Transcriptome And Proteome Of Cassava(Manihot Esculenta Crantz)Leaves Under Cold Stress

Cassava(Manihot esculenta Crantz)belongs to the Euphorbiaceae family and has about 100 species.Cassava is grown in over 90 countries and is the third most important source of calories in the tropics,after rice and maize,because of its high productivity,even in extreme conditions.Its large,starchy,roots and edible leaves provide food for 800 M people globally,many of whom subsist on it,in part because it is drought tolerant and requires little in the way of inputs.It is described as "classic food security crop" because it offers the advantage of a harvest even in situations of erratic rainfall and infertile soils.The high starch content(20-40%)makes cassava a desirable energy source of commercial animal feed,fiber for paper and textile manufacturers,starch for food and pharmaceutical industries,and bio-fuel applications.Cassava now is a very important raw material for starch and alcohol industry in China but production is very limited due mainly to the small cultivated areas and lack of cold resistant varieties.Cassava originated in tropical America and was first introduced into China in the 19th century.Cassava is sensitive to low temperature and easy to get chilling injury under the stress of low temperature.Improving cassava's tolerance to cold is important to help increasing yields in the south subtropical and tropical regions in China where cassava as an essential starch crop for its uses in feed,fuel,and starch industry.Cassava's natural stress tolerance can be substantially improved by breeding,especially by marker-assisted selection of key physiological traits associated with cold tolerance.But the molecular basis for this tolerance is poorly understood.Therefore,the research on the mechanism of cold tolerance for cassava is very important.In this study,we used cassava accessions SC8 and MS5 as materials to study the gene regulation under cold stress and the molecular mechanisms of cold tolerance by using the methods of physiological and biochemical indexes,transcriptome and proteomics.The main research results of this study were shown as follows:1.Dynamic analysis of physiological indexes shows that:(1).Cassava accessions SC8 and MS5 were taken as test materials.After with five different time(0d,7d,10d,12d,15 d)of treatments at 5? low temperature stress,the relative conductivity,soluble protein content,soluble sugar content,proline content,MDA content,enzyme activity of SOD and POD were measured.The experimental result reveals that as the extension of stress time,cassava seedling leaves' relative conductivity rate,soluble sugar content and MDA content present a constantly rising trend while soluble protein content,proline content,enzyme activity of SOD and POD firstly increased and then decreased finally.(2).under cold stress,cassava relative conductivity rate was very significantly positively correlated to MDA and soluble sugar content,while very significantly negatively correlated to enzyme activity of SOD and soluble protein content.Soluble protein content was significantly negatively correlated to MDA content and very significantly positively correlated to proline content and enzyme activity of SOD and POD,and SOD activity was very significantly negatively correlated to soluble sugar content and very significantly positively correlated to POD activity.(3).Principal component analysis indicates that the relative conductivity rate,SOD activity and soluble sugar content account for the largest amount of information in the three principal components,closely related to cassava cold tolerance,which could be used as physiological in dices to identify cold resistance of cassava accessions or varieties.MS5 had much stronger cold-resistance than SC8 by using the Comprehensive analysis of biological data.2.Analysis of 7-transcriptome sequencing data reveals:(1).According to the cassava genome AM560 that a total of 47,443 annotation of transcripts,Based on the shared locus of variable shear we cluster exons of cassava transcript and clustering of the localization overlap information according to each of single exon cassava transcript on the genome,a total of 43,277 genes were obtained.(2).Clustering analysis of gene expression patterns reveals that a dynamic change of cassava transcriptome performance in the leaves through regulating the expression of genes in response to low temperature stress.Compares to the normal temperature,the number of gene expression in the two Cassava accessions are increased significantly after low temperature stress,the increased expression of genes with high expression genes and low expressed genes.Cassava SC8 leaves open more low expressed genes at low temperature treatment for 7 days.While there are more the low expression of genes were closed and more high expression genes were opened after low temperature treatment.Cassava MS 5 leaves open more low expressed genes at low temperature treatment for 7 days,while there are more the high expression of genes were closed and more low expression genes were opened after low temperature treatment recovering to the normal temperature conditions.(3).Analysis of genes differentially expressed was found that the change of temperature affecting the regulation of gene expression.There are 7009 genes participated in low temperature stress regulation from room temperature to low temperature stress and 8273 genes participated in low temperature stress regulation from low temperature stress to normal temperature in cassava SC8 leaves,while cassava MS5 leaves are 6751/6343 respectively.There are 4060 genes generally involved in the regulation of different temperature in the two cassava accessions leaves,these genes may be a basic regulation of transcriptome base on the low temperature stress.Compared with the two cassava accessions transcriptome,MS5 has less number of regulation genes than SC8 involved in low temperature stress,the result shows that the number of low temperature stress regulation genes eventually embody in the cold tolerance differences of individuals.