Analysis And Mutant Creation Of SvSHR Gene Family In Setaria Viridis

Vascular tissue is known to be distributed among the roots,stems and leaves of plants,which not only plays a role in transporting materials,but also plays a role in supporting the plant body.The development or senescence of vascular tissue will directly affect the morphogenesis and yield of plants.Therefore,the exploration of genes related to vascular development of C₄ crops is a prerequisite for the creation of crop varieties with higher yield.GRAS family of transcription factors has been reported to be involved in regulating plant growth and development,morphogenesis and stress response.Its subfamily members have been reported in the development of root cortex of Arabidopsis thaliana and maize and the formation of root nodules in legumes.However,the function of SHR in the development of vascular organs of C₄ crops remains to be further explored.The creation of shr mutants in the C₄ model plant Setaria viridis is of great reference significance for exploring the possible biological functions of SHR in C₄ crop vascular development.In this study,according to the published ZmSHR1 and ZmSHR2 genes in maize,seven homologous genes in Setaria viridis were found,and the evolution,phylogenetic relationship and tissue-specific expression analysis were performed.CRISPR/Cas9 knockout vector and pGPro8-GUS vector were constructed in Setaria viridis,and positive plants were obtained by genetic transformation.This study provides an important reference and material basis for studying the role of SHR in vascular development in C₄ crops.The main results are as follows:（1）Based on the published ZmSHR1 and ZmSHR2 genes in maize,we searched for homologous genes in Arabidopsis,rice,maize,foxtail millet,soybean and Setaria,and constructed phylogenetic trees.The results showed that the phylogenetic tree could be divided into four groups.Soybean SHRs genes belonged to group IV,Setaria viridis SHRs genes belonged to groups Ⅰ,Ⅱ and Ⅳ,and Arabidopsis,rice,maize and foxtail millet SHRs genes were distributed in groups Ⅰ,Ⅱ,Ⅲ and Ⅳ,indicating that SHRs genes were relatively conserved during the evolution and differentiation of C₃ and C₄ crops.In group Ⅳ,rice,maize,millet and setaria were clustered in the same clade,while soybean and Arabidopsis were clustered in another clade.It was suggested that there were some differences in SHRs genes between monocot and dicot crops after differentiation.Seven homologous genes of Setaria viridis were distributed in groups Ⅰ,Ⅱ and Ⅳ,among which SvSHR2 and SvSHR7 in group Ⅳ had the highest homology with ZmSHR1 and ZmSHR2 in maize.（2）Tissue specific expression of SHR homologous proteins in Arabidopsis thaliana,rice and maize was analyzed using data provided by eFP and MazieGDB,and it was found that the expression sites and expression trends of SHR were similar in these species.Single cell data analysis showed that SHR cluster during vascular development in maize.Through the protein similarity analysis of corn and Setaria viridis,it was determined that the feasibility of creating shr mutant in Setaria viridis and functional verification was high.All seven SvSHR genes were associated with leaf development,but only SvSHR2 and SvSHR7 expression decreased gradually with leaf maturity,which may be related to early leaf morphogenesis.GO enrichment of these seven homologous SvSHR proteins was predicted,and it was found that all seven SvSHR were enriched in the biological process of vascular development.（3）Multiple SvSHR gene editing vectors were constructed using CRISPR/Cas9 technology and transformed into Setaria viridis by Agrobacterium tumeticum AGL1 genetic transformation method.Through tissue culture,SvSHR1,SvSHR2,SvSHR4,SvSHR5,SvSHR7 and SvSHR8 positive plants were obtained in T0 generation.Identification of the T1 generation showed that only shr7 had significant fragment deletion.Statistical analysis of the representative type of the obtained shr mutant T2 showed that compared with the wild type,shr2 had the phenotype of short plants and thick unfixed roots.shr4 showed earlier flowering and fruiting.shr7 had the phenotype of shorter and more lateral roots.The double process shr8 of SvSHR2 and SvSHR7 showed the phenotype of short stature and short roots.There are phenotypic inconsistencies among shr mutants,and further analysis is needed to clarify the reasons for these phenotypic differences.（4）2071bp upstream of ATG was intercepted and the pGPro8-SvSHR7pro::GUS vector was constructed.Transient transformation of tobacco confirmed that the length promoter was active and expressed in both vein and mesophyll.Positive plants were obtained from transformation of Setaria viridis,which provided experimental materials for further determination of the expression site of SHR in Setaria viridis leaves.