Analysis On Transcriptional Profiles And Salt Responsive Genes In An Introgression Cultivar Of Common Wheat

As an important agronomic trait in crop plants, salt stress tolerance is controlled by quantitative trait loci (QTLs). Salt stress condition may activate multiple signaling pathways, and enhance or inhibit downstream effect genes, including genes related with metabolism, defense, ion influx and transport, reestablishing homeostasis, biosynthesis of osmoprotectants and some cellular structures, etc. All of these genes make full functions coordinately to keep plant growing and developing regularly. Therfore, it is necessary to analysis the whole genome transcriptional profile of plant under salinity stress.A new somatic hybrid introgression line Shanrong No.3 (hereafter SR3) has been generated in our lab from hybridization of common wheat Jinan 177 with Thinopyrum ponticum, a salt and drought tolerant grass. Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor Th. ponticum were introgressed into this line. SR3 had a significantly higher yield than its parent JN177 and the salt-tolerant control cultivar in salt- alkali soil of Shandong, China. It has passed Shandong provincial regional yield trial for new salt-enduring wheat cultivar (Lu-Nong-Shen-Zi No. [2004]030). The result of SSR marker analysis suggests that a major salt tolerance gene and some microgenes controlled the salt tolerance of SR3.In order to investigate the mechanism of SR3 response to salinity, we analyzed genome-wide transcriptional analysis of two genetically related wheat genotypes (SR3 and JN177). The data come from wheat genome array of different tissues at different times under a gradually imposed salinity stress. Based on the analysis from transcriptional profiles between the SR3 and JN177. as well as by using the RACE method, three full-length cDNA related to salt stress were cloned from SR3 and their expression characters and functions were analyzed. The main research contents and results achieved in this work were summarized as follows. 1. Comparative transcriptional profiling of SR3 and JN177 under salinity stress using the affymetrix wheat genome arrayWe used the Affymetrix wheat genome array containing 61127 probe sets to explore the difference in transcriptome of the wheat salt-tolerant genotypes SR3 and it wheat parent JN177 under control and salinity-stressed conditions during vegetative growth. A totally of 6,504 probe sets, on behalf of 5,577 transcripts were identified with differential expression patterns between SR3 and JN177 under salt stress or no-salt stress conditions. These genes were mainly involved in the process related to material transport, ribosome, membrane, calcium ion related signal transduction, environment stress response, transcript regulation as well as cell wall organization and biosynthesis. Bioinformation data shows that under salt stress, the differential expression probe sets in root are probably two times of that in leaf blade. Because the root of plant faces the salinity stress directly, a lot of the difference expressed genes in root were involved in the ion absorption and transport, mainly include many water channel proteins, the calcium-dependent potassium ion channel and non-selective channel proteins. Salt specific responsive probe sets includes some famous salt tolerance related genes, e.g. Na⁺/H⁺ antiporter gene (NHX1), PPase and TaHKT1, that was induced or inhibited in SR3. This implies that the sodium absorption and ion compartmentation in vacuole play an important role in the SR3. It was also found that some probe sets showed differential expression between SR3 and JN177 also involved in the transcriptional response to salt stress. Some probe sets responsive to salt stress were further analyzed including: ion transporter. ABA metabolism, signal transduction pathway and responsive genes, histone, proline synthesis and metabolism pathway. Some genes in the hormone signal transduction pathway like ABA, JA and GA were induced after salinity treatment, which suggested that there is a crosstalk between the hormone and abiotic stress. Histone may induce or suppress the expression of many function genes through the post-transcription processing, which were generally suppressed in SR3 after salt stress with a specific pattern and worth for further study.2. Cloning and functional analysis of the full-length cDNA of TaCHP gene involved in salt stress The full-length cDNA of TaCHP was cloned and sequenced from SR3 and JN177. Expression of TaCHP in SR3 is down regulated under salt stress but up-regulated in JN177 based on the array data analysis. RT-PCR result revealed that the transcription of wheat TaCHP gene was suppressed in salt-tolerant line SR3 under saline stress and ABA treatment and showed low-expression level in JN177, but no-affected under drought and H₂O₂ treatment. That implied that TaCHP gene acts as a co-regulator of the salt and ABA response pathway. Analysis of in situ hybridization shows that TaCHP gene expresses in the cortex and vessels of xylem in the maturation region of root of SR3 under control condition, while no signal was detected in the root after 24h salt stress.The deduced amino acid sequence of TaCHP gene