| Currently, inevitable increase in energy consumption consequently leads to continuous decrease in fossil fuel reserves and serious problem of global environment worsening. To reconcile this conflict, it is necessary to look for a form of ecological, sustainable and renewable energy source. Using bioenergy plants to produce biodiesel and bioethanol is nowadays a hot spot. Jatropha curcas L., a woody shrub or small tree belonging to the Euphorbianceas family,has attracted extensive attention, due to its high oil content in seed and easy conversion into biodiesel, lipid composition similar to fossil fuel, no competition with food crops and advantages for improving the ecological environment. Nevertheless, because of its origin from the tropical MidAmerican zone, J. curcas confers an inherent but undesirable characteristic(low cold tolerance) that may seriously restrict its large-scale popularization. This adaptive flaw can be genetically improved by elucidating the mechanisms underlying plant tolerance to cold.The newly developed Illumina Hiseq? 2000 RNA-seq is a deep high-throughput sequencing approach, and preferentially used for cold-resistance related transcriptome analysis of J. curcas in this work. After discarding the low quality reads, de novo transcriptome assembly with clean data was carried out by the short reads assembling program of Trinity. The results showed that the final transcriptome of J.curcas covered 55,112,142 clean reads(4,960,092,780 nt total), consisting of 106,749 contigs(36,062,865 nt total, average length of 338 nt) and45,251 Unigenes(35,713,278 nt total, average length of 789 nt). The average G+C content of the clean data was 42.16%. Blastx and ESTscan analyses of 45,251 Unigenes’ sequences showed that 34,274 had reliable coding sequences(CDS)(33,362 Unigenes were aligned with database by Blastx and 912 Unigenes were predicted by ESTscan). 35,791(79.09%) Unigenes have been successfully annotated to different protein databases, the remaining 9460(20.91%)Unigenes not aligned were recognized as new genes. Among these unigenes, 27,293 can be categorized into 61 functional groups of GO, 11,887 with COG-annotations were functionally classified into at least 25 molecular families, 18,787 were possibly involved in approximately128 known metabolic or signaling pathways in KEGG. Moreover, 6,665 SSR loci were found in5,922 Unigenes, with an average length of 12 bp, occurrence frequency of 13.09%, and the major types of mononucleotide, dinucleotide and trinucleotide(accounting for 94.49%).Gene expression of J.curcas after chill hardening at 12℃ for 12 h, 24 h, 48 h were investigated through the transcriptome–referred DGE analysis. Based on the criteria for screening the differentially expressed genes, a total of 4185 J. curcas genes from the referencetranscriptome as mentioned above were found to be differentially expressed after three cold treatments(12℃ for 12, 24, and 48 h) in comparison to the untreated control(constant exposure at 26℃). Therein, 1915, 2085, and 3213 genes corresponded to 12, 24, and 48 h of cold exposure, respectively, with 553 genes in common. After 12 h, 24 h, 48 h of chill hardening at12℃, 1211, 1302 and 2516 genes were upregulated for expression, respectively, and concomitantly accompanied with the downregulation of 704, 783, and 697 genes. From the 457 commonly upregulated genes in all three cold treatments, candidates were identified for their association with electron transportation, redox balance, hydrolases, mini-molecular transfer families, DNA & RNA binding proteins, and transcription factors such as AP2/ERF, ABC, etc.Under chill hardening, the starch catabolism changes dramatically, the sugars(analogues)inositol, galactinol and/or sucrose, instead of the well-known trehalose, are probably the main osmotic substances. In the ABA-independent signal transduction pathways, most of genes such as those encoding the membrane proteins for signal transduction, ICE, CBF, kinases, and transcription factors were upregulated, implying that the CBF signalling system may play an important role in chilling tolerance of J.curcas. Furthermore, 238, 160, and 330 novel genes were individually induced after 12, 24, and 48 h of cold treatment, with 61 genes commonly expressed at all three time points. Among them, the most highly expressed novel genes possibly encode the DNA binding proteins, receptor proteins, oxidordeuctases, and etc.According to the data of DGE, 22 