Quantitative RNA-seq Analysis Of The Sugarcane Response To PEG And The Identification Of Differentially Expressed Genes

Sugarcane(Saccharum officinarum L.) is one of the more important sugar and energy crops of the world. Cane sugar accounts for more than70%of the sugar production in the world and90%of that in China. Moreover, sugarcane fuel ethanol accounted for about40%of total biomass fuel ethanol production in the world. Drought is one of the most important yield-limiting factors of sugarcane plants. In China, due to85%of the sugarcane planting in saline or sloping dryland, the problem of insufficient water supply became more prominent.Mining for drought tolerance gene sources is an important work for sugarcane resistance breeding. Identification of the genes responsible for drought tolerance by monitoring the changes in gene expression at the transcriptional level is an effective method to target drought-tolerance genes. Erianthus arundinaceus is a closely related genus of S. officinarum L. With its outstanding stress-tolerance and high rooting ability, E. arundinaceus has proved to be one of the every important genetic resources for improvement of stress-tolerance in sugarcane. Although conventional hybridization is an effective way, and the BC3or BC4fertile hybrids from intergeneric crosses between S. officinarum L. and E. arundinaceus have been generated over the past ten years of work. Still, there are several difficulties in the process of making use of the outstanding stress-tolerance gene resources of E. arundinaceus and "noblelization" backcross, such as pollen sterility, flowering asynchronism, and target trait polymerisation problems, etc. Thus, it usually takes more than ten years for a cycle of sugarcane breeding to develop a good variety. In this study, an intergeneric BC2hybrid (S. complex) with good comprehensive characters named YCE05-179from S. officinarum X E. arundinaceus, was taken as the experimental material in imitation of drought stress. Analysis of molecular mechanism of sugarcane in response to drought stress and mining important functional protein genes and transcriptional regulatory elements are our main purpose. A high-throughput technology of RNA-Seq based on Illumina/Solexa platforms (Illumina GA/HiSeq) was used to produce a substantial expressed sequence tags dataset from the roots of YCE05-179under the treatment of PEG (25%PEG8000,24h). The main results and conclusions are as follows:1. RNA-seq based on the Illumina GA/HiSeq, was used to generate an extensive map of YCE05-179(S. complex) roots transcriptome under the treatment of PEG. In total,514,274,992basepairs raw sequence data corresponding10,495,408clean reads and556,342,815basepairs raw sequence data corresponding11,353,935clean reads were generated from the treatment and the control samples, respectively. The quality of these sequence reads was assessed by multiple analyses, and the information indicated that the high-quality RNA sequencing data in this study could fully cover the expressed genes in cells from each sample.2. Clean reads were aligned to sorghum genome and unigenes. In total,2.18million reads were aligned to the genome and unigenes, and23,550genes were identified. A total of12,035genes,6,480up-regulated genes and5,555down-regulated genes, were found to be differentially expressed in roots. Among them,597genes (329up-regulated genes,268down-regulated genes) were defined as highly significant differential expression filtered by conditions on FDR≤0.001and|log2Ratio|≥1. In this study, three types of gene including dirigent-like protein gene, metallothionein gene and R2R3-Myb transcription factor gene, which were clustered based on gene ontology (GO) terms, possess functional signatures identify as secondary metabolism (lignin synthesis), heavy metal binding (oxygen and reactive oxygen species metabolic process) and regulation of transcription, respectively, and all these genes were picked out for further functional verification.3. Dirigent and dirigent-like family proteins contain a number of proteins involved in lignification or in the response to pathogen infection and abiotic stress in plants. In this study, a full-length cDNA sequence of a dirigent-like gene designated ScDir (GenBank Accession Number JQ622282) was obtained from sugarcane based on RNA-Seq and bioinformatics analysis using the data of sugarcane stem full-length cDNA library. The ScDir gene was819bp long, including a564bp ORF encoding187amino acid residues. The protein N-terminus contained signal peptides at amino acid residues of1to25and trans-membrane regions at7to26aa. A His-tagged ScDir protein with an estimated molecular mass of27.4kDa was expressed in Escherichia coli system. The expressed ScDir protein had increased the host cell’s tolerance to PEG and NaCl. When an endogenous GAPDH gene was used as the internal control, results from Real-time qPCR demonstrate that the ScDir mRNA amount in sugarcane stalks was significantly higher than that in the roots, leaves and buds, as18.64±0.48-,25,635.16±2,966.03-,721.50±8.17-fold, respectively. The ScDir transcript