showed homologous to CHP-rich zinc finger protein-like of Arabidopsis and rice, with 3 divergent C1 domains that only found in plant proteins. This short domain is rich in cysteines and histidines and probably also binds to two zinc ions. The function of proteins with this domain is uncertain in plant; however this domain may bind to molecules such as diacylglycerol and take part in the PLD/PKC signal transduction pathway. The transgenic Arabidopsis lines over-expressed TaCHP gene exhibited enhanced resistance against salinity stress.3. TaWRSI5, a Bowman-Birk type protease inhibitor, is involved in the tolerance to salt stress in wheatA salt responsive gene TaWRSI5 was characterized from salt tolerant cultivar SR3, which was induced after salt stress in wheat genechip and the silver staining mRNA differential display. The peptide encoded by TaWRSI5 contains a Bowman-Birk domain sharing a high level of sequence identity to monocotyledonous protease inhibitors. When expressed in vitro, the TaWRSI5 gene product exhibited trypsin, but not chymotrypsin inhibition. The expression level of TaWRSI5 was increased in SR3 roots exposed to salt, drought or oxidative stress while with different peak time of H₂O₂ (1h), salt (6 h), AlCl₃ (24 h) and PEG (24 h) in roots. That suggested H₂O₂ may act as an upstreaming regulator of TaWRSI5 and take part in abiotic stress. In situ hybridization showed that it is induced in the endodermal cells of the mature region of the SR3 root tip, with no signal detectable in the corresponding region of the salt susceptible cultivar JN177. SR3 has a higher selectivity for K⁺ over Na⁺. and therefore limits the transport of Na⁺ from the root to the shoot. When over-expressed in Arabidopsis thaliana, TaWRSI5 improves the ability of seedlings to grow on a medium containing 150 mM NaCl. We suggest that TaWRSI5 plays an important role in regulating long-distance Na⁺ transport or the sodium distribution in SR3 plants exposed to salt stress.4. Isolation and characterization of the TaSKC1 gene from SR3 involved in salt stressAn important mechanism of salinity resistance in wheat is adjusting the sodium-selective transport from root to shoot and retaining low Na⁺ density in shoots. No candidate gene has been reported in wheat for this process, and there are not any corresponding probe sets on the wheat genechip. Homologous cDNA of OsSKC1 in wheat- TaSKC1 was cloned using the RACE methods. The deduced amino acid sequence exhibits 71% identity to that of OsSKC1 and the next to AtHKT1. To determine the TaSKC1 function, we expressed the gene in Saccharomyces cerecisiae mutant strain G19, which had defect in the Na⁺ efflux system and show Na⁺ sensitive phenotype when grows in medium with NaCl. The result showed that G19 strain with TaSKC1 overexpression displays increased Na⁺ sensitivity phenotype than the control with empty vector. This result suggested that TaSKC1 could mediate Na⁺ uptake in yeast mutant strain G19. The transcription of TaSKC1 gene is down-regulated in both SR3 and JN177 under saline stress, oxidative stress and ABA treatment, with a different degree. The putative mechanism was that: there are massive Na⁺ accumulated in the root cell after salt stress and lots of stress responsive genes were induced, leading to the negative regulate to TaSKC1 gene and suppressing its expression, then reducing the Na⁺ uptake from the roots to shoots so as to keep low Na⁺ content in shoots of wheat.5. Somatic hybridization could promote the mutation in genome sequence and differental expression of geneWe cloned the TaCHP1 and TaSKC1 genes homologous to the parents of SR3, JN177 and Thinopyrum ponticum. Sequence analysis showed that TaCHP1 and TaSKC1 genes of SR3 have the same cDNA sequences with its wheat parent, JN177. It implies that the difference expression among SR3 and JN177 is possibly decided by the different upstream regulation sequence. This result provides a clue to the suggestion that somatic hybridization can affect and regulate the expression pattern of genes in acceptor. As for the third genes, half part of cDNA sequence of TaWRSI5 shows high similarity to that of Thinopyrum ponticum and another half are homologous with that of JN177, with several dot mutations. This implies that TaWRSI5 of SR3 is a new gene derived from the recombination of homolog from SR3 and JN177 in the somatic hybridization by a similar mechanism to the HMW-GS genes that we reported. It gives a molecular proof that somatic hybridization both affects the genome constitution and regulates the expression of some functional genes. This kind of change may promote novel gene formation and enhance wheat germplasm diversity.In summary, the transcriptional profiles of somatic hybrid SR3 shows great difference with its wheat parent JN177 under salinity treatment. This could be the result of the introgression of Thinopyrum ponticum chromatin to wheat in the process of somatic hybridization. Among the different expression genes, some salt specific responsive ones could function in the salinity enhancement of SR3. Of these genes, TaCHP, TaWRSI5 and TaSKC1 studied in this work likely play an important role in the complex network controlling the salt tolerance of wheat variety SR3.