Identification And Functional Analysis Of Candidate Genes For Glume Wax Deficiency Using Wax-deficient Mutant Cer-GN1 In Barley(Hordeum Vulgare L.)

To adapt to drought stress,plants have formed a series of complex mechanisms of drought resistance from molecular to phenotypic during the long evolutionary process,among which the wax deposited on the plant surface is an important barrier to prevent water loss and reduce the harm of drought stress.Identifying the key genes regulating cuticle wax and analyzing the mechanism of wax formation can provide gene resources and theoretical basis for breeding new varieties of drought-resistant crops.Our previous studies obtained Cer-GN1,a spike glumes wax-deficient barley mutant,by EMS mutagenesis.Based on this,the mutant Cer-GN1 and its original parent WT were used as materials in this study.Mendelian genetic rule analysis,metabolomics and transcriptomic analysis were used to identify the candidate genes of wax-deficient in the mutant Cer-GN1,and the function of the candidate genes for waxy deletion of glume was preliminarily verified.The main results are as follows:1.The barley mutant Cer-GN1 showed a wax-deficient phenotype in spike glumes,which began to appear after 8-12 days of heading stage became more obvious at filling stage;The spike glumes wax-deficient phenotype of Cer-GN1 was controlled by a recessive single gene,and the mutant was more sensitive to drought stress,which resulted in a significant decrease in 1000-grain weight,spike length and spike width after moderate drought treatment(P<0.05).2.The analysis of metabolites in Cer-GN1 spike cuticular revealed that only 16-hydroxyhexadecanoic acid(16-hydroxypalmitate)identified in the NEG model was significantly enriched in the cutin,suberine,and wax biosynthesis pathways,with a fold change of 0.04-fold that of WT.The 16-hydroxypalmitic acid is one of the predominant C16 and C18 fatty acid-derived cutin monomers.It is speculated that the drastic decrease of its content may lead to the blockage of the synthesis long chain fatty acids and their derivatives,resulting in the decrease of the accumulation of Cer-GN1 spike glumes wax.3.After transcriptome sequencing of Cer-GN1 mutant and WT spike glumes collected at 4,8,12 and 16 days after heading,we identified the candidate genes HvMSTRG.29184 and HvMSTRG.29185 in Cer-GN1 mutant that regulate spike glumes wax-deficient.These two genes encode long-chain fatty acid omega-monooxygenase(CYP704B1)and are significantly down-regulated in the glume of Cer-GN1 mutants after heading(P<0.05),thereby inhibiting the conversion of 16-palmitic acid to 16-hydroxyhexadecanoic acid in keratin metabolism.4.Both HvMSTRG.29184 and HvMSTRG.29185 genes are located on the 4H chromosome of barley.There were 423 bp repeats in the 563743203-563743626 bp region of the two genes,and HvMSTRG.29185 may be a part of the 3 ’end of HvMSTRG.29184,which belonging to the same gene.The HvMSTRG.29184 gene has a total length of 2097 bp,a CDS region located at 145-1800 bp,with a length of 1656 bp.It encodes 551 amino acids,the relative molecular weight is 60.61 KDa,and the theoretical isoelectric point is 8.02.This protein was compared in uniport database and found to have CYP704B1 activity.Compared with WT,HvMSTRG.29184 gene has four base mutations of C > T,T > C,C > T and C > G at the 62,684,953 and 1176 bases in CDS region,respectively.Due to the degeneracy of codon,the encoded protein has one amino acid difference from WT,that is,alanine(A)> valine(V)was mutated in the 318 th amino acid.5.Based on the differences in phenotype,wax composition,regulatory genes and location information of 82 barley waxy mutants published in the International Barley Gene and Barley Genetic Resources Database,Cer-GN1 was preliminarily identified as a new type of wax-deficient in barley spike cuticular obtained by EMS mutagenesis.6.The subcellular localization of Arabidopsis protoplast showed that HvMSTRG.29184 gene was located on the endoplasmic reticulum,which was consistent with the predicted result.It can be preliminarily determined that the HvMSTRG.29184 gene encodes CYP704B1 protein.The down-regulated HvMSTRG.29184 gene lead to the blocking of the conversion of 16-palmitic acid into16-hydroxypalmitic acid in spike glumes of the mutant Cer-GN1,which affected the whole synthetic biology process of the mutant Cer-GN1 wax,and thus lead to the mutant Cer-GN1 spike glumes wax-deficient phenotype.