Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.).

Calcium-dependent protein kinases (CDPKs) are crucial sensors of calcium concentration changes in plant cells under diverse endogenous and environmental stimuli. We identified 20 CDPK genes from bread wheat and performed a comprehensive study on their structural, functional and evolutionary characteristics. Full-length cDNA sequences of 14 CDPKs were obtained using various approaches. Wheat CDPKs were found to be similar to their counterparts in rice in genomic structure, GC content, subcellular localization, and subgroup classification. Divergence time estimation of wheat CDPK gene pairs and wheat-rice orthologs suggested that most duplicated genes already existed in the common ancestor of wheat and rice. The number of CDPKs in diploid wheat genome was estimated to be at least 26, a number close to that in rice, Arabidopsis, and poplar. However, polymorphism among EST sequences uncovered transcripts of all three homoeologous alleles for 13 out of 20 CDPKs. Thus, the hexaploid wheat should have 2-3 fold more CDPK genes expressing in their cells than the diploid species. Wheat CDPK genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H(2)O(2)), salt, drought, powdery mildew (Blumeria graminis tritici, Bgt), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA). Each CDPK gene often responded to multiple treatments, suggesting that wheat CDPKs are converging points for multiple signal transduction pathways. The current work represents the first comprehensive study of CDPK genes in bread wheat and provides a foundation for further functional study of this important gene family in Triticeae.

[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.

[Isolation, characterization and expression analysis of male sterility gene homology sequence in wheat].

One 134 bp fragment was amplified in anthers of male sterile and fertile wheat using one pair of degenerated primer designed based on the conserved domain of MS2 gene in Arabidopsis thaliana and Oryza sativa, and one 1604 bp male sterility gene homology sequence was extended by in silico cloning based on the 134 bp fragment. The amino acids encoded by the male sterility gene homology sequence include a 200 amino acid conserved domain of male sterility, and this sequence expressed only in wheat male fertile anthers; no expression in male sterile anthers, roots and leaves. This research demonstrated that the cloned male sterile homology sequence is specific to wheat anther development.

Dynamics of the evolution of orthologous and paralogous portions of a complex locus region in two genomes of allopolyploid wheat.

Two overlapping bacterial artificial chromosome (BAC) clones from the B genome of the tetraploid wheat Triticum turgidum were identified, each of which contains one of the two high-molecular-weight (HMW) glutenin genes, comprising the complex Glu-B1 locus. The complete sequence (285 506 bp of DNA) of this chromosomal region was determined. The two paralogous x-type ( Glu-1-1 ) and y-type ( Glu-1-2 ) HMW-glutenin genes of the complex Glu-B1 locus were found to be separated by ca. 168 000 bp instead of the 51 000 bp separation previously reported for the orthologous Glu-D1 locus of Aegilops tauschii, the D-genome donor of hexaploid wheat. This difference in intergene spacing is due almost entirely to be the insertion of clusters of nested retrotransposons. Otherwise, the orientation and order of the HMW glutenins and adjacent genes were identical in the two genomes. A comparison of these orthologous regions indicates modes and patterns of sequence divergence, with implications for the overall Triticeae genome structure and evolution. A duplicate globulin gene, found 5' of each HMW-glutenin gene, assists to tentatively define the original duplication event leading to the paralogous x- and y-type HMW-glutenin genes. The intergenic regions of the two loci are composed of different patterns and classes of retrotransposons, indicating that insertion times of these retroelements were after the divergence of the two wheat genomes. In addition, a putative receptor kinase gene near the y-type HMW-glutenin gene at the Glu-B1 locus is likely active as it matches recently reported ESTs from germinating barley endosperm. The presence of four genes represented only in the Triticeae endosperm ESTs suggests an endosperm-specific chromosome domain.

[ESTs analysis of resistance to powdery mildew in wheat at primary infected stage].

A wheat line, Bai Nong 3217 x Mardler BC5F4 with resistance to powdery mildew was used in the study. A suppression subtractive hybridization cDNA library was constructed from wheat leaves challenged by Erysiphe graminis DC at primary stage. Seven hundred and sixty ESTs were acquired, and the ESTs similarity analysis based on BLASTx software was finished by comparing sequences in nr database of GenBank. Two hundred and seventy one ESTs' functions were identified in the total ESTs. The results showed that GTP-binding proteins associated signal pathway, salicylic acid pathway, MAP pathway etc were supposed to involved in the disease resistance reaction. SAR genes were rich not only in varieties but also in quantity, including five kinds of phyogenesis-related proteins, induced defence-resistance genes, heat shock proteins and genes induced by abiotic-stresses etc. There are lots of evidence to testify PAL pathway, cell wall modification, cell survive system serving in the disease resistance. In the function unknown ESTs, many homologous ESTs were found from other biotic and abiotic-stresses selected cDNA libraries after BLASTn analysis, the stresses included pathogen, salt, drought, cold, high temperature etc. The novel ESTs was 16.6% in total ESTs.

[cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

[Research on the preparation of bacterial artificial chromosome (BAC) vector DNA].

Bacterial artificial chromosome (BAC) library plays a pivotal role in genomics studies. A crucial step in BAC library construction is preparation of BAC vector DNA. Preparation of highly purified vector DNA is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, the BAC vector pECBAC1 was digested by the restriction enzyme of BamHI and dephosphorylated by HK phosphatase respectively. In order to improve the ligation capability of vector DNA, gel purification of linearized vector DNA was also conducted in our study. At the same time, we did a series of experiments to get high quality of vector DNA for construction of BAC library. They included the optimal concentration of restriction enzyme, optimal digestion time, the type of phosphatase and gel purification of linearized vector DNA.