Establishment Of Cell Cycle Synchronization Method And Mining Of Related Genes In Gossypium Arboreum

In the process of ontogeny of higher plants,the formation of organs is a post-embryonic development process,and the cells in the meristem divide to form new organs.Cell cycle flexibly controls the occurrence and morphological changes of organs,which is very important to ensure the normal growth,organ development and reproduction of crops.As the most important economic crop in the world,the yield and quality of cotton are of great significance in production.The lateral root growth and fiber development of cotton are closely related to the cell cycle,so it is necessary to explore the key genes related to the cell cycle in cotton.In this study,an optimal method for cell cycle synchronization of cotton root tip was established by using Gossypium arboretum variety SXY1,and the key genes related to cell cycle were mined by transcriptome sequencing in the critical period of mitosis.A preliminary study was conducted on the regulation mechanism of cotton cell cycle.The main conclusions are as follows:(1)The optimal system of cell cycle synchronization of root tip cells of G.arboretum based on flow cytometry was established.According to the factors such as the concentration and time of treatment of DNA synthesis inhibitor HU and mitotic spindle inhibitor Oryzalin,and the recovery time,the optimal scheme of cell synchronization in the metaphase of root tip mitosis of G.arboretum was determined: the best buffer was treated with LB01,4.5 m M HU for 18 hours,then restored growth with Hoagland solution for 2 hours,and then treated with 10 μM Oryzalin for 2 hours.At this time,the mitotic metaphase cell index was as high as 47.5%.(2)Transcriptome analysis of critical period of root tip mitosis in G.arboretum based on cell cycle synchronization.Through transcriptome sequencing of root tips enriched to G1/S stage(after HU treatment)and G2/M stage(after Oryzalin treatment),4684,4869 and 1919 differentially expressed genes were identified in three different groups of comparative combinations.Through GO enrichment,the results showed that these differential genes were mainly enriched in eight components: cell division,cytokinin biosynthesis,DNA replication,double strand break repair,lateral root morphogenesis,microtubule,mitotic cell cycle and root development regulation.The results of KEGG enrichment showed that these differential genes were mainly enriched in two signal transduction pathways of plant hormone and MAPK,and the key candidate genes related to cell cycle could be mined through the above enrichment information.(3)Mining and screening of genes related to root tip and cell cycle in cotton.According to the GO enrichment results,it was found that the tubulin gene family accounted for the largest proportion of differential genes.In addition,we also screened the following key genes: ERF1,SYP111,IPT5,LOG1,RNR1,TSO2,PARP2,XRCC2,AIR1,HSL3,NACK1,TPX2,CYCB1;2,CYCD2;1,AGP30 and ZAT12.These genes can be used for further study.According to the results of KEGG enrichment,it was found that23 genes in the two signaling pathways of MAPK and hormone were enriched simultaneously by two pathways,including PYR/PYL,PP2 C,Sn RK2,MYC2,PR1,EBF1/2,ETR/ERS and MPK3/6.More importantly,MPK3/6 is the key connecting gene between each hormone pathway and MAPK.In addition,auxin-related genes AUX/IAA and SAUR account for the largest number,indicating that auxin pathway plays an important regulatory role in the process of cell cycle,and AUX/IAA and SAUR gene families play an important role in the process of cell cycle.In this study,the cell cycle synchronization system of G.arboretum root tip was established based on flow cytometry,and the gene expression and transcriptional dynamics of critical period of mitosis in cotton root tip were studied by RNA-seq technique,and the key genes related to cotton cell cycle were screened.This study revealed the differences of gene expression in different cell cycle of cotton for the first time.These results not only laid the foundation for further understanding the molecular mechanism and interaction network of cotton cell cycle regulation,but also provided data resources for breeding new high quality cotton varieties.