Cloning And Functional Analysis Of Genes Encoding Fructan Biosynthesis Enzymes In Triticeae Plants

Fructans, being widespread in temperate and sub-temperate herbaceous and grass plants, which biosynthetic and degraded processes characterize one of major metabolic pathways of wheat carbohydrate, can enhance the resistances of plants to drought, salinity, low temperature and other abiotic stresses. As an important allohexaploid plant and a staple crop, wheat has received great attention in the study on FBEs gene structure features, expression characteristics, genetic features, the mechanism of wheat fructan accumulation, the analysis of the relationship between fructan synthesis-related genes and the resistances, which shows great significance on the enhancement of the resistance of wheat and other crops of important agricultural traits. In this study, three diploid species (Triticum urartu Thum: AA; Aegilops speltoides Gren: SS; Aegilops tauschii Coss: DD), one tetraploid accession (Triticum turgidum: AABB) and one hexaploid variety (Triticum aestivum: AABBDD) were chosen as plant materials for FBEs isolation. Touch-down PCR technique were employed to clone genes coding for fructan biosynthetases: sucrose: sucrose-1-fructosyltransferase, 1-SST; fructan:fructan-1-fructosyltransferase, 1-FFT and sucrose: fructan-6-fructosyltransferase, 6-SFT. Sequence alignment and structural analysis were also performed among them and those of different origins. Using I-PCR and TAIL-PCR technology, partial length of promoters for FBEs were identified from common wheat cv. Yangmai 6. Functional elements were predicted through online bioinformatics. Functional elements were confirmed through the construction of fusion vector with truncated promoter fragments and GUS and then transformation into the wheat immature embryos using biolistics. Prediction on the gene copies in common wheat was carried out using Southern blotting analysis. Six common wheat cultivars were chosen for expression profile assay by real-time technique under drought stress at different time intervals. Agrobacterium-mediated transformation was adopted to characterize the function of the cloned wheat 6-SFT in tobacco (Nicotiana tobacum) under drought, salt and low temperature stresses. Main results are covered as the following:1. High conserved 1-SST in Triticeae plants. The full length of gDNA and cDNA were 3326 bp and 1989 bp, respectively, containing four exons and three introns in composition with 406 bp, 9 bp, 872 bp, 702 bp in exon length and 161 bp, 146 bp, 1030 bp in intron length. At the amino acid level, AABB-originated 1-SST shared the similarity of 95.62% and 96.83% with the AA- and SS-originated. In AABBDD genome, it had 97.89%, 99.09% and 99.24% identity with that in AA, SS, and DD genomes at the amino acid level, respectively.2. The gDNA and cDNA lengths of 1-FFT were the same from AA, SS, AABB, AABBDD genomes with 2652 bp and 1947 bp in length, respectively, composed of four exons and three introns. The sizes of four exons were 367 bp, 9 bp, 869 bp, 702 bp and the sizes of the 3 introns, 156 bp, 377 bp, 172 bp, respectively. In the DD genome, gDNA length was 2603 bp and cDNA length, 1935 bp consisting of 4 exons: 355 bp, 9 bp, 869 bp and 699 bp, and 3 introns: 160 bp, 393 bp and 118 bp. At the amino acid level, 97.84% and 99.38% similarities were found between AABB vs AA and AABB vs SS. Compared to AABBDD genome-originated, the similarity was 99.07%, 99.07%, 88.63% in AA, SS and DD genome, respectively. 3. 6-SFTs from Triticeae plants were more active during evolution. Different gDNA lengths were found to be 3122 bp, 3302 bp, 3100 bp, 3138 bp and 3146 bp in AA, SS, DD, AABB, and AABBDD genome, respectively. And the cDNA sizes were 1851 bp. 6-SFT also contains four exons and three introns. High efficiency of insertion/deletion mutation in the first exon accounted for the full-length difference in Triticeae plants. At the amino acid level, 97.08% and 98.54% identity was found between AABB vs AA, AABB vs SS, respectively. 97.24%, 98.70% and 96.92% similarity was shared by AABBDD-originated 6-SFT with AA-, SS-, and DD-originated at the amino acid level, respectively.4. Multi-copies of FBEs in Triticeae were found. For 1-SST, there were three copies in the AA group and the DD group, while in the SS group, at least one copy was found. In the AABB and AABBDD genomes, there were 5 to 6 copies and more than 7 to 8 copies, respectively. In the SS and DD groups, there existed three copies of 1-FFT, while in the AA genome, at least two copies existed. Then in tetraploid wheat, AABB, there were six copies and in the common wheat, AABBDD, at least seven copies were present; 6-SFT in AA group, there are 2 or 3 copies; in the SS group, at least one copy existed; in the DD group, there were three copies and at least 5 to 6 and 7 to 8 copies were present in AABB and AABBDD genomes, respectively.5. Successful isolation of promoter for FBEs from wheat cv. Yangmai 6, designated as PSFT, PSST and PFFT with 1064 bp, 1927 bp and 2142 bp in length, respectively. Functional elements prediction online showed the presence of TATA box and CAAT element, stress response elements, such as MYB, LTR, ABRE and GARE and Circadian element responsible for circadian rhythm in plants and Skn-1 components controlling endosperm-specific expression (except PFFT) in all the three promoters. Functional truncated assay on PSST and PFFT demonstrated that they were abiotic stress-responsive.6. Expression pattern was characterized on FBEs under different time-interval drought stresses. The highest expression levels of 1-SST and 1-FFT reached at seven days, while that of 6-SFT reached after 26-day treatment. Further analysis showed different expression characteristics in different wheat varieties. As for 1-SST expression, Yangmai 6 was high, followed by Yangmai 12, while during the whole process of drought stress in common wheat cv. Chinese Spring, the expression remained at the same level with relatively lower expression, which may answer for the low drought-tolerance in part. 1-FFT and 1-SST shared the same expression pattern with the highest expression in Hanxuan 10 at 7-day drought stress. 6-SFT responded to drought stress slower than 1-SST and 1-FFT with the same highest expression level presenting in Hanxuan 10.7. Biolistics-mediated transformation of onion epidermal cell with fusion vector 16318-hGFP::6-SFT. Transient expression of GFP showed 6-SFT may be located at cell membrane using confocal microscope.8. Obtaining transgenic tobacco lines with wheat 6-SFT. When treated with no watering, 300 mM NaCl and -20℃for 30 min, transgenic tobaccos showed strong tolerance to drought, salt and low temperature stresses while wild types display wilting, growth retardment and turn white.