Identification Of WOX Genes And Their Functions And Mechanisms In Improving Genetic Transformation Efficiency In Triticum Aestivum L.

Low genetic transformation efficiency and strong genotype dependence are important factors restricting the development of wheat genetic engineering.The mining and application of wheat regeneration-related genes,improving wheat genetic transformation and regeneration efficiency,and overcoming genotype dependence are of great significance to the promotion of wheat genetic engineering research.WOXs are a group of plant-specific transcription factors,which play important regulatory roles in plant embryo development and function maintenance of meristem,and is closely related to plant regeneration.GRF are a type of transcription factors which take part in the regulation of plant growth and development,hormone signal transduction.In this study,genome-wide identification,phylogenetic evolution and expression analysis of wheat WOX family genes were carried out,and the genes with the highest correlation with regeneration function were screened out.By overexpression these genes in callus,genetic transformation efficiency of wheat was improved.The effects of key regeneration genes on the phenotype were analyzed,possible interaction genes were screened.The main results obtained in this study are as follows.1.The WOX genes in 6 Triticeae plants were identified.Phylogenetic,conserved domains and expression patterns were analyzed.43 WOX transcripts in Triticum aestivum,30 in Triticum turgidum,25 in Triticum dicoccoides,17 in Hordeum vulgare,13 in Aegilops tauschii,and 8 in Triticum urartu were identified.The phylogenetic analysis and classification of WOX proteins in 6 Triticeae plants was carried out.Conserved domain analysis of these WOX proteins was performed,the conserved domains such as HOX,WUS-box and EAR in the WOX protein sequence are predicted.The expression patterns of Ta WOX genes were analyzed,the genes closely related to differentiation and regeneration such as Ta WUS,Ta WOX8,Ta WOX9,Ta WOX10 and Ta WOX14 were screened out.2.Two key genes for wheat regeneration,TaCB1 and TaCB2,were cloned.Two coding gene sequences of 633 bp and 927 bp were cloned,tentatively named TaCB1 and TaCB2.The other copies of TaCB1 and TaCB2 in T.aestivum,the homologous of TaCB1 in Triticum monococcum and Aegilops speltoides were cloned and submitted to the Gen Bank.3.Overexpression of TaCB1 in the callus induced from immature embryos greatly improves the regeneration efficiency of wheat.Through overexpression of TaCB1,the transformation efficiency of wheat varieties BS366,Ningchun 4,Aikang58 were successfully increased from 0% to 83.5%,29.3%,21.8%;the transformation efficiency of Jimai 22,Sumai 3 and Zhoumai 18 were successfully increased from 5.8%,2.67%,11.8% to 55.4%,57.4% and 92.6%,respectively.The transformation efficiency of Fielder and CB037 increased from about 50% to 90-100%,which basically overcomes the genotype dependence in wheat transformation.Overexpression of TaCB1 did not affect root regeneration while improving the transformation efficiency,so there is no need to eliminate its expression after transformation.4.Identified proteins that may interact with TaCB1.Through RNA-seq,yeast library screening,protein immunoprecipitation and other methods,candidate interacting proteins and their coding genes were screened.The interaction proteins of TaCB1 such as Elongation factor 1?,Importin ?1b,Uridine Kinase-like protein,Ta GRF1,Ta GRF3 and Ta GRF4 has been verified through yeast two-hybrid and Luciferase complementation assay.5.Overexpression of TaCB1 can significantly promote plant regeneration through immature embryo callus culture.Immature embryo culture and main agronomic and physiological traits identification showed that the regeneration ability of immature embryos of homozygous TaCB1-OE lines was generally significantly higher than that of wild-type controls,while there was no negative effect to the main agronomic traits such as hight,tiller number.The main phenotypic changes,such as increased leaf width,leaf folds,split of flag leaf,can be used as a means to identify positive transformed seedlings.These findings lay a foundation for the application of TaCB1 overexpression to improve the transformation and regeneration efficiency.6.The candidate interaction protein GRF family and their co-acting factor GIF genes were identified.The wheat GRF and GIF genes were identified,mapped to the chromosome group,and analyzed for phylogenetic,conserved domains and expression patterns.29 Ta GRF genes and 8 Ta GIF genes were identified in the wheat genome.According to the phylogenetic analysis of Ta GRF protein,they are divided into three clades.There are QLQ and WRC conserved motifs in Ta GRF,and QLQ conserved motifs in Ta GIF protein.The expression patterns of Ta GRF and Ta GIF were analyzed by transcriptome data and q PCR,and the expression trends of Ta GRF family genes in different organs were detected.It was found that Ta GRF3,Ta GRF4,Ta GRF6 and Ta GRF10 may participate in the formation and rapid development of callus in wheat immature embryos.