Function Of WOX And SPL Genes On Adventitious Root Formation In Walnut (juglans Regia L.)

Walnut is one of the non-wood products tree species and is difficult to root.This limits the industrialization process of excellent walnut varieties.By means of continuous grafting and buried treatment,the cuttings obtained high rooting rate.Previous RNA-seq studies have found that SPL and WOX were two key transcription factors in walnut adventitious root formation（ARF）.However,the function of these transcription factors has yet to be demonstrated.Due to the fact that some reseach have been successfully performed on several marker genes transformation in walnut somatic embryo（SE）,SE could be used as a good receptor for the study of gene function in woody plants.In this study,we carried out bioinformatics analysis on SPL and WOX transcription factor families,and analyzed their expression patterns in the process of ARF.Finally,some specific gene were selected for genetic transformation.The main research results were as follows:1.The elishment of somatic embryogenesis and plant regeneration systems of walnut.Two early-bearing walnut young fruits were sampled during 42-77 days after blooming.Somatic embryogenesis percentages were counted to screen the optimum devolepmental stage of the fruit.After subcultured several times,somatic embryos distortion percentage was calculated and then the optimum ABA concentration was tested.Somatic embryos were desiccated respectively for 0,24,48,72,96 and 120h with saturated NH₄NO₃,and the germination rate of somatic embryo can be obtained.The results showed that the highest somatic embryogenesis percentage of’Zao Lin Xiang’and’Zhong Lin 6’reached 88.32%and 86.7%when the young fruits were sampled at 49d and 56d after flowering,respectively.Moderate concentration of ABA suplement not only reduced the rate of somatic embryos aberration,resulted from multiple subcultivation,but also increased the rate of somatic embryogenesis,and the optimum ABA concentration was 1mg·L^-1.As dehydration time was prolongated,water loss was gradually increased,while germination rate increased first and decreased later on.After dehydrated 72h,the germination rate of‘Zao Lin Xiang’and‘Zhong Lin 6’were up to the highest（56.67%and 53.33%）,while the water loss was 38.73%and 40.56%,respectively.2.The establishment of genetic transformation system of walnut.Using the somatic embryo of‘Zhonglin 6’as receptor explant,the effects of somatic embryo pretreatment,agrobacterium concentration and infection time on the genetic transformation of walnut were explored.The orthogonal test showed that the effect of the somatic embryo pretreatment on walnut embryo infection guy reached significant level,but the influence of Agrobacterium concentration and infection time were not significant.After 1 month of pretreatment（changed DKW medium/7d）and 0.6 agrobacterium concentration isinfect for 15min,the results of infection was the best.The urvival rate of infection reached 77.78%.3.Genome-wide identification of WOX gene family and expression analysis during rejuvenational rhizogenesis in walnut.In this study,12 genes were identified and clustered into three groups based on phylogenetic,special domains and conserved motif.The gene structure and conserved motif were relatively conserved,while the 12 sequences of the JrWOXs domain were diversified.Gene expression in root,stem,leaf,female flower,immature fruit and zygotic embryo revealed that the expression level of JrWOX4a,JrWOX4b,JrWOX5,JrWOX11 and JrWOX13 in root were significantly higher than that of other JrWOXs,while only the expression of JrWOX11 was exclusive in root organ.Additionally,rejuvenation treatment significantly induced almost all JrWOX genes,except JrWOX4a,JrWOX4b,and JrWOX13（Rc 0 vs Mc 0）.During the ARF process,the transcripts of JrWOX11 and JrWOX5 were consecutively increased on a significance level,on the contrary,the transcription levels of the other JrWOXs decreased or changed no significantly.The phenotype and histology observation indicate that rejuvenation treatment made the base of the stem expand,and reduced thickness and density of sclerenchyma between the cortex and phloem.That might provide the conditions for new meristem niches formation.4.The investigation of the effect of 3 walnut JrWOX genes on ARF.Overexpression of JrWOX4,JrWOX5 and JrWOX11 genes in poplar’84K’could promoted the formation of ARs.JrWOX11 and JrWOX5 advanced the formation of root primordium by 2d and 1 d,respectively,and that root number and root lengt were advanted.However,JrWOX4 shortend and thickened AR,and increased the number of ARs.JrWOX11 could also improve the resilience to drought and salt stress.Furthermore,JrWOX11 gene was overexpressed and gene edited in walnut.The phenotype observation showed that plant height,diameter and pith width were overexpressed plants>control plants>gene edited plants,cambium width was overexpressed plants>control plants>gene edited plants,and xylem width was overexpressed plants<gene edited plants<control plant.Moreover,JrWOX11 could also promote the AR formation of walnut.5.The identification of JrSPL family gene and expression analysis in the process of ARF in walnut.Using bioinformatics methods,28 SPL family genes were identified from walnut genome,and they were not evenly distributed on the 14 chromosomes.All JrSPLs were clusterred into the subgroupⅠ-Ⅹ.The gene structures of each subgroup were quite different,and members of similar phylogenetic line have similar intron/exon structure,gene length and conserved motif,which may have similar functions.All 28 SPL members contained conservative SBP domains,including 2 Zn-fingers and a nuclear localization signal sequence（NLS）.The expression of most JrSPL genes were inhibited in the rejuvenation cuttage.The expression levels of JrSPL1.1 and JrSPL7.2 genes changed significantly in the process of ARF.6.The analysis of biological functions for two walnut SPL genes.Subcellular localization indicated that JrSPL1.1 was localized in the nucleus,while JrSPL7.2 was expressed in the nucleus and cytoplasm.Overexpression of JrSPL7.2 in tobacco inhibited the formation of adventitious and lateral roots,but promoted the elongation of principal root and flowering process.Additionally,overexpression of JrSPL7.2 in walnut,plant growth was inhibited.However,the overexpression of JrSPL1.1 in walnut could significantly improve plant height and leaf size.The preliminary rooting experiment indicated that JrSPL1.1 promoted the root length of walnut,but it still needed further verification.