Analysis And Application Of Genes Controlling Rhizome Development In Leymus Secalinus Based On Transcriptome Sequencing Technique

Leymus secalinus is a perennial rhizoatous plant of gramineous,which is a heterotopic tetraploid species(2n=4x=28).Tillering is a unique branching phenomenon of monocotyledonous plants,which has special significance in developmental biology.The rhizomatous pasture play an important role in grassland and community structure,which can clonally grow for asexual reproduction.Many excellent pasture possess developed underground rhizomes,and forming complex rhizome net by continuous tillering.The rhizome apical buds of Leymus secalinus has two kinds of growth direction,horizontal and vertical.In this study,RNA-seq technique was used to investigate the associated gene and pathways in different growth states.Combined with the morphological analysis and the analysis of key gene validation,to reveal the mechanism and intrinsic molecular mechanism of rhizome apical bud development.The main results are as follows:(1)The data of 49.7Gb were measured in RNA-seq of the horizontal and vertical rhizome buds.The clean Data volume of each sample were reached 7.84 Gb and the percentage of Q30 base was more than 94.76%.After assembling,we got 76098 Unigene,and there are 22881 Unigene which the length is more than 1 kb.The results of annotations for Unigene show that a total of 44769 Unigene annotations were obtained in comparison with NR,Swiss-Prot,KEGG,COG,KOG,GO and Pfam databases.In total,5397 SSR markers were obtained by the analysis of genetic structure for the Unigene library.The analysis of CDS prediction?SNP and gene expression were also performed.The annotations of KEGG suggest that vertical growth of rhizome buds in the growth direction grow from horizontal extension to reverse growth(vertical),although not yet out of the ground,but genes related photosynthetic functional have been activated early expression.(2)The results showed that there were 4906 differential genes at the vertical and horizontal rhizome gene expression in Leymus secalinus.Meanwhile,1970 genes were up-regulated in horizontal rhizomes and 2936 genes were up-regulated in vertical rhizomes.These differential genes were significantly different expression in photosynthesis;photosynthetic antenna protein;leucine metabolism;glutathione metabolism;biotin metabolism;glutathione biosynthesis process pathways.32 transcription factors and 132 kinases were found in the 4906 different genes of vertical and horizontal rhizome of Leymus secalinus by gene annotation.However,many transcription factors and kinases were related to the growth and development of plant rhizomes and stress resistance.The results suggest that the rhizome buds at the level of rhizome grass may first be aware of biological or non-biological threats during the extension and extension of the lines and initiate the corresponding stress metabolic processes.At the same time,this result also reflects the horizontal rhizome buds are more complex in stress feedback than in vertical rhizome buds.(3)The real-time fluorescence quantitative PCR was used to verify the results of RNA-seq,and 12 differentially expressed genes were selected randomly for qRT-PCR analysis.The qRT-PCR analysis results were highly consistent with the results of RNA-Seq,indicating that the sequencing data's accuracy is highly and the results of bioinformatics analysis is credible.(4)it's found: The epidermis cells are larger and the cell wall are thicker,the layers composed of thin-walled parenchyma cells ring are more,the rings of vascular bundle are more in horizontal rhizomes,comparing with the vertical rhizome by resin slicing the vertical rhizome tip and horizontal rhizome tip of Leymus secalinu.This may be related to the physiological functions that the horizontal rhizomes growth needs to go through the obstacle in sandy soil,therefore,the horizontal rhizome tip have coat to be protected.At present,little study of rhizome growth status of Leymus secalinu by bioinformatics techniques such as RNA-seq.It is of great theoretical significance to understand the growth and development systematically,metabolic pathway,physiological function and resistance mechanism of Leymus secalinus by the transcriptome studies.In this study,the transcriptome data of horizontal and vertical rhizome were measured by Illumina Hiseq2500,and the data of transcriptome have verified and analyzed.Meanwhile,it's also lay the foundation for exploring the rhizome metabolic pathway,related enzyme gene,growth and development related genes in rhizome,and stress resistance study etc.