Cloning, Expression And Functional Analysis Of The SlWHY2 In Tomato

In the whole process of growth, plants will be affected by various kinds of environmrntal stimuli, such as drought, salinity and high temperature, and so on. In the process of long-term evolution, plants have formed their own defe nse mechanisms against various biotic stresses and abiotic stresses. Drought stress, as a common abiotic stress in the north of C hina, can affect the growth and development of plants, and lead to the changes of morphology, physiology and chemistry. Disease damage is one of the factors that affect the normal growth of tomato. WHIRLY transcription factor, as a class of plant specific transcription factor, can respond to the stimulation of drought and disease, regulate the encoding levels of different antioxidant enzyme genes and expression levels of stress-related genes and defense-related genes to enhance the resistance of plants to drought and pathogen.In this experiment, we cloned a WHIRLY transcription factor Sl WHY2 from tomato. O ur objective is mainly to explore the function and potential mechanisms of Sl WHY2 gene in reactive oxygen species?ROS? scavenging under drought stress and in pathogen defense response. The results are as follows:?1? We isolated the sequence m RN A of Sl WHY2 gene according to NCBI and the Gen Bank accession number is XM₀10314783. The full length m RNA of Sl WHY2 is 1162 bp, and the open reading frame?ORF? is 726 bp encoding 241 amino acids.?2? The expression of Sl WHY2 in tomato by q RT-PCR showed that the expression level of Sl WHY2 gene in leaf was higher than that in other tissues. The m RNA expression levels of Sl WHY2 was induced by PEG, Na C l, SA and H2O2. In addition, the m RNA expression level of Sl WHY2 was also induced by bacterial pathogen.?3? p BI121 vector of Sl WHY2 was constructed. Sl WHY2 overexpression tobacco plants were screened by PCR and q RT-PCR, and three overexperssion lines?OE-1, OE-3, OE-5? with diffent expression levels were choosed for subsequent experiment.?4? Under normal growth conditions, there were no significant difference between the wild-type?WT? and OE tobacco plants. Under drought conditions, the growth of OE lines were better than that of WT plants. The seedling of WT and Sl WHY2 overexpression plants were staining by DAB and NBT. The results showed that leaf colour of WT was deep than that of transgenic plant after drought treatment. And the contents of H2O2 and O2?- showed that the accumulation of ROS in WT plants were higher than that in the OE lines. The activitises of antioxidant enzymes and the expression levels of related genes in O E lines were higher than that in WT. These results indicated that the overexpression of Sl WHY2 increased antioxidant capacity, reduced the oxidation toxic to plants and enhanced the drought resistance of tobacco.?5? Relative water content?RWC?, relative electrolyte leakage?REL?, MDA content and free proline content of O E lines and WT leaves were determinated after drought treatment. The results showed that the REL and MDA content in WT were significantly higher than that in OE lines, while the RWC and free proline content in WT were lower than that in the OE lines, which showed that the over-expression of Sl WHY2 can improve the ability of resisting osmotic stress.?6? OE lines and WT were treated by injecting and spraying of Pseudomonas solanacearum, repectively. DAB and trypan blue staining showed that H2O2 accumulation and the cell death number in WT was much more than that in O E lines. The transcript levels of known defense-related genes?Nt PR1 and Nt PR2? were nvestigated by q RT-PCR. Results showed that transgenic plants enhanced the resistance to P. solanacearum infection by SA dependent pathway.