| Cassava (ManihotesculentaCrantz), a typical cash crop in the tropics and subtropics, is not only one of the most important sources of energy for humans and animals, but also an important raw material for industrial production of starch and fuel ethanol. The major growing areas of cassava are located in the subtropical regions and northern margin of tropics in China, drought and low-temperature become the major abiotic stress to its growth and development as well as geographic distribution, so the research on drought and cold tolerance of cassava has an important biological and economic significance. The ability of plant drought and cold resistance is not because the role of a single gene or a few ones, but by the role of complex gene network. In this study, a digital gene expression profiling of cassava SC124subjected to drought and cold stress was conducted using high-throughput sequencing technology on the level of transcriptome. At the same time, the transcripts data was analyzed by system biology methods and the means of physiological, biochemical and molecular analysis. As a result, the complex cassava drought and cold resistance coordination mechanism was explored in depth. The main results are as follows:(1) The gene expression profiling of cassava SC124under the treatment of drought acclimation hurt and control, cold acclimation injury and control, was conducted in the study. In total, around3.7×106tags were obtained from each sample library. Among the gene expression profiling data, the copy number which is above100accounts for about1.25%and2%of the total tags, respectively; the copy number which is51to100accounts for about1.5%and2%of the total tags, respectively; the copy number which is21to50accounts for about3%and3.5%of the total tags, respectively; the copy number which is11to20accounts for about3.25%and4%of the total tags, respectively; the copy number which is6to10accounts for about5.25%and6%of the total tags, respectively; the copy number which is2to5accounts for about24.25%and24.25%of the total tags, respectively; the copy number which is below2accounts for about60.5%and54.2%of the total tags, respectively.(2) In this study,2235differentially expressed genes were identified from the gene expression profiling of SC124unfolded leaves subjected to drought acclimation hurt at the4th,6th and10th day;the up-regulated differentially expressed genes accounts for59.8%of the total differentially expressed genes, and the number of specific expressed gene is518,221,322corresponds to the4th,6th and10th day; the down-regulated differentially expressed genes accounts for40.2%of the total differentially expressed genes, and the number of specific expressed gene is262,113,183corresponds to the4th,6th and10th day.3266differentially expressed genes were identified from the gene expression profiling of SC124folded leaves subjected to cold acclimation injury at the6th,24th and48th hour; the up-regulated differentially expressed genes accounts for54.5%of the total differentially expressed genes, and the number of specific expressed gene is521,128,473corresponds to the6th,24th and48th hour; the down-regulated differentially expressed genes accounts for45.5%of the total differentially expressed genes, and the number of specific expressed gene is305,88,382corresponds to the6th,24th and48th hour.(3)GO functional analysis indicated that drought-stress and cold-stress related biological pathways, molecular function, cell composition, such as "response to stimulus","metabolic process","transcription regulator","antioxidant" and "organelle", have obvious response to the stress. The drought-stress related differentially expressed genes with the "cell killing","metallochaperone","protein tag" function are up-regulated completely."Virion" responses only in the up-regulated differentially expressed genes to chilling injury stress. The cold-stress related differentially expressed genes with the "cell killing","protein tag"function are down-regulated completely, unlike the result of drought stress.(4) Metabolic pathway analysis showed that drought stress and cold stress can significantly influence metabolic pathways including photosynthesis, respiration, secondary metabolites, citric acid cycle, cell wall metabolism, amino acid metabolism, lipid metabolism, starch metabolism and so on. In addition, drought stress and cold stress significantly affected regulatory pathways such as phytohormone metabolism, redox metabolism, transcription factors, protein metabolism, calcium regulation and kinase metabolism.(5) The plant important physiological and biochemical activities during drought and cold resistance process are closely related to stomatal movement. Stomatal opening and closing on the lower unfolded leaf surface of SC124(drought-resistant) and C4(not drought-resistant), also on the lower folded leaf surface of SC124(cold-resistant) and KU50(not cold-resistant), were observed by scanning electron microscopy. The results showed cassava stomatal movement was significantly affected by drought and cold stress. The drought-resistant and cold-resistant SC124closed the stomata quickly when plant felt the stress to reduce the loss of water effectively, gradually opened when the plant adapt to the environment in order that the plant can ensure the normal basal metabolic activities. The drought and cold acclimation plant can respond to stress and adapt to external environment more quickly.(6) The content of endogenous phytohormone (ABA, IAA, GA, ZR) in the unfolded leaves of SC124(drought-resistant) and C4(not drought-resistant), also in the folded leaves of SC124(cold-resistant) and KU50(not cold-resistant), both under acclimation and non-acclimation treatment, was determined by enzyme-linked immunosorbent assay (ELISA) based on the analysis of phytohormone metabolic pathway and the importance of hormone to regulate stomatal movement. The result showed the content of ABA in drought and cold resistant SC124unfolded leaves increased, while the content of IAA, GA, ZR decreased under drought and cold stress. Drought and cold acclimation enhanced SC124tolerance to stress, and increased the amplitude of accommodation.(7) Osmotic adjustment plays an important role in plant drought and cold resistance, the content of soluble sugar including glucose, fructose, sucrose and so on is an effective indicator of drought and cold resistance. The content of glucose, fructose and sucrose in the unfolded leaves of SC124(drought-resistant) and C4(not drought-resistant), also in the folded leaves of SC124(cold-resistant) and KU50(not cold-resistant), both under acclimation and non-acclimation treatment, was determined by HPLC-ELSD. The result showed that the content of glucose, fructose and sucrose (in the early stage) increased in drought and cold resistant SC124unfolded leaves under drought and cold stress, drought and cold acclimation can enhance the accumulation of glucose and fructose, the accumulation of soluble sugar in C4(not drought-resistant) was not obvious. Cold stress can also effectively improve the content of glucose, fructose and sucrose in cassava folded leaves, and cold acclimation can enhance the accumulation of soluble and cold tolerance.(8) Multiple of gene expression patterns related to cassava cell wall metabolism, protein metabolism, secondary metabolism, stress-responsive and important transcriptional factors were identified by real-time quantitative PCR based on the analysis of metabolic pathways and regulatory pathways under drought and cold stress, to further explain the molecular mechanism of cassava drought and cold resistance.(9) The2,000bp upstream sequences relative to the transcription starting site of all of the differentially expressed genes from each co-expression module under drought acclimation hurt and cold acclimation injury was analyzed to find out the common cis-acting elements. Finally, we obtained drought and cold resistance related trans-acting factors like ACIPVPAL2, EMBP1TAEM, SGBFGMGMAUX28, MYB1AT, AGCBOXNPGLB,CMSRE1IBSPOA, RBENTGA3, etc.Some transcription factors like ACIIPVPAL2. AGMOTIFNTMYB2〠MYCATERDlwere related to drought and cold resistance at the same time, which proved the cross talk between the drought-resistance and cold-resistance mechanism. |
