The Functional Identification Of The Trehalose-6-phosphate Synthasell And Three Late Embryogenesis Abundant Genes

Plants are sessile organisms and hence they cannot moving to escape from unfavourable environmental conditions, such as high salinity, drought, water stress and high or low temperature. These biotic and abiotic stresses not only harmful for the growth and development of plants, but also threaten the quantity and quality of the crops. During the long evolutionary history, plants have evolved finely tuned signaling and resistance mechanisms. LEA proteins and trehalose play important roles to protect plants from stresses.Based on the Solex transcriptional profile, we isolated and identified one GhTPS gene and three GhLEA genes, named GhTPSl1, GhDHN, GhLEA₁8and GhLEA₄, respectively. We used quantitative RT-PCR and the reverse-genetic method to study the expression pattern and biological function of the four genes. The results are as following:（1） Sequence alignment of the amino acids showed that the GhTPS11had high homology with the TPS and TPP domain, and had high homeology with the AtTPS11. The three GhLEA showed high homeology with the Dehydrin, PvLEA18and LEA₄, respectively. So they were named GhDHN, GhLEA18and GhLEA₄, respectively.（2） Results of quantitative RT-PCR showed that the GhTPSll, GhLEA₁8, GhLEA₄were expressed in roots and leaves, with higher transcripts in leaves. But the GhDHN was only detected in roots. Under abiotic stresses, the expressions of the GhTPS11and three GhLEAs were induced by different stimuli, such as GA treatment, heat stress, cold stress and PEG treatment. High salinity and PEG treatment induced the expression of GhTPS11and GhLEAs significantly. In addition, the four genes could be induced by ABA treatment, suggesting that GhTPS11, GhDHN, GhLEA₁8and GhLEA₄might play important roles in high salinity and osmotic responses and probably related with ABA signaling. （3） When GhTPSll, GhDHN, GhLEA₁8and GhLEA₄overexpressed in Arabidopsis seedlings of the overepression lines did not show observed different phenotypes after high salinity, PEG and ABA treatment. Only under cold （4℃） treatment, the seeds of OEGhTPS11were germinated slower than the wild type （WT）, suggesting that GhTPS11overespresion could lead to chilling sensitivity of Arabidopsis during seed germinatin.（4） By analysis of cold related genes between the OEGhTPS11lines and WT, we found that the overexpression of GhTPS11increased the expression of CBF1and CBF2, while decreased the expression of ICE1and CBF3. These data indicated that sensitivity of OEGhTPS11to cold might dependent on the ICE1-CBF signaling pathway.（5） Further analysis showed that the contents of the T6P and trehalose in OEGhTPS11lines were higher than those in WT, indicating that T6P and trehalose might be the reasons that caused the different gene expression in OEGhTPSll lines and WT.（6） In addition, we also cloned the promoter of the GhDHN by the TAIL-PCR, and obtained a1679bp fragment. Analysis using the PLANTCARE promoter element tool, indicated that the GhDHN promoter contained many abiotic-stress responsive elements Together its root specific and abiotic induced expresseion patterns, we thought that the GhDHN promoter could be used as a specific inductive promoter in gene engineering.