Genome-wide Identification Of LBD Genes In Foxtail Millet (Setaria Italica) And Functional Characterization Of SiLBD21

Setaria italica,commonly known as “ millet ”,is called “ su ” in the five grains of China.It have a long history of cultivation in China and have played an important function in the origin and development of agriculture,comparable to that of rice.Rencently,with the completion of the sequencing of the foxtail millet genome and the establishment of genetic transformation systems,foxtial millet are also considered to be an ideal C4 model crop.Lateral organ boundary domain(LBD)transcription factors play an important role in plant growth and development,yet no cloning of LBD genes has been reported for foxtail millet.In this study,we identified members of the SiLBD gene family at the genome-wide level,combined multiple bioinformatics tools to analyse the evolution,localisation,duplication and predicted physicochemical properties of SiLBD genes,and quantified their expression patterns,cloned the gene SiLBD21 associated with lateral root development and transformed it into the model plants Arabidopsis and rice to verify its function.It is the first time to identify and clone members of the LBD gene family in foxtail millet and to verify their functions,which has laid the foundation for enriching the molecular genetics of foxtail millet.The main results are as follows:(1)By screening the foxtail millet whole genome database,we identified 33 members of the SiLBD gene family,which are unevenly distributed on the nine chromosomes of foxtail millet,and we named them SiLBD1-SiLBD33 according to their order of appearance on the chromosomes.Physico-chemical property analysis and subcellular localization prediction of the 33 members showed that the SiLBD genes and protein sequences vary in length,with large differences in molecular weight and isoelectric point,and the majority of the proteins are unstable proteins,all with subcellular localization in the nucleus.The phylogenetic tree was constructed by combining the reported LBD members of Arabidopsis and rice.The SiLBD genes was divided into two major classes,Class I and Class II,with Class I having five clades,Class Ia,Class Ib,Class Ic,Class Id,and Class Id,and Class II having two clades,Class IIa and Class IIb.In addition,the 12 SiLBD genes constitute six segemental duplication events,and no tandem replication events occurred during the expansion of the SiLBD family.The SiLBD genes of the same clade share similar gene structure and protein motif composition,and have a relatively simple gene composition with no more than three exons.The prediction results of cis-acting elements showed that a large number of cis-acting elements appeared in the promoter region of millet LBD gene,with a total of 47 species and 800 sites,more specifically divided into three categories and 12 subtypes.(2)The results of expression pattern analysis suggest that LBD gene family members play an important role in millet growth and development,stress and hormone response.The results of qRT-PCR analysis of 28 SiLBD genes in different tissues at the filling stage showed that millet LBD genes have the characteristics of tissue expression specificity,such as five genes expressed specifically in the root,two genes expressed specifically in the stems,one gene expressed specifically in the leaf,and four genes expressed specifically in the seed.The 2-week-old seedlings were treated with simulated abiotic stress,and their expression patterns were quantitatively analyzed.The results showed that the members of SiLBD gene family had the expression characteristics of responding to abiotic stress,such as the up-regulation of SiLBD4 was extremely significant under heat stress,and the up-regulation of SiLBD22 was extremely significant under exogenous ABA treatment.(3)In order to verify the function of the root-specific expression gene SiLBD21,a 35 S over-expression vector was constructed and transformed into Arabidopsis(Col-0)and rice(Nipponbare,NIP).Positive lines were screened by PCR and those with high expression by semi-quantitative PCR,and phenotypic observations were made on Arabidopsis seedlings and plants at T3 generation;and on rice seedlings and plants at T2 generation.The results showed that overexpression of SiLBD21 reduced the primary root length and increased the number of lateral roots in seedlings;it reduced the length and width of leaves and plant height at maturity.In summary,this research mainly used bioinformatics,molecular biology and plant physiology to identify the members of the LBD gene in foxtail millet,explore their physicochemical properties,evolutionary relationships and cis-element predictions,analyse the expression patterns of the SiLBD genes and the preliminary functional validation of SiLBD21,and provide a certain theoretical and experimental basis for subsequent studies.