| Plants are constantly challenged in their life cycles by series of adverse environmental factors such as drought, salt, cold, which severely affect plants’growth and development. To deal with these adverse environments, plants have developed complex and precise signal response mechanisms. The signal pathway generally includes secondary messenger, receptor protein and responsive proteins. Ca2+, as a second messenger, plays important roles in signaling pathway in responding to abiotic stresses in plants. The calcium signaling can be perceived by receptor proteins and transmitted to downstream to launch a series of responding mechanisms. Calcineurin B like (CBL) protein is unique calcium recognition receptor protein in plants, which can specifically interact with CBL-interacting protein kinase (CIPK) and activate a series of downstream responsive mechanisms through the process of target protein phosphorylations.In the model plant such as Arabidopsis, the functional studies of CIPKs in response to abiotic stresses have been widely documented. However, little information is available until now about their functions in wheat, an important cereal crop. Bread wheat possesses heterologous hexaploid genome, having a huge and more complex genome compared to diploid plants. Although major progress has been made in wheat genome sequencing, it is still unable to provide complete genome sequence and physical map. In this study, we identified 79 TaCIPK genes (belonging to 29 gene clusters) and amplified the representative genes from 20 gene clusters in wheat. We systematically analyzed the expression patterns of TaCIPKs and the TaCBLs/TaCIPKs specific interactions. The functional analysis of TaCIPK24 in transgenic Arabidopsis was verified. We obtained the transgenic wheat plants overexpressing TaCIPK10 and TaCIPK10-RNAi via particle bombardment method. The main results are as the follows:1) A total of 79 TaCIPK genes were identified in wheat genome using bioinformatic methods. All the genes can be classified into 29 gene clusters, in which genes are the alleles located on heterologous chromosomes of the subgenome and share high similar nucleotide sequence. The gene clusters were thus named by following the rice homologous OsCIPKs. We adopted the new naming rules to distinguish the heterologous gene on chromosome (such as TaCIPK2-A, TaCIPK2-B and TaCIPK2-D). Among those identified TaCIPKs,42 of which had complete structure information, which contain all the sequence from the start code to stop code. We found that 23 TaCIPKs contain no intron,3 TaCIPKs contain one intron, while other 16 TaCIPKs contain 5-14 introns. The results of gene sequences comparison among Triticum urartu, Aegilops tauschii and Triticum aestivum showed that the TaCIPKs shared high similar sequences with genes of other two species. Considering that the physical maps of current wheat genomes do not cover all the chromosome arms, it is difficult to know whether these potential un-identified TaCIPK genes are resulted from gene retention, duplication or whether these wheat specific genes are the results of the loss of CIPK after polyp loidization. In addition, a total of 20 TaCIPK genes were amplified from wheat by RT-PCR method.2) We analyzed the expression profiles of 17 TaCIPKs in 10 wheat tissues or organs using the semi-quantitative RT-PCR method. To study the expression of 23 TaCIPKs during the whole life cycle of wheat, we analyzed public microarray data that were obtained from tissues at various developmental stages. The results showed that TaCIPKs had different expression levels in different organs or at development stages, which reflects that they may be widely involved in the process of wheat growth and development. To further clarify the roles of TaCIPKs during seed germination, we analyzed public microarray data representing the transcription patterns during the seed germination process. The results showed that the expression of eight TaCIPKs were significantly changed during seed germination process. In the process of pollen development, TaCIPKs displayed different expression patterns. It is worth noting that the six TaCIPKs were expressed only in the mature pollen with high expression levels, which suggests that they may function in the process of pollen germination and pollen tube elongation. In addition, we analyzed the expression patterns of 17 TaCIPKs in response to ABA, GA, MeJA, ACC, low temperature, PEG, H2O2, NaCl and high-temperature treatments in the roots and leaves of wheat seedlings using the method of semi-quantitative RT-PCR, and randomly selected five genes(TaCIPK1, TaCIPK15, TaCIPK24, TaCIPK31 and TaCIPK32) to be analyzed by real-time fluorescence quantitative RT-PCR method in wheat seedlings treated with ABA, PEG, low temperature, H2O2 and NaCl. Results showed that most of TaCIPKs responded at different levels to at least one of the phytohormones and/or abiotic stresses.3) We analyzed the physical interactions between 7 TaCBL and 20 TaCIPK proteins using the Yeast Two-hybrid method. Furthermore, seven combinations were randomly selected for Bimolecular Fluorescence Complementation confirmation in the epidermis cells of tobacco. Analyses of a series of C-terminal deletion mutants of TaCIPK11 showed that there were diverse interaction patterns between TaCIPK11 mutants and TaCBLs, suggesting that the C-terminal flanking sequence of TaCIPK proteins has important effects on their interactions with the TaCBLs. These changes in the patterns of interaction may result from changes in protein structure after the deletions. In terms of this deletion assay, we proposed that the interaction between CBLs and CIPKs followed a "concave-convex" model. According to this model, the nonsynonymous sequence variations of alleles from different wheat subgenomes might result in aberrant spatial structures that affect the patterns of interaction, which was verified by interaction patterns of TaCIPK17-A and TaCIPK17-B1.4) Overexpression of TaCIPK24 in Arabidopsis could enhance plant tolerance to salt stress. The results showed that there was no significant difference of K+ contents in control and transgenic plants, while the transgenic plants significantly accumulated less Na+ contents than control plants. TaCIPK24 may activate the SOS pathway in Arabidopsis, by excluding more Na+ to reduce the damage Arabidopsis caused by excessive Na+. By analyzing the hydrogen peroxide content, we found that the transgenic plants accumulated less reactive oxygen substances. Further determining the enzyme activities of catalase, peroxidase and superoxide dismutase showed that the transgenic plants may reduce oxidative damage caused by salt stress through activating peroxide scavenging system.5) Using the Weighted Total Expression Network analysis method, we constructed TaCIPK co-expression network during seed germination and pollen development process in wheat. Enrichment of gene function analyses showed that eight TaCIPKs participated in the carbohydrate and lipid metabolism and nucleosome assembly process during seed germination, and six TaCIPKs, which are highly and specifically expressed in the mature pollens, are involved in stress response, carbohydrate and nitrogen metabolism, electron transfer and ion transport processes.6) We found that the expression of TaCIPK10 in leaves was significantly higher than other tissues. Using Pearson Correlation Coefficient method, we screened 65 genes which had the same expression patterns with TaCIPK10. Meanwhile, no other TaCIPK had similar expression pattern with TaCIPK10, indicating that the TaCIPK10 may play a special role and there exists less possibility of functional redundancy by other TaCIPKs. Therefore, we constructed TaCIPK10-overexpressing and TaCIPK10-RNAi vectors to generate transgenic wheat plants by particle bombardment. Eight TaCIPK10-overexpressing and nine TaCIPK10-RNAi transgenic wheat plants were separated in T1 generation, which provided basis and materials for further functional study. |
PDF Full Text Request