levels in sugarcane seedling increased under H2O2, PEG or NaCl stress. The expression level of ScDir was significantly upregulated under PEG stress, and the highest level (>35-fold) was observed at12h after stress. Thus, both the ScDir hosted cell performance and the enhanced expressions in sugarcane imply that the ScDir gene is involved in the response to abiotic stresses of drought, salts, and oxidation. The transcription of the ScDir gene is highly stalk-specific, as revealed by Real-time qPCR.4. Metallothioneins (MTs) are cysteine-rich, low-molecular-weight, metal-binding proteins. Due to their ability to reversibly bind both toxic and essential metal ions, previous studies indicated that plant MTs play important roles in detoxification, metal ion homeostasis and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3(GenBank Ace:JQ627644), was obtained from sugarcane based on RNA-Seq and bioinformatics analysis using the data of sugarcane stem full-length cDNA library. ScMT2-1-3gene was700bp long, including a240bp ORF encoding79amino acid residues. A His-tagged ScMT2-1-3protein with an estimated molecular mass of19.01kDa was successfully expressed in Escherichia coli system and was identified at the same time by MALDI-TOF-TOF MS. The expressed ScMT2-1-3protein had increased the host cell’s tolerance to Cd2+, Cu2+, PEG and NaCl. The expression of ScMT2-1-3was up-regulated under Cu2+, H2O2, PEG and NaCl stress, but down-regulated under Cd2+stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3gene in bud and root were over14times higher than those in the stem and leaf. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3gene act as antioxidants when it functions in sugarcane response to drought, high salinity and oxidative stress. Moreover, ScMT2-1-3gene is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells need more testification though its expressed protein could abviously increase the host E. coli cell’s tolerance to Cd2+.5. R2R3-MYB gene is a principal member of MYB transcription factor superfamily, which has been showed to play an important role in secondary metabolism and abiotic stress responses. Based on high-throughput RNA sequencing, three sugarcane R2R3-Myb genes/sub-family genes were isolated in the present study. (1) Genomic DNA sequence of Sc2RMybl gene (GenBank Acc:JQ823165) was1,807bp in length, including3exons and2introns, and the complete coding sequence was1,248bp encoding427amino acids. The recombinant protein with an estimated molecular mass of52kDa was produced in positive prokaryotic strain induced by IPTG The recombinant E. coli cells exhibited better growth than the negative strain in liquid LB medium with addition of NaCl. When25srRNA gene was used as the internal control, the expression profiles of Sc2RMybl gene responsive to different stresses were detected by Real-time qPCR in sugarcane. The transcript levels of Sc2RMybl gene in sugarcane seedling decreased under both H2O2and NaCl stresses. Moreover, when treated with PEG, Sc2RMybl gene was only slightly up-regulated at the treatment time point of12h and down-regulated visibly in the other six treatment time points. It suggested that the Sc2RMyb1gene, as a negative adjustment factor, may involve in the response to salt stress, etc.(2) Sc2RMyb2gene was able to generate at least two distinct transcripts, Sc2RMyb2S1and Sc2RMyb2S2, by alternative splicing. Sc2RMyb2S1gene was1,066bp long, including a687bp ORF encoding288amino acid residues. Sc2RMyb2S2had no significant open reading frame and did not encode a functional protein. Sequence analysis showed that series introns structure in the gDNA sequences of Sc2RMyb2gene was the structure foundation for alternative splicing. The alternative splicing of Sc2RMyb2gene is responsive to PEG stress, by means of regulating the relative transcript level of Sc2RMyb2S1and Sc2RMyb2S2in cells. The transcript level of Sc2RMyb2S1gene was inhibited by PEG with obvious down-regulation during the whole period of treatment. The tobacco leaves turned yellow when the Sc2RMyb2S1gene expressed transiently in it, which suggested that the Sc2RMyb2Sl gene was involved in cellular senescence induced by PEG(3) In the present study,7genes termed as Sc2RMyb3-1to Sc2RMyb3-7were cloned and classified into the same sub-family genes:Sc2RMyb3s. Three of them, Sc2RMyb3-1, Sc2RMyb3-2and Sc2RMyb3-3, were chosen for a transient expression assay in the tobacco leaf. The transient expression of Sc2RMyb3-1or Sc2RMyb3-2gene in the tobacco leaves, did not cause any significant phenotypic changes, such as colors. On the contrary, the tobacco leaves turned yellow notably when the transient expression assay of Sc2RMyb3-3gene was performed under the same conditions. It was suggested that the Sc2RMyb3-3gene was involved in cellular senescence induced by PEG, and the complex role played by Sc2RMyb3s sub-family members in regulating multiple aspects of different stresse responses. Sequence analysis showed that the C-terminal amino acid sequence may play an important role in determining the function of Sc2RMyb3s transcription factors in sugarcane.