Expression Profiles Of Wheat Somatic Hybrid Introgression Line Shanrong No.3 Under Salt Stress And Functional Analysis Of Salt Responsive Genes

As salt-stress tolerance characters in plants are controlled by quantitative trait loci,a comprehensive resolution of salt-tolerance mechanisms has been complicated.Microarrays have become powerful tools for high throughput screening of salt-stress responsive genes. In model plants,such as Arabidopsis and rice,tens of thousands of gene expression patterns in response to salt stress have been monitored.But for wheat,the information was still limited.A new somatic hybrid introgression line Shanrong No.3(SR3) has been generated in our lab from hybridization of common wheat Jinan 177(JN177) 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 T.ponticum were introgressed into this line.SR3 had a significantly higher yield than its parent JN177 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 results of SSRs analysis suggest that salt tolerance of SR3 is controlled by a major salt tolerance gene and some microgenes.To investigate the salinity tolerance mechanism of SR3,we have developed a new long oligo-DNA microarray based on the EST sequences either from SR3 or from public wheat EST database,which harbors 15,000 unigenes.Using it,the transcriptomes of salt and osmotic stress responses were fully analyzed in SR3 and JN177. Additionally,four stress responsive genes were cloned from SR3 and functionally analyzed.The main research contents and results achieved in this work were summarized as follows.1.Probe design of oligo-DNA microarrays for SR3For the special genetic background of SR3,it may have transcripts other than in common wheat which may be related to the salt tolerance of SR3.To harbor these transcripts in the microarray,we generated a SSH cDNA library between SR3 and JN177 under salt treatment.One thousand sequences from the library were clustered into 180 unigenes.We also get 2,539 unigenes from a complete cDNA library of SR3 which was constructed before.Based on these sequences,together with another 12,281 unigenes from DFCI Wheat Gene Index,probes were designed and synthesized in situ on glass slides by Agilent Technologies.2.Transcriptomes comparison between SR3 and JN177 under salt stressUsing the microarray,root transcriptomes were compared between SR3 and JN177 under time course salt treatment,and 836 probes were identified to be expressed with significant difference.By analyzing the expression patterns and potential functions of the probes,we found that:Genes related to transport(of metal ions,water and nutrient),antioxidant production, flavonoid biosynthesis,some ABA pathway components and defense,were expressed more in SR3 than in JN177.While genes involved in photosynthesis and defense were expressed less.Based on the responsive patterns of regulatory genes(including transcription factor genes and genes involved in hormone biosynthesis and metabolism) and transporter genes under salt stress,it was found that more of these genes were up-regulated in SR3 than in JN177,and the up-regulation was also greater in SR3.However the down-regulation was more moderate in SR3.This may indicate that the adaptational response is stronger in SR3. Expression of the key genes involved in JA and GA biosynthesis were greatly suppressed and over-expressed respectively in SR3 than in JN177.This suggested the difference of the hormone balance between the two lines.Finally,54 differently expressed probes which represented new ESTs were highlighted. They were all from SR3 cDNA libraries and might be exogenous or mutant genes which resulted from the somatic hybridization.These findings can not only help to reveal the salinity tolerance mechanism of SR3 and provided lots of candidate salt-tolerance related genes,but also confirmed the great impression on transcriptome by somatic hybridization and further indicated the worth of applying this technique on salt tolerant crop breeding.3.Transcriptome analysis under NaCl and PEG treatments with equal osmotic potential revealed the mechanism of early salt responsesTo investigate the mechanism of early salt responses in wheat roots,especially the relationship between osmotic and ionic stress during salt stress,we treated SR3 and JN177 with NaCl and PEG which conferred equal osmotic stress.After analyzing the transcriptome data of microarray,we found that:The early salt responses of wheat root transcriptome were mainly composed of the up-regulation of regulatory genes and the down-regulation of transporter genes.The transcriptome changes under NaCl and PEG treatment shared common in a large extent,but more genes were affected under PEG treatment in comparison with NaCl treatment,and the regulatory genes(genes involved in transcriptional regulation or hormone biosynthesis and metabolism) also changed greater under PEG treatment.Some probes only responded to NaCl but not to PEG and some transporter genes were more suppressed under NaCl treatment than under PEG treatment.These findings confirmed that osmotic stress was the main stress conferred by salt treatment at the earlier stage.It was surprising that osmotic stress itself caused larger transcriptomal changes than the combination of osmotic and ionic stress.This suggests a salt stress specific way to moderate the osmotic impaction.The differently expressed genes may be involved in early salt signal transduction or ionic stress specific signal transduction.4.Cloning and functional analysis of early salt responsive genes TaDi19A and TaDi19B from SR3Based on the differential screening of SSH cDNA library,TaDi19A and TaDi19B were cloned from SR3,which belonged to Di19(DROUGHT INDUCED 19) gene family.TaDi19A was localized in long arm of the chromosome 3B and its protein products mainly presented in nucleus.It was constitutively expressed in both the roots and leaves of wheat seedlings grown under non-stressed conditions,but was substantially up-regulated by the imposition of stress(salinity,drought and cold),or the supply of stress-related hormones (ABA and ethylene).The heterologous over-expression of TaDi19A in Arabidopsis thaliana increased the plants'sensitivity to salinity stress,ABA and mannitol during the germination stage.Root elongation in these transgenic lines showed a reduced tolerance to salinity stress and a reduced sensitivity to ethophon.Flowering was accelerated in the transgenic lines when stressed with H₂O₂.The expression of the ABA signal pathway genes ABI1,RAB18,ERD15 and ABF3,and SOS2(SOS pathway) was altered in transgenic lines.These results suggest that TaDi19A plays a role in the plant's response to abiotic stress,and some possible mechanisms of its action are proposed.For TaDi19B,we analyzed its sequence character,subcellular localization and expression patterns.The heterologous over-expression Arabidopsis transgenic lines were generated.TaDi19B protein was found to be presented in nucleus and was transcriptionally up-regulated by salt,osmotic and cold stress.Its stress response was greater in SR3 than in JN177.This suggests that it may contribute to the salinity tolerance of SR3 and is involved in abiotic stress response.5.Cloning and preliminary functional analysis of TaERD15A and TaERD15B, two stress responsive genes from SR3Also based on the differential screening of SSH cDNA library,TaERD15A and TaERD15B were isolated from SR3.By homology analysis of their protein sequences,it was found that this type of proteins was less conserved among species.The only known homologous gene in Arabidopsis is ERD15A(EARLY RESPONSE TO DEHYDRATION 15), a negative regulator of ABA pathway.Gene structure,subcellular localization,expression patterns of the two genes were analyzed and heterologous over-expression Arabidopsis transgenic lines were generated respectively.TaERD15A localized on the short arm of chromosome 1A and the protein presented in nucleus.It was up-regulated by NaCl and PEG treatments and more expressed in JN177 than in SR3.It also responded to cold but not to exogenous ABA.The salinity tolerance of transgenetic Arabidopsis seedlings was reduced.These results indicate that TaERD15A is a negative regulator of salinity tolerance and functions in an ABA independent manner.Its less expression in SR3 under salt stress may results in the significant salt tolerance.The subcellular localization of TaERD15B was similar to TaERD15A.It was also induced by PEG and cold treatment but not by NaCl treatment.Under PEG treatment,its transcripts accumulated more in JN177 than in SR3.These imply that it is involved in osmotic and cold stress response.