Genome Survey Of Transcription Factor Family And Gene Characterization Related To Stress Resistance In An Introgression Cultivar Shanrong No.3of Common Wheat

Wheat is one of the most important crops worldwide, but wheat growth and productivity are adversely affected by abiotic stresses, including low temperature, drought and high salinity, urging us to breed stress-tolerant wheat cultivar. In our previous work, a new somatic hybrid introgrcssion line Shanrong No.3(SR3) was generated using common wheat Jinan177(JN177) and Thinopyum ponticum, a salt and drought tolerant grass. SR3has dramatically higher stress tolerance than JN177, and their transcriptional and proteomic profiles also show great difference under stressful and non-stressful conditions, suggesting the unique characteristics of stress toleranee mechanism of SR3. In order to investigate the mechanism of SR3in response to stress, in this work, a bioinformatics study on evolutionary characteristics and stress-responsive expression patterns of CBF and WRKY transcription factor families from wheat was conducted; two stress tolerance associated transcription factors, TaZF13and TaCBFB27, were cloned and their functions involved in stress toleranee were analyzed. The main research contents and results achieved in this work were summarized as follows.1. The allelic variation and expression patterns of WRKY gene family in wheatUsing multiple BLAST searches against the wheat databases with the WRKY domain sequences as query,129WRKY protein coding genes in wheat were identified, and almost all of their deduced amino acid sequences contained one or two WRKY domains as well as C2H2zinc finger domains. The unroot-phylogenetie-trees of WRKY family were depicted using WRKY domains and WRKY genes respectively by the CLUSTAL X program, both of which similarly showed the evolutionary relationship among WRKY family. Based on the clustering features of WRKY domains and their phylogenies, the wheat WRKY gene family can be classified into three groups (Ciroup Ⅰ,Ⅱ,Ⅲ) and eight subgroups, which was similar to the cluster classification of WRKY families from the rice (Oryza.saliva L.) and Arahidopsis thaliana. The sequence variation analysis showed that some WRKY genes in group Ⅲ were evolved more actively than others in a species-specific manner, suggesting their special roles in stress response.More interestingly, in comparison with JN177, sequences of homoeotic genes of WRKY family in SR3were found to have allelic variations, including SNP, short fragment delete and crossover, indicating the fast evolution of WRKY family during somatic hybridization.In order to address the effect of allelic variation on transcription in response to stress,18WRKY genes in SR3and JN177were randomly selected to conduct real-time RT-PCR analysis. Of them, six were induced by high salinity, drought and exogenous ABA. Firstly, these genes showed different expression patterns to different stress. WRKY62was induced by ABA earlier than other stress, whereas WRKY95was more sensitive to PEG and ABA than NaCl. Secondly, these genes had different expression patterns under a certain stress. When treated by PEG, WRKY62,94,13and82had the highest expression level after12hours, while WRKY31and95had a transcription pick at1hour. Thirdly, these genes showed expression patterns in SR3and JN177. For example, WRKY23was more sensitive to stress in SR3than in JN177. These results demonstrated that the allelic variation of WRKY family in SR3offered the alternation of its expression modulation in response to stress.2. The allelic variation and expression patterns of CCAAT-binding transcription factor (CBF-TF) family in wheatWith the same method, using the amino acid sequences of several CBF genes as query probes,54CBF-TF genes were identified in wheat. In combination with CBF-TFs in rice and Arabidopsis, these54genes were phylogenetically classified into three subfamilies based on amino acid sequence, containing a lot of paralogy genes. This result hints that the evolution of this family may happen in a species-specific manner, and new members were produced mainly through tandem duplication events. As above, allelic variation such as SNP and indel of this family between SR3and JN177was also found.Of9CBF-TF genes, four were found to be stress-responsive. CBF40had stronger mRNA abundances under drought stress and ABA treatment than salinity stress. The transcript of CBF46had a sharp peak at1hour during stressful conditions, whereas those of CBF51and12were higher during the whole stressful course than the control. Besides, CBF12had significantly different ABA-responsive patterns between SR3and JN177, with a more sensitive response in SR3. These results displayed the diverse roles of CBFs in stress tolerance, and the putative correlation between the allelic variations of CBFs with high salinity tolerance of SR3.3. Cloning of TaZF13and its function involved in salt stressA RanBP zinc finger protein gene TaZF13and its allelic variant gene tazf13were cloned from JN177and SR3, respectively. The cDNA of tazf13had a27bp insertion in comparison with TaZF13. The alignment among genomic sequences of TaZF13and tazfl3and their homolog gene of Th. ponticum showed that the27bp insertion had an absolutely identity to the TaZF13homolog gene of Th. ponticum. This indicated that tazfl3was a mosaic gene of its homolog genes from biparent of SR3.RT-PCR analysis indicated that the transcriptions of TaZF13and tazf13were both up-regulated by salt and drought stress, and kept constant under the ABA treatment. Except for flower, TaZF13and tazf’13were expressed in the whole seedlings of wheat. Their products localized in nuclei and cytoplasm. The transgenic Arabidopsis and wheat lines over-expressing tazf13exhibited enhanced salinity tolerance. The expression of some marker genes involved in ABA-independent signal transduction pathway in transgenic Arabiclopsis was obviously altered.4. Cloning of TaCBFB27and its function involved in salt stressBased on above CBF family analysis, TaCBFB27was cloned from SR3. Its genomic sequence of SR3showed SNP and insertion variation in comparison with that of JN177. Unlike TaZF13, however, the cDNAs between SR3and JN177were absolutely identical. The transcription of TaCBFB27gene is up-regulated in both SR3and JN177under saline stress, oxidative stress and ABA treatment. HMSA result showed that TaCBFB27had specific binding ability to CCAAT motif. The ectopic expression of TaCBFB27in tobacco improved the tolerence to salinity, in addition, the proline contents in transgenic plants were obviously higher than those in wildtype plants under normal condition, implying that such salinity tolerance enhancement may be achieved through ABA-dependent pathway.In summary, phylogenetic analysis of WRKY and CBF transcription factor families in wheat as well as characteristics of their variation and transcription in SR3and JN177gave us a family-level view into the mechanism of SR3’s stress tolerance. From these results, two stress-responsive transcription factors, tazf13and TaCBFB27were identified and confirmed to have positive roles in salinity tolerance.