Over-expression Of SlJA2 Decreases Heat Tolerance Of Transgenic Tobacco Plants Via Salicylic Acid Pathway

With the increasing global warming,heat stress has become an important factor limiting plant distribution and yield.Heat stress induced a series of physiological and biochemical reactions,including stomatal closure,water loss,inhibition of cell growth and photosynthesis,degradation of photosynthetic pigments,accumulation of reactive oxygen species,increased membrane lipid peroxidation,protein degradation and loss of enzyme activity,leading to severe cell damage,and even cell death.Tomato is thermophilic vegetables,but it is not tolerant to high temperature.In summer and autumn,the abnormal high temperature in some areas seriously inhibits the growth and development of tomato.That how to alleviate the heat stress has become a top priority.Therefore,it is with great theoretical significance and practical value to explore the molecular mechanism of tomato to heat tolerance.NAC transcription factor is one of the largest family of transcription factors.It plays an important role in regulating plant growth and development,organ formation and responses to stress and hormone.About 102 NAC transcription factors were isolated from the tomato genome by bioinformatics analysis.However,the function of most NAC transcription factors has not been investigated.In this study,an NAC transcription factor?SlJA2?was isolated from tomato.Over-expressing SlJA2 transgenic tobacco plants were used to analyze the growth of plants under heat stress and the effect of SlJA2 on high temperature resistance.The main results are as follows:?1?The full-length cDNA of SlJA2 was obtained by PCR reaction using tomato cDNA as a template and using specific primers designed by DNAMAN 6.0 according to the sequence from NCBI?Accession number: NM₀01247043?.The full length of SlJA2 gene was 1567 bp and the open reading frame was 1050 bp encoding 349 amino acids.?2?Transient expression of p1300-SlJA2-GFP fusion protein in tobacco was observed by laser scanning confocal microscopy.The green fluorescence was observed only in the nucleus,indicating that SlJA2 was localized in the nucleus.Transcriptional activation of SlJA2 was detected by yeast two-hybrid system,and that the C-terminal was found to possess transcriptional activation activity,suggestting that SlJA2 functions as a transcription factor.?3?The expression pattern of SlJA2 in tomato was analyzed by real-time quantitative PCR?qRT-PCR?,and the expression of the gene was induced by 4 °C,42 °C,ABA,SA,PEG and MV.The results of tissue-specific expression analysis and GUS histochemical staining showed that the expression level of SlJA2 was the highest in leaves and stems.?4?pBI121-SlJA2 eukaryotic expression vector was constructed,and Agrobacteriummediated leaf disc transformation was used to obtain transgenic tobacco over-expressing SlJA2.The transgenic tobacco was screened by PCR and qRT-PCR.OE-5,OE-7 and OE-18 were selected for further experiment.?5?Under normal conditions,growth of wild-type and transgenic tobacco has no difference.The growth of wild-type plants and transgenic lines was inhibited under heat stress,but transgenic plants were more severe than wild-type plants.The transgenic tobacco plants had lower seed germination rate and fresh weight,higher stomatal aperture and water loss,leading to more severe wilting.Under heat stress,the content of salicylic acid in transgenic tobacco decreased,resulting from the down-regulation of salicylic acid synthesis gene and the up-regulation of salicylic acid degradation gene;the content of abscisic acid in transgenic tobacco was increased because of the up-regulation of abscisic acid synthesis-related genes.Exogenous application of salicylic acid and abscisic acid can delay and alleviate the wilting degree of transgenic tobacco under heat stress.?6?After heat stress,the Pn,Fv/Fm and D1 protein levels of tobacco plants were decreased,with higher decreased degree of transgenic tobacco.Heat stress resulted in decreased chlorophyll content,with lower level in transgenic tobacco.?7?Under heat stress,contents of H₂O₂ and O₂^·- increased significantly,and the accumulation of H₂O₂ and O2·-in transgenic tobacco was higher than that of wild-type tobacco.The activities of antioxidant enzymes such as catalase?CAT?and ascorbate peroxidase?APX?decreased in both transgenic and wild-type plants.However,wild-type tobacco plants maintained relatively higher antioxidant enzyme activities compared with transgenic plants.In addition,the expression levels of genes encoding antioxidant enzymes?NtCAT,NtAPX?were lower in transgenic tobacco than that in wild type tobacco.Compared to wild-type tobacco,MDA content and REL level of transgenic tobacco were higher than those of wild type under heat stress,which indicated that the damage of transgenic tobacco cell membrane was more severe.After treated with high temperature,expression levels of stress-related genes such as high temperature response genes and proline synthetic gene in transgenic tobacco were significantly lower than those in wild-type plants.At the same time,the accumulations of proline and soluble sugar in transgenic tobacco were significantly lower than those in wild type.