Cloning And Expression Analysis Of Key Genes Of Phosphorus Responses In Sugarcane

As one of the necessary fertilizers for the growth and development of sugarcane,phosphorus plays an important role in all stages of sugarcane growth and development.For example,phosphorus can not only promote the formation of sugarcane roots,stems and leaves,but also can effectively improve the sugarcane stem diameter,effective stem number,single stem weight,and thus contribute to the increase in sugarcane yield.Researchers have found that not only the available phosphorus content in most sugarcane areas is still at moderate ground level,but also the utilization rate of phosphorus in the sugarcane is relatively low,only 6.7%-15.3%.In recent years,people through the study of Arabidopsis thaliana and rice,it has been found that the use of molecular means to carry out innovation and screening of phosphorus efficient germplasm materials and identification of differences in the nutritional needs of different stages of the plant can greatly improve the status quo.In this study,we used the unigene database what was established by transcriptome sequencing to explore the genes involved in sugarcane phosphorus response.And the specific cloning primers were designed by using the sequence obtained from the transcriptome database,and the cloning and expression patterns of some key genes will be performed and analyzed.The aim of this study was to reveal the corresponding genes inthe regulation process of sugarcane in sugarcane,and lay the foundation for the next step in the identification and selection of phosphorus efficient sugarcane germplasm.The main results and conclusions are as follows:Had been established unigene database were used to explore the genes involved in sugarcane phosphorus response.From it,there are 130 pieces of unigene information were noted by us,and seven pieces of ScPHR1-3,twelve pieces of ScSPX1-6,four pieces of ScPAP2,thirteen pieces of ScPHTl,five pieces of ScPHO1;1-1;3 and so on.The genes of ScPHR1-3,ScSPX1-6 were cloned by using PCR technique.The cloning results showed that the cDNA sequence of ScPHR1-3 and ScSPX1-6 was 1539bp,1520bp,1351bp,1639bp,2050 bp,892bp,1893bp,889bp,1533bp in length and encode 442AA,417AA,393AA,310AA,278AA,259AA,336AA,262AA and 260AA,respectively.The prediction results of the primary structure of ScPHR1-3 and ScSPXl-6 proteins shows that all of them are hydrophilic and unstable protein.Isoelectric point prediction shows,in addition to ScSPX3 protein was alkaline,the rest are acidic.And the results of domain analysis show that ScPHRl-3 genes belong to the MYB-CC gene family and contain both MYB-DNA and Coil-colied(CC)domains.ScSPXl-6 genes belong to the SPX family and contain one SPX domain.The results of homologyand genetic evolution showed that sugarcane had a close genetic distance with sorghum,maize and rice.Protein secondary prediction resultsshowed that sugarcane ScPHR1-3 and ScSPX1-6 proteins do not contain any transmembrane structures and both them have a same function for Transport and binding.Application of the sugarcane tissue materials,the expression patterns of ScPHR1-3 and ScSPX1-6 were analyzed by the qRT-PCR technique.The results showed that the nine genes of sugarcane can express in different tissues of sugarcane.The expression of ScPHRl gene was the lowest in the stem,followed by root,leaf and bud;ScPHR2 was the highest in the root,and the expression of ScPHR3 was the highest in the stem,the lowest expression,followed by stems,buds,leaves.The analysis of the SPX gene family showed that the expression of ScSPX6 was the lowest in the shoot except for the lowest expression level of the bud.In addition to ScSPX1 in the leaves of the highest expression,the rest are the highest expression in the stem.The subcellular localization analysis of ScPHR1-3 and ScSPX1-6 genes showed that ScPHR1 and ScPHR2 were located in the nuclei of tobacco mesophyll cells,and ScPHR3 was located on the cell membrane and cytoplasm of tobacco mesophyll cells.ScSPXl and ScSPX2 were located in the cytoplasm and cytoplasm of tobacco mesophyll cells;ScSPX4 and ScSPX5 were located on the cell membrane and nucleus oftobacco mesophyll cells,and ScSPX5 was also expressed in the cytoplasm.Combined with structural and functional predictive analysis results found that ScPHRl and ScPHR2,ScSPXl and ScSPX2 genes may be involved in the transport and anchoring of substances in the nucleus.While other genes in this feature are presented as diversity,as they are expressed on the cell membrane,indicating that They may be involved in the transport and anchoring of the membrane inside and outside,and ScSPX3,ScSPX5,ScSPX6 are also found in the nucleus,indicating that they may also be involved in the transport and anchoring of the nucleus.In addition,ScPHR3,ScSPX4 and ScSPX5 are also expressed in the cytoplasm,so they may also be involved in the exchange of organelles.The results of prokaryotic expression analysis showed that the nine genes could express the target protein under 10h,18 ? and 0.5mM IPTG.The target protein size of ScPHRl-3 was about 53KDa,50.5KDa and 48KDa,and the target protein size of ScSPXl-6 was about 39.5KDa,37KDa,42KDa,33.5KDa and 36KDa,which are consistent with the predicted protein size.And the expression of proteins are further to lay a foundation for the gene functions discussed.