Molecular Basis Study Of Somatic Cell Fusion In Sugarcane

Sugarcane is the most important sugar crop in China.It is a kind of warm crop.Sugarcane ROC22 has good agronomic traits such as high yield and high sugar,but its cold tolerance is poor.GT28 is a cold-tolerant variety.By somatic cell fusion with ROC22 and GT28,hybrid progeny with stronger cold tolerance and excellent agronomic traits can be obtained.However,there are still great difficulties in regenerating plants of sugarcane hybrid cells.As a high-quality material for gene function analysis,protoplasts are expected to provide a new way to study the molecular mechanism of somatic cell fusion and solve the problem of plant regeneration in hybrid cells.In this study,ROC22 and GT28 sugarcane varieties were used as materials to explore the reasons for difficult extraction of sugarcane protoplast RNA,and the extraction conditions and methods of sugarcane protoplast RNA were optimized.ROC22,GT28 sugarcane young leaves and protoplasts were used as materials.The differentially expressed genes before and after enzymatic hydrolysis were screened by transcriptome sequencing technology,and the expression of key genes related to regeneration of sugarcane hybrid cells at different hormone concentrations was analyzed.Research indicates:(1)The two major factors affecting the extraction of sugarcane protoplast RNA are protoplast RNase activity and osmotic pressure.Protoplast RNase activity is significantly negatively correlated with protoplast activity,so obtaining high viability of protoplasts is an important guarantee for successful extraction of sugarcane protoplast RNA.The osmotic pressure directly affects the protoplast RNase activity,and can also affect the protoplast RNase activity indirectly by affecting the protoplast activity.The osmotic pressure is significantly positively correlated with the protoplast RNase activity.To extract high-quality total RNA from sugarcane protoplasts,it is necessary to ensure the vitality of protoplasts is at least 70%.The total RNA of sugarcane protoplasts extracted by modified Trizol method has good purity and highest yield.(2)In this study,transcriptome sequencing of sugarcane young leaves and protoplasts after enzymatic hydrolysis was carried out,and the differences in gene expression before and after enzymatic hydrolysis in sugarcane somatic cell fusion were analyzed.A total of 117,411 Unigene,43460 differentially expressed genes were obtained,of which 21,123 were up-regulated and 22,337 were down-regulated.Among them,GO term closely related to developmental process?growth?cell proliferation?transcription regulator activity?signal transducer activity?antioxidant activity?oxidative stress?kinase activity?cell cycle?cell differentiation?plant hormone signal transduction and so on.(3)The sugarcane hybrid cells were cultured at different hormone concentrations,and the growth of sugarcane hybrid cells under optimal and worst hormone combinations and differential expression of key genes were analyzed.The results showed that the regeneration rate of sugarcane hybrid cells in the optimal hormone combination culture was significantly higher than that of the worst hormone combination.In the early stage of culture,the optimal hormone combination was more favorable than the worst hormone combination for the proliferation of sugarcane hybrid cells.Splitting,and the plateau rate was higher under optimal hormone combination culture conditions when cultured for 30 days;cell wall synthesis(GAUT,CESA),cell cycle regulation(Cyclin B,Cyclin A),cell proliferation(PSK),growth Eight key genes of cytokinin signaling pathway(Aux/IAA,Cyclin D3,CDC2)were analyzed,and the differential expression of eight key genes in the optimal and worst hormone combination of sugarcane hybrid cells was analyzed.It is indicated that hormones play a crucial role in the regeneration of hybrid cells by regulating key genes involved in hybrid cell regeneration.The results of this study will provide important technical support and scientific basis for studying the molecular mechanism of somatic cell fusion in the future and solving the difficulties of hybrid cell regeneration in somatic cell fusion.