| Stylosanthes guianensis originally grown in tropical and subtropical regions of the America, the most appropriate growth latitude between north latitude 30° to 30° south of the equator. S.guianensis, a kind of tropical legume forage, which has the largest cultivation acreage in tropical regions, and most widely is used for animal’s green fodder, hay meal production, soil and water conservation and grazing. In addition, it likes live in the high temperature and high humidity environment, also can grow in the drought and low fertility region. It has the heat-resistance,pest-resistance and the resistance to poor, low phosphorus and acidic soil, but sensitive to low temperature. In the breeding process of S. guianensis, we fond a new strain named as 89-1 from S. guianensis var. intermedia, which has better cold resistance. We used Illumina sequencing technology in S. guianensis var. intermedia under the treatment of cold(4℃) and appropriate temperature(28℃). On this basis, we performed the gene cloning and functional analysis of Sg NAC1, Sg NAC2 and Sg GH3. The main research results are as follows:(1) Treated at 4℃ for 0h, 24 h and 48 h, the stylo TPRC2001-1 was sensitive to low temperature. The results showed that no obvious difference between stylo 89-1 and TPRC2001-1 at 0h. But after 48 h of cold treatment(4℃), the relative conductivity and malondialdehyde content of TPRC2001-1 were 2.10-fold and 1.57-fold that of 89-1, and the content of proline, soluble sugar and soluble protein of 89-1 were 1.45-fold, 1.24-fold and 1.28-fold that of TPRC2001-1 respectively. This study showed that 89-1 had stronger resistance to the low temperature than TPRC2001-1.(2) After completed the filtration, splicing, assembly and annotation of the sequence from transcriptome sequencing, we got 89,868,386 and 91,942,130 high-quality reads from V CK and LT. These reads were assembled into 222,717 contigs(length from 201 to 12,267nt). The contigs were assembled into 83,873 unigenes. A total of 10,7349, 63,990 and 53,797 contigs were functionally annotated in NR, Swiss-Prot and KEGG database respectively. Based on similarity comparison with other plants, the plant that has the highest similarity matching degree is Glycine max. As a result, we used Gene Ontology to classify functions of the annotated genes, 42.90% contigs were categorized to three GO trees which contained 54 functional groups. By means of using Clusters of Orthologous Groups database, 25.57% contigs were categorized to 24 functional groups. In addition, 21.50% contigs were identified with pathway annotation, and they were functionally assigned to 238 KEGG pathways. A total of 42,588 significantly changed unigenes were detected between cold-treated and control group, and 41.18% unigenes were up-regulated. The differentially expressed unigenes were categorized to 18 pathways, the primary biochemical pathway was ‘cutin,suberine and wax biosynthesis’ which were related to the synthesis of unsaturated fatty acid. Futhermore, we identified 9 classes of transcription factors from all differentially expressed unigenes, and most of them were involved in signal transduction pathways.(3) According to the transcriptome sequencing data, we obtained part of the CDS sequence of Sg NAC1 and Sg NAC2, and also got CDS full-length sequence of Sg GH3. Then, we cloned the CDS full-length sequences of Sg NAC1 and Sg NAC2 by hi TAIL-PCR technology. The CDS full-length sequences of Sg NAC1, Sg NAC2 and Sg GH3 were 909, 777 and 1809 bp respectively. Sg NAC1 and Sg NAC2 contained three exons and two introns, and the total length of their genomic DNA were 1185 and 951 bp respectively. Sg NAC1 and Sg NAC2 protein contain a conservative NAM domain structure. So, we speculated that they may be a NAC transcription factors, and verified their transcriptional activity through a single yeast hybrid. For another Sg GH3 which had the characteristics of plant GH3 proteins, and the multiple sequence alignment and evolutionary tree showed that it belonged to a member of the family of GH3 associated with hormone synthesis. Gene expression analysis illustrated that Sg NAC1 and Sg NAC2 responded to low temperature in the early, but Sg GH3 responded to cold in the later.(4) In this research, we builded a protoplast isolation system of S.guianensis. The results showed that the best dissociation liquid composition included 1.5% cellulose, 0.4% macerozyme, 0.4 mol/L mannitol and 20mmol/L MES, the most suitable digestion time was 8h. According to the system, we obtained the number of protoplasts approximately 3.0×106 / g FW, and the energy of the protoplasts was higher than 80%. To determine the subcellular localization of Sg NAC1 and Sg NAC2, the GFP-Sg NAC1 and GFP-Sg NAC2 driven by the Ca MV 35 S promoter were transiently expressed in protoplasts of S.guianensis. This study indicated that Sg NAC1 and Sg NAC2 were nuclear proteins.(5) We builded the Sg NAC1, Sg NAC2 and Sg GH3 gene overexpression vectors. The recombinant plasmids were introduced into Agrobacterium tumefaciens EHA105 respectively. Tobacco transformation was performed by co-cultivation of leaf discs. We studied the expression of Sg NAC1, Sg NAC2 and Sg GH3 in response to low temperature in transgenic tobaccos through q RT-PCR. The transcript level of Sg NAC1 and Sg NAC2 increased by 2.89-fold and 2.68-fold after 6h of cold treatment, and gradually decreased thereafter. The expression of Sg GH3 was no obvious change during the first 12 h under cold treatment, then achieved the highest level in the 24 h, which increased by 6.46-fold and then decreased by 8.30% at 48 h. The results showed that Sg NAC1 and Sg NAC2 as transcription factor, were earlier in response to low temperature, while Sg GH3 probably was regulated by the other genes in the later. On the other hand, we found that after cold treatment for 48 h,the relative conductivity and malondialdehyde content of WT increased by 1.32-fold, 1.25-fold and 1.48-fold for transgenic tobaccos. The content of proline, soluble sugar and soluble protein in WT was lower than transgenic plants, which only reached 71.95%, 74.40% and 63.11% of transgenic lines such as Sg NAC1-OE, Sg NAC2-OE and Sg GH3-OE. These results suggest that the over expression of Sg NAC1, Sg NAC2 and Sg GH3 reduced the damage of cell membrane under low temperature and promoted the accumulation of intracellular osmotic regulation substances, and improved the cold resistance of tobacco. |
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