Study On The Function Of SlJMJ18 And SlJMJ23 Genes In Tomato Resistance To Heavy Metal Cadmium Stress

Plants will be affected by a variety of abiotic stresses in the process of growth and development,including heavy metal stresses such as cadmium（Cd）,mercury（Hg）and copper（Cu）.Cd as a non-essential and toxic element for plant growth in the soil,which lower content will cause toxic effects on plants.As one of the important vegetable crops,tomato（Lycopersicon esculentum Mill.）has high nutritional value and certain medicinal value.With the continuous expansion of facility production scale in China in recent years,the soil quality of cultivated land has deteriorated seriously,and the massive accumulation of heavy metals in crops has seriously affected food safety and endangered animal and human health.Therefore,it is imminent to study the molecular mechanism of tomato response to cadmium stress and to screen cadmium resistancerelated genes in plants.Histone demethylase（JHDM）is a family of proteins containing only Jmj C domain.In recent years,there are many studies on its participation in abiotic stress（drought,chilling injury,salt stress）,but there are few reports on heavy metal stress.In this paper,the CDS region and promoter region of SlJMJ18 and SlJMJ23 genes were cloned respectively using tomato as the experimental material,and Arabidopsis was genetically transformed to preliminarily explore the role of the gene in tomato response to heavy metal stress.This provides theoretical basis and genetic resources for studying the molecular mechanism of histone demethylase genes（SlJMJs）in tomato resistance to heavy metal stress.The results of this research show that:1.Screening the response genes of JMJs gene family in tomato under Cd stress Related genes of the same subfamily in tomato（9 genes in total）,which based on the reported functions of JMJs family-related genes in Arabidopsis and rice were selected for research.Six genes,SlJMJ2,SlJMJ9,SlJMJ18,SlJMJ20,SlJMJ23 and SlJMJ524,responded to heavy metal cadmium stress were identified by semi-quantitative and realtime PCR.SlJMJ18 and SlJMJ23 genes were finally selected for in-depth study in this paper.2.Cloning and bioinformatics analysis of SlJMJ18 and SlJMJ23 genes The tomato SlJMJ18 and SlJMJ23 genes were obtained by cloning.The sequence analysis showed that the CDS regions of these two genes were 1 134 bp and 1 530 bp in full length,encoding 377 and 509 amino acids respectively.These two proteins have complex tertiary structures,and both contain Jmj C conserved domains,which are in line with the structural features of the Jmj C domain-only gene family.Further phylogenetic tree analysis showed that these two proteins belonged to different subfamilies,and members of the same subfamily were relatively conserved in the sequence and structure of the Jmj C domain.3.Expression analysis of SlJMJ18 and SlJMJ23 genes under abiotic stress The results showed that SlJMJ18 and SlJMJ23 genes were expressed in different degrees in various tissues of tomato.The expression of SlJMJ18 gene was the highest in flowers and SlJMJ23 gene highest in young leaves.Moreover,these two genes are responsive to the stress of heavy metals including Hg,Pb and Cu.In addition,bioinformatics analysis showed that the total length of the promoters of SlJMJ18 gene and SlJMJ23 gene were 2 053 bp and 2 022 bp respectively.The promoter sequences contained a large number of regulatory elements related to abiotic stress（ARE,GCmotif,LTR）,plant hormone response（ABRE,P-box,TGACG-motif）and light response（AE-box,Box4,G-box）.Further GUS staining analysis showed that the expression of SlJMJ18 gene was enhanced under Cd,Hg and Cu stress for 6 h,and the expression of SlJMJ23 gene was induced at different time points under Cd,Hg,Cu,drought,mannitol and salt stress.4.Functional verification of SlJMJ18 and SlJMJ23 transgenic Arabidopsis plants4.1 SlJMJ18 transgenic Arabidopsis plants showed early flowering at seedling stage SlJMJ18-OE showed cadmium tolerance phenotype at germination stage and seedling stage,but showed early flowering phenotype at seedling stage.Through the analysis of hormone（Strigolactone,Brassinolide,Abscisic Acid）related gene expression,it is speculated that the antagonistic effect of Brassinolide and Abscisic Acid in overexpressed plants may be related to the abnormal growth of plants.4.2 SlJMJ23 transgenic Arabidopsis plants have different responses to cadmium at different stages of developmentSlJMJ23-OE showed cadmium sensitive phenotype at germination stage（opposite to SlJMJ18-OE phenotype）,while it showed tolerance to cadmium stress at seedling stage and adult seedling stage.Further research showed that the expression of metal transporter-related genes in SlJMJ23-OE was significantly reduced in the seedling stage,and the hormone content in the body also changed significantly,which may affect the absorption of cadmium by plants to a certain extent.In addition,SlJMJ23-OE may resist cadmium stress by increasing antioxidant capacity and increasing the synthesis of metabolites（total phenols）in the seedling stage.