[Sequence polymorphism and mapping of wheat Ca2+-binding protein TaCRT-A gene].

Taking thirty-seven hexaploid wheat (AABBDD) accessions with different drought resistance at seedling stage, three wheat species with A genome (AA), and three tetraploid wheat species (AABB) as test materials, and by direct sequencing the single nucleotide polymorphism (SNP) in TaCRT-A, this paper analyzed the relationships of the SNP with the drought resistance of wheat ( Triticum aestivum) at its seedling stage, and mapped the TaCRT-A on the chromosome of wheat. The full-length sequence of the TaCRT-A genomic DNA was 3887 bp. A total of 202 nucleotide variant loci were observed in the full length sequence of 167141 bp, among which, 165 SNP and 37 InDel with the frequencies of 1 SNP/1013 bp and 1 InDel/4517 bp were detected, respectively. The nucleotide diversity (pi) in coding region of TaCRT-A was lower than that in non-coding region, suggesting that the selection pressure in coding region was stronger than that in non-coding region. The 43 accessions could be classified as 14 haplotypes (H1-H14) by haploid analysis, among which, H1, H2, and H13 all contained one accession which was the donor species of A genome in common wheat, H16 and H7 had one high drought-resistant accession, H8 comprised tetraploid wheat, drought-resistant accessions, and drought-sensitive accessions, whereas H11 included the wheat accessions with drought-resistance and medium-drought resistance. Though the expression of TaCRT was induced by water stress, no significant relationship was identified between TaCRT-A polymorphism and drought resistance. Using a population of recombinant inbred lines derived from a cross of Opata 85 x W7984, the TaCRT-A was mapped between SSR markers Xmwg30 and Xmwg570 on chromosome 3A, and the genetic distances were 10.5 cM and 49.6 cM from the flanking markers, respectively.

Cloning the PvP5CS gene from common bean (Phaseolus vulgaris) and its expression patterns under abiotic stresses.

A full-length cDNA denominated PvP5CS for Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in the biosynthesis of proline, was cloned from common bean using a candidate gene approach. PvP5CS contains an open reading frame encoding a 716 amino acid polypeptide. Sequence analysis showed that PvP5CS shares 95.1% homology in nucleotide sequence and 93.2% identity in amino acid sequence with the mothbean (Vigna aconitifolia) P5CS. The expression patterns of PvP5CS in common bean treated with drought, cold (4 degrees C), and salt (200 mM NaCl) stresses were examined using real-time quantitative PCR. These abiotic stresses caused significant up-regulation of the expression of PvP5CS in leaves. The PvP5CS mRNA transcript increased to 2.5 times the control level after 4d drought stress. A rapid up-regulation of PvP5CS, to about 16.3 times the control at 2h post-treatment was observed under salt stress. A significant increase in PvP5CS expression (11.7-fold) was detected after 2h of cold stress. The peaks of proline accumulation appeared at 8d for drought, 24h for cold and 9h for salt stress, somewhat later than the peaks of PvP5CS expression. These results suggest that PvP5CS was a stress-inducible gene regulating the accumulation of proline in plants subjected to stress. Finally, subcellular localization assays showed that the PvP5CS protein was present in the nucleus and at the plasmalemma.

Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

[Comparison of methods to construct a full-length cDNA library].

The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library.

[Construction of a full-length cDNA library of Aegilops speltoides Tausch with optimized cap-trapper method].

To discover new genes in a throughput manner,the cap-trapper method published previously was optimized for raising the efficiency in the construction of full-length cDNA library. Using the optimized protocol,we successfully constructed a full-length cDNA library of Aegilops speltoides,which contained 3.0 x 10(6) clones and more than 99% of plaques were recombinant phages. Sequence analysis results indicated that more than 89% of the clones were full-length.