CDNA-AFLP Screening And Functional Analysis Of Low Phosphorus-stress Responsive Genes For Wheat

Phosphorus plays an important role in the growth and development of plants, the lack of effective phosphorus in the soil severely limits the crop growth, yield increase and quality improvement. In order to find the key genes and the mechanisms of low phosphorus stress resistance in wheat, and to foster the wheat varieties with high phosphorus efficiency, we took use of six main varieties of wheat in the Huang-Huai Wheat Region and cDNA-AFLP technology, at the transcriptional level, to screen co-expression genes that responding to the low phosphorus stress in different wheat varieties under low phosphorus stress condition. And the differential fragments are screened, cloned and sequenced. Bioinformatics analysis and functional identification are followed. Then qRT-PCR was used to verify the cDNA-AFLP patterns of selected low phosphorus response genes. In addition, this research was based on the cloning and bioinformatics analysis of screening TaRBPMS-like, TaLC1314 and TaSNXl gene fragments. This research offered a better understanding of these genes’ functions in wheat. Major conclusions were as follows:(1) The screening of differentially expressed genes in six varieties of wheat under different phosphorus concentrations condition was done by cDNA-AFLP technique.216 primer combinations were used for selective amplification, and 246 transcript derived fragments(TDFs) were selected for their differential expression, of which 83 target genes were recovered, cloned and sequenced.The analysis of these target genes by using Blastx homology showed that 45 genes were highly homologous in the database. Among the 45 homologous proteins, there were 13 predicted proteins with unknown function, and the target genes’homologous proteins included:RNA binding proteins, Aspartate aminotransferase(AAT), Glucan endo-1,3-beta-glucosidase, Glutathione transferase, Basic region/leucine zipper protein(bZIP), Peroxidase, CCCH-type zinc finger proteins, Ty3-gypsy subclass protein, Salt-inducible protein, Vacuolar protein sorting-associated protein(VPS), U-box domain-containing protein,60S ribosomal protein, Putative auxin influx carrier, Dynamin-related protein, Methylmalonate-semialdehyde dehydrogenase(MMSDH), RRNA intron-encoded homing endonuclease, Chloroplastic thylakoidal processing peptidase and so on. In addition, there were 16 differentially expressed genes that were found no similar sequence in the GenBank database, they may be new genes.According to the results of bioinformatics analysis,16 genes, which were selected from differentially expressed fragments separated by cDNA-AFLP approach, were used for qRT-PCR experiments:relative quantitative analysis of the target genes were tested with a standard of reference gene (26s). The results showed that their expression abundance was consistent with what cDNA-AFLP polyacrylamide gel electrophoresis displayed. Screening genes were significantly induced the expression by low phosphorus stress at the transcriptional level, and their functions in the low phosphorus stress metabolic network of wheat would be worth making further study.(2) On the basis of the wheat genomes and transcriptome information, cDNA sequences of TaRBPMS-like, TaLC1314, and TaSNXl gene and gDNA sequences of TaSNXl gene were isolated from common wheat cultivar Zheng-mai 9023. And the systematic analysis was made for their sequence characteristics and evolutionary relationships.The results showed that the complete ORF of TaRBPMS_like was 843bp and it encoded a putative protein that was composed of 280 amino acids. The estimated molecular mass of TaRBPMS-like is 30.2 kDa and its isoelectric point is 8.57. The sequence of N-terminal has a RNA Binding Domain and the signal peptide sequence. There were some phosphorylation sites and glycosylation sites and apparent transmembrane regions on this protein sequence. The protein is similar to RBPMs, so it may have the function of RNA binding proteins in plants, and it can adjust the interactions between protein and RNA, and protein and protein.Sequence analysis showed that the complete ORF of TaLC1314 was 996bp and it encoded a putative protein that was composed of 331 amino acids. The estimated molecular mass and isoelectric point of TaLC1314 are 36.4 kDa and 7.86 respectively. This protein belongs to 17th family of glycosyl hydrolyses, and it has the typical amino acid sequence of this family:LIVM-X-(LIVM-FVW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. There were also some phosphorylation sites on this protein sequence. TaLC1314 is forecasted to have the function of β-1,3-glucanase which plays an important role in disease resistance, stress tolerance of plants according to the existing reports.Bioinformatics analysis showed that TaSNXl contained 10 exons and 9 introns within the genomic DNA sequence and had an open reading frame of 1203bp encoding a protein with 400 amino acids. The estimated molecular mass of TaSNXl is 46.0 kDa and isoelectric point is 6.17 respectively. The protein contains typical phosphoinositide binding sites PX and Bin/Amphiphysin/Rvs (BAR) of SNX gene family. The sequence of N-terminal has a signal peptide sequence. There are some phosphorylation sites on this protein sequence. In 12 wheat varieties for the genetic analysis of TaSNXl gene, single nucleotide differences found that a total of 33 bases of TaSNXl gene mutated and resulted in six amino acid changes. According to the existing reports of AtSNX1 gene function, TaSNXl is predicted to participate the polar auxin sorting and PIN2 protein sorting process, which provides specific position and orientation information for the various parts of the plant body cells.(3)The analysis of gene single nucleotide difference for TaSNXl gene in the 12 wheat varieties indicated that a total of 33 bases of TaSNXl gene mutated and resulted in six amino acid changes. TaSNXl protein was found deletions and insertions comparing with related proteins in wheat Triticum urartu(A-genome) and Aegilops tauschii (D-genome).These changes may be adjustments made by the species to adapt to the low phosphorus environment. The further study for the effects on the gene function caused by the amino acid changes will contribute to a better understanding of the molecular basis of wheat adaptations to low phosphorus stress.