought stress,and the content of MDA was significantly reduced.Under ABA treatment,the stomatal opening of leaves,seed germination rate and seedling root length was significantly inhibited in transgenic plants.Meanwhile,the expression of stress-responsive genes is obviously promoted in Vc MYB108overexpression lines.