Mining Of UV-resistant MYB Genes In Upland Cotton And Preliminary Exploration Of MYB4 Functio

Large amounts of ozone depletion caused by human activities in recent years has disrupted the dynamic balance of the ozone layer,so the increase in ultraviolet radiation will become an important threat to global crop production in the future.To ensure agricultural safety,it has become the direction of future agricultural research to further explore the genetic potential of ultraviolet rays(UV)resistance and improve the breeding efficiency of crops.As the inherent antagonist of plants themselves,anthocyanins can not only absorb UV,but also have the ability to resist oxidation and scavenge free radicals,so anthocyanins play important roles in the adaptation of plants to UV,especially to UV-B radiation.At present,based on model plants,many key structural genes in the anthocyanin biosynthesis pathway have been identified,but the corresponding regulatory mechanism is far from clear.Previous studies have shown that many MYB transcription factors(TFs)are involved in this pathway.However,MYBs belong to a large family with diverse functions and are often species-specific.So,how the MYB TFs regulates anthocyanin biosynthesis and their roles in UV-B stress resistance requires further investigation.Gossypium hirsutum L.,as an economic crop widely grown in the world,occupies an important position in world agricultural production.Therefore,how to improve the UV,especially the UV-B tolerance in G.hirsutum and meanwhile improve its yield and quality has received extensive attention.In this study,based on a comprehensive analysis of the characteristics of the MYB gene family in G.hirsutum,10 cotton MYB genes related to anthocyanin biosynthesis and UV resistance were identified by using gene co-expression network association analysis,quantitative real-time PCR comparison,and transcriptome examination.Furthermore,taking one of the negative regulatory transcription factors,GhMYB4,as an example,the function of TF was preliminarily studied.The main results are as follows:1.Using the genomes of tetraploid G.hirsutum,diploid G.herbaceum L.and G.raimondii Ulbr.,812,349,and 348 MYB gene family members were identified,most of which belonged to the R2R3-MYB gene subfamily.According to sequence homology and phylogenetic analysis,89% of the MYB genes in the tetraploid G.hirsutum retained homoeologous copies from their biparental diploids.The collinearity analysis showed that the whole genome duplication was the main reason for the expansion of MYB gene family in G.hirsutum.2.According to the transcriptome data,31 MYBs in G.hirsutum were predicted to be involved in the regulation of anthocyanin biosynthesis by gene co-expression association analysis.Further real-time PCR quantification and transcriptome sequencing confirmed that 16 MYBs(9 positively correlated,7 negatively correlated)were involved in the anthocyanin biosynthesis pathway,and 10 of which were responsive to UV-B stress.3.Taking the MYB4 gene of G.hirsutum(GhMYB4)as an example,the genetic changes of the gene after polyploidization and domestication,and its functional characteristics in response to UV-B were preliminarily analyzed.(1)By cloning and sequencing,the two subgenomic homoeologs of the GhMYB4 gene,GhMYB4-At and GhMYB4-Dt,were identified.The CDS lengths of the two homoeologs are 768 and 762 bp,respectively.No length variation was found between the subgenomic homologous pairs of the gene in cultivated and wild cottons.However,a non-synonymous mutation at the position 539 bp of the GhMYB4-Dt coding region was found between cultivated cotton and wild cotton.Real-time quantitative PCR analysis showed that both homoeologs were expression in multiple organs and tissues,and the expression levels in the cultivated cotton were higher than that in wild cotton.Meanwhile,GhMYB4-At was always more dominantly expressed than GhMYB4-Dt in different organs and tissues.Furthermore,the expression levels of both homoeologs were significantly decreased under UV-B stress.(2)Transgenic Arabidopsis experiments showed that resistance of the GhMYB4 overexpressed plants to UV-B stress was significantly reduced against wild type.(3)After silencing GhMYB4 by VIGS,the anthocyanin contented was significantly increased in G.hirsutum leaves,the expressions of structural genes in the anthocyanin biosynthesis pathway were also up-regulated.Combined with UV-B treatment and transcriptome sequencing,12 structural genes in the anthocyanin biosynthesis pathway that can respond to UV stress and are regulated by GhMYB4 were preliminarily screened.In conclusion,MYB TFs that both regulate anthocyanin biosynthesis and respond to UV stress in G.hirsutum were deeply explored in this study.The sequence and expression characteristics of these genes were clarified,and the structural genes regulated by GhMYB4 in this metabolic pathway was preliminarily determined.These results provide an important reference for understanding the regulation pattern of anthocyanin biosynthesis in G.hirsutum and offer a direction for breeding new high quality cotton varieties with UV-B resistance.