J. curcas gene candidates were ascertained with large correlation to cold tolerance and characterized for their expression information by semi-quantitative RT-PCR. All 22 genes have the fundamental expression in root, with a common expression tendency of initial downregulation and then upregulation with the duration of cold hardening. Generally, nsLTPA, GA20 ox, ADMT, DXS, PIP2, DHN2 and LEA5 reached the maximum expression level after 24 h of 12℃ exposure. In stem, 18 of the 22 genes expressed with a basal level under chilling hardening indistinctively from the control, while other genes such as GSTs, APX1, LEA5 clearly showed cold induction, except for PDI expressed slightly. All 22 genes notably showed differential expression in leaf, gradually upregulated following the chilling hardening and reached the maximum level after 24 h of 12℃exposure.Meanwhile, a part of chilling-induced novel genes(e.g. DNAj, CHS, LRR-RLK, ADMT, DXS,and APX1), had no fundamental expression under the control condition, consistent with the results of DGE analysis.To further gain insight into the mechanism of chilling tolerance developed in J. curcas, we selectively cloned 8 genes, including GS3 and nsLTPA with notably differential expression, coldnewly induced CHS and GA20 ox, PDI and MSRA responsible for ROS elimination and macromolecular functional stability, and CBF1, CBF2 of the CBF family. Protein sequence analysis indicated that both of GS3 and nsLTPA belonged to the multiple gene families and exhibited a salient evolution, with the conserved active center composed by 7 parall β-sheets and 8 amino acid residues for GS3, and a motif consisting of 4 α-helixes that are sustained by 4disulfide bonds for nsLTPA.CHS belongs to the PKS family, with the classical active centers of chalcone synthase and styrene ketone synthase, product binding site, and malonyl-CoA binding site. GA20 ox belongs to 2-ODD family, with the structure of double-layer β-barrel and H-D-H motif. Both of them are evolutionarily conserved and have more than 70% sequence similarity to their homologues in other plants. PDI obtained in our study, belongs to the L-type of thioredoxin family, with the-a-b-b′-a′- domain arrangement, two-CGHC- catalytic motifs, and the endoplasmic reticulum anchoring signal residues-KDEL-, which is in charge of disulfide bond oxidation, reduction and isomerization. MSRA belongs to the A subfamily of MSR family,it has no homology in amino acid sequence and higher structure with MSRB, fRMSR subfamily,while it is featured with the conserved-GCGWP- sequence of A subfamily and α/β barrel structure consisting of 6 β-sheets and 3 α-helixes. The obtained CBF1 has the classical AP2 motif, responsible for binding the CRT/DRE cis-acting element in COR genes, the conserved Val-Glu-Glu residues were however not found. Further analysis of CBF1 promoter revealed that it had TATA box, CAAT box, ICE binding element, MYB binding element, and MYC binding element, implying J.curcas CBF1 can be induced by multiple hormones and involved in a number of stress responses. In addition, the recombinant Saccharomyces cerevisiae strains with GS3, nsLTPA, CHS, and PDI also grew better than the control under cold conditions, implying a considerable relevance of those genes with chilling-tolerance and a putative application potential for genetical improvement in J. curcas.In conclusion, we have obtained a chill-hardening transcriptome data of J. curcas through the high-throughput sequencing approach, disclosed the expression profiles and main metabolism pathways for cold-resistance genes through DGE analysis, and experimentally evaluated the expression of 22 selected chilling tolerance genes in root, stem and leaf of J.curcas. In addition, we have cloned 8 genes from J.curcas, followed with an extensive bioinformatic analysis and functional identification. This study provided a preliminary elucidation of the molecular mechanism for cold resistance formation in J. curcas, with remarkably more number of nucleotide sequences than all previous relevant deposits in the public databases such as GeneBank. The results allowed us to decipher the key genes coding for chilling tolerance from this sequence library, and could improve our current understanding ofthe mechanisms underlying plant chilling tolerance and favor the screening of crucial genes for genetically enhancing the chilling tolerance in J. curcas. |
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