Gene Expression Profile Of TaLEA-introduced Transgenic Populus Simonii×P.nigra Dwf1

Primary symptoms of salt damage caused by salt stress comprise low osmotic potential effect and ion toxicity. Metabolic disorder of plant will occur due to lack of water when the plant absorbs too much salt from the soil. During that process, a series of proteins stored in plant cells will protect the cells from dehydration, among which the LEA protein is the most common one. Though it is believed that the LEA protein plays an important role in reducing dehydration stress, its physiological and biochemical functions and stress resistance mechanism are not clear yet. The TaLEA (DQ663481) gene was introduced into Populus simonii×P. nigra by Agrobacterium tumefaciens-mediated transformation. It was totally obtained 11 independent transgenic lines. Among them, a dwarf mutant (named dwfl) was found. Salt resistance analysis showed that the dwfl plant had stronger salt tolerance than the control group, and mutation symptoms like dwarf, change of leave shape, faster ageing of leave. To further research molecular mechanism causing mutant phenotypes of the dwfl plant and the salt resistance mechanism of the TalEA protein, we had studied the dwfl from the respective of DNA, mRNA and protein.Two flanking sequences of TaLEA gene were cloned from the dwfl by TAIL-PCR. Bioinformatics analysis showed that the two sequences were located in a retroposon and the promoter regions of an unknown gene (homologous with hypothetical protein of grape, XM₀02267539.1) respectively. After amplifying the reverse transcriptase of the retroposon by RT-PCR, the target band was not found in dwfl or wild type, so we supposed that the transposon was inactive. Fluorescence quantitative PCR was used for testing expression of the unknown gene and found that expression of the gene was up-regulated in the dwfl up to 2.22 times the expression of the control group. We presumed that the insertion may affect the expression of the gene. The function of the gene is still unknown. Whether the mutant characters of dwfl are related with the up-regulated expression of the gene shall be determined by further functional verification.Differential transcription expression profiling of the dwfl and wild type control group was carried out by Afrymetrix microarray in non-stress condition. Differential genes were screened by SAM software and 537 differential genes were selected on the basis of q-value (%)<5 and Fold Change>2 or<0.5.280 genes have up-regulated expression and others have down-regulated expression. Real-time fluorescence quantitative PCR verification showed the same results with the microarray chip analysis, thus the microarray chip analysis was reliable. Except 43 genes which have unknown functions or cannot be classified, the differential genes can be classified into 11 groups, including metabolism, stress, protein synthesis and degradation, kinase, cell wall, hormone, signal transduction, transcriptional regulation, transportation, cellular structure and cell fate. Analysis showed that most stress-induced genes had up-regulated expression, such as Osmotin, RD22, chitinase, beta-1,3 glucanase, peroxidase, chalcone synthase, glutathione S-transferase, WRKY, bZIP, AP2/EREBP, NAC, serine/threonine kinase, stress-induced receptor-like kinase, lipid transfer protein and so on. In normal growth condition, high level expression of these stress-induce genes provides the molecular basis for the salt tolerance of dwfl. Moreover, many genes, such as Cullin-1, DWF5, BAK1, AIL5, JAZ10 that were related to hormone synthesis and regulation in the dwfl also expressed significantly differently. These genes are very likely to influence the level of hormone. Hormone change may impact on growth of the plant, causing the mutant phenotypes of dwfl like dwarf.Differential protein expression profiling of the dwfl and wild type control group was carried out by Label-free LC MS/MS in non-stress condition. Differential proteins were screened and 99 differential non-redundant proteins were selected on the basis of Score value>200, q-value (%) <5 and Fold Change>1.3 or<0.75.32 proteins have up-regulated expression and 17 have down-regulated expression.33 proteins Specifically expressed in dwfl and 17 proteins Specifically expressed in WT. Except 43 proteins which have unknown functions or cannot be classified, the differential proteins can be classified into 8 groups, including metabolism, stress, protein synthesis and degradation, transcriptional regulation, cell fate, transportation, cell wall, and cytoskeleton. The results of Label-free LC MS/MS are compared with that of Affymetrix microarray. Analysis finds that mRNA and protein expression abundant of the genes in the glycosis, inositol phosphate metabolism, nitrogen metabolism pathway is similar. While mRNA and protein expression abundant of the genes that are related to signal transduction, transcription factor, hormone, ribosomal protein and translation initiation factor is significantly different. The inconsistency of the transcription and protein expression profiles may provide important clues to the research of post-transcriptional regulation of stress resistance genes.