Screening And Expression Analysis Of Salicylic Acid (SA） Induced HR-related Genes In Cucumber

Cucumber(Cucumis sativus L.)as a genus Cucurbitaceae which is the major vegetable crops in China. It is susceptible to some pathogen infection so that what causes serious loss to cucumber production. Salicylic acid(SA) as a endogenous signal molecules that can induce hypertensive response(HR), systemic acquired resistance(SAR) to enhance the disease resistance of plants and the expression of several genes related to disease-resistant. This study attempted to treat the leaves of cucumber with different concentrations of salicylic acid. To find the proper concentrations of SA(10mM) that induces HR in cucumber leaves under the same environmental conditions. 10 mM SA was used to treat cucumber leaves in following experiment:1.Identification of cucumber PCD: The four true leaf aged cucumber seedlings were used as experimental materials. 10 mM SA was dropped on the leaves to induced treatment. In situ detection of ROS, Trypan blue staining, PI and FDA staining and TUNEL(chromogenic and fluorescence) assay are used in this study. The results illustrated that 10 mM SA induced PCD and accompanied by the accumulation of ROS and H2O2; to use blade epidermal cells were treated by 0.2% MeJA treatment which were obtained from four true leaf aged cucumber seedlings to determine cucumber leaves can occur PCD under the treament of 0.2% MeJA through TUNEL(fluorescence)assay.2.The SA induced screening of cucumber differentially expressed genes( DEGs):chloroplast is one of the important organelles in plant photosynthesis, also play an important role in plant defense responses and hypersensitive response(HR). Therefore,49 chloroplast genes associated with HR were screened via annotation and analysis of1759 differentially expressed gene(DEGs) which was obtained by Illumina high-throughput sequencing technology, then primers were designed as well. The four true leaf aged cucumber seedlings treated by 10 mM SA were used as experimental materials. Total RNA was extracted and reverse transcription was used to obtain cDNA,49 genes were validated by RT-PCR and 15 genes of them were confirmed. The result suggested the 15 genes were differently expression in plant PCD.3.Expression analysis of SA induced HR-related DEGs in cucumber chloroplast:The result of semi-quantitative RT-PCR expression analysis of 15 genes suggested they were expressed under the treatment of both SA and MeJA. In addition, the majority of them presented similar to the express trend, what indicated that SA and MeJA signal may appear crossing phenomenon and Collaborative work during SA-induced PCD in cucumber. To analyze the expression trend of chloroplast DEGs via Real-time fluorescent quantitative PCR, classifying that it based on different express trend of 15genes:(1) The sharp decline tendency:A2(Betaine aldehyde dehydrogenase 1)(2) The descending firstly than upgrade latter tendency:D6(Protochlorophyllide reductase), F7(2-Succinylbenzoate-Co A ligase), E6(Alpha-glucan water dikinase 1)(3) The upgrade firstly than descending latter tendency:A6(Chlorophyll(ide) b reductase NYC1), E3(Fructose-bisphosphate aldolase 3), E8(Allene oxide cyclase 4),D1(6-Phosphogluconate dehydrogenase), G3(CBS domain-containing protein 1),C3(Sigma factor binding protein 1), A5(Pheophorbide a oxygenase), E7(ATP-dendent zinc metalloprotease FtsH), B3(Thylakoidal processing ptidase 2), A4(Calcium sensing receptor), E4(Nicotinamide adenine dinucleotide transporter 1).