(4).Clustering of gene difference expression patterns found the regulation of cassava transcriptome mainly related to the experimental conditions(low temperature stress).The same test conditions materials gathered in the first category,second is the material sources of differences.(5).By using of GO(Gene Ontology)functional enrichment analysis of gene difference expression,obtained three types of biological process,cellular component and molecular function and 16 branch.The same test under the condition of gene regulation in the same direction,also performance on the regulation of gene function.Differentially expressed genes under different experimental conditions were enriched in the nine functions:membrane-enclosed lumen,metallochaperone activity,anatomical structure formation,macromolecular complex,cell,cell part,organelle,organelle part and metabolic process.Regulation of the gene in cassava leaves involved in low temperature stress was enriched in the functions of nutrinet reservoir avtivity.Regulation of gene in cassava leaves and the various experimenal conditions involved in low temperature stress were enriched in the function of growth.These results indicate that cassava leaves in response to low temperature stress mainly through regulating the basic function of cells.(6).A total of 13 metabolic pathways enrichment of DEGs(Differentially Expressed Genes)was obtained by using KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis.DEGs were enriched in the 8 metabolic pathways under different experimental conditions;they were RNA transport,Ribosome biological origin,Carbon fixation in photosynthetic organisms,Spliceosome,Photosynthesis antenna proteins,Ribosome,Photosynthesis,Carbon metabolism respectively.DEGs almost were enriched in Glyoxylate dicarboxylic acid metabolism and Legionellosis pathway under different experimental conditions.Estrogen signaling pathway and Methane metabolism were found after low temperature stress to normal temperature,while DEGs occurred in low temperature stress and the regulation of genes induced under different experimental conditions was enriched in Proteasom.(7).Based on the transcriptome data,an analysis of Reactive Oxygen Species and Antioxidants in Cassava leaves at low temperature stress was found that APX activity in MS5 leaves has not been suppressed,while there has been a marked inhibition in SC8 leaves APX activity in cassava played a very important role in response to low temperature stress,the strength of the active direct embodiment on the cold tolerance of differences of individuals.3.Proteomic analysis of low-temperature-induced protein result display:(1).After with 7d treatment at 5? low temperature stress,42 differences protein expression level in more than 2 times were found in the 45d old cassava tissue culture seedlings of SC8 leaves,among which 11 up-regulated and 31 down-regulated protein spot.Mass spectrometry analysis helped to identify 37 spots representing 11 up-regulated and 31 down-regulated proteins.These differential expressed proteins involved in the functional group on Photosynthesis related proteins,Carbohydrate and energy metabolism associated proteins,Protein biosynthesis,Defense,Detoxifying and antioxidant,Signal transduction mechanisms,Chaperones,DNA binding protein,Inorganic ion transport and metabolism and Amino acid metabolism.In addition,it also includes 2 Function unknown proteins.(2).With the same treatment,59 differences protein expression level in more than 2 times were found in MS5 leaves,among which 10 up-regulated and 49 down-regulated.Mass spectrometry analysis assisted to identify 52 protein spots representing 10 up-regulated and 42 down-rcgalated.These differential expressed proteins involved in the f unctional group on Photosynthesis related proteins,Carbohydrate and energy metabolism associated proteins,Protein biosynthesis,Defense,Detoxifying and antioxidant,Chaperones,DNA binding protein,Inorganic ion transport and metabolism and Amino acid metabolism.In addition,5 Function unknown proteins was also included.(3).Compare the low temperature induced proteins to cassava accessions SC8 and MS5,34 differential expressed proteins has co-expressed in both of them,of which 9 proteins were up-regulated and 29 were down-regulated,and the same protein points has the same expression characteristics in the two cassava accessions,These co-expressed proteins involved in the functional group on photosynthesis,carbohydrate and energy metabolism,Protein biosynthesis,amino acid metabolism,chaperones,defense,inorganic ion transport and DNA binding protein and 2 Function unknown proteins.There were 3 differential expressed proteins expressed only in SC8 and involved in photosynthesis,carbohydrate and energy metabolism and signal transduction mechanisms,and there were 18 differential expressed proteins expressed only in MS5 and involved in photosynthesis,carbohydrate and energy metabolism,protein biosynthesis,defense,detoxifying and antioxidant,signal transduction mechanisms and chaperones,3 function unknown proteins was also included.In summary,this study,based on physiology,starts from transcriptom and proteomics to analyze the mechanism of cassava cold tolerance,a large number of genes and proteins involved in cold response were found.Especially the APX2 gene/protein plays a very important role in cassava to adapt to the low temperature stress.These results will be useful for identifying cold respond genes and proteins associated with cold tolerance.And these resources should also allow us to accelerate understanding of basic biology of cassava resistance to low temperature.