Response And Function Of Lycopersicon Esculentum Mill.SlSGR2 Gene Under Cadmium Stress

Tomato（Lycopersicon esculentum Mill.）is one of the most important economic crops in China,which is rich in various nutrients.At present,nearly one fifth of the arable land in China is seriously polluted by cadmium（Cd）,which seriously affects crop yield and quality and safety.Meanwhile,Cd stress can cause severe damage to the chloroplast structure and function of plants.SGR2 is a stagnant green-related gene,which has functions of regulating senescence,responding to abiotic stress,regulating seed and fruit maturity.In this experiment,the tomato cultivar"Hong Luocheng"was used as the test material.The SlSGR2 gene was cloned and analyzed by bioinformatics.The GFP subcellular localization vector was constructed to transform Arabidopsis protoplasts under a laser confocal microscope.The subcellular localization of SlSGR2protein was carried out;the expression pattern of SlSGR2 gene was analyzed by treating tomato with Cd;the p CAMBIA2300 overexpression vector was constructed;and Agrobacterium-mediated genetic transformation was used to infect Arabidopsis to obtain SlSGR2 transgenic Arabidopsis Simultaneously,sgr2 Arabidopsis mutants were identified by three primers.The phenotypes of wild-type,over-expressed,mutant Arabidopsis strains under Cd stress were observed.It was the degradation process of tomato SlSGR2 gene under Cd stress.Role in providing theoretical support.The main experimental results are as follows:（1）CdCl₂ solution with 0～100μM concentration was used to treat tomato five-leaf stage plants and observe the phenotypic changes of tomato leaves.The test results show that with the increase of treatment time,the yellowing degree of tomato leaves gradually deepens;the greater the treatment concentration,the greater the degree of yellowing.The above phenotype was more obvious on the fifth day treatment group.Observation of the submicroscopic structure of the third and fourth leaves of tomato on the fifth day of treatment showed that with the increase of Cd concentration,the degree of chloroplast structure was gradually increased.It indicated that cadmium stress caused yellowing of tomato leaves and caused damage to chloroplast structure.（2）The gene coding region of SlSGR2 gene from tomato was obtained by gene cloning technology,with a total length of 819bp and encoding 272 amino acids.Analysis of the physicochemical properties of the protein showed that the protein was an unstable hydrophilic protein,showing weak basicity;analysis of the secondary and tertiary structure of the protein showed a large number ofα-helixes and irregular coils;amino acid sequence alignment showed the highest similarity to the SlSGR2protein It is the potato SGR protein.The phylogenetic tree indicates that the protein belongs to the monocotyledonous subfamily and belongs to the same branch as the potato St SGR protein.Conservative domain analysis shows that the N-142^Phe to N-600^Ala are the conserved regions of the SlSGR2 protein,belonging to the Stay-Green superfamily;plotting the visual logo shows that the N-terminus andC-terminus of each subfamily motif are well conserved;analysis of the 2500bp promoter region upstream of the SlSGR2 gene reveals that it has two cores,TATA-box andCAAT-box Promoter elements,light-responsive elements,elements involved in anaerobic breathing elements,gibberellin and abscisic acid,and TC-rich repeats cis elements involved in stress and stress.（3）Construction of the p CAMBIA1302-SlSGR2 green fluorescent protein GFP fusion expression vector.Protoplasts were selected from Arabidopsis grown in unextracted under short daylight,and the empty vector and the recombinant vector plasmid were respectively transformed into protoplast cells,with the empty vector as a control The position of SlSGR2 gene was observed under a laser confocal microscope.The empty vector was found to be expressed in all tissues,and the SlSGR2 gene mapping coincided with the spontaneous red light of the chloroplast,indicating that the SlSGR2 gene was mapped to the chloroplast.（4）CdCl₂ solution with 0～100μM concentration was used to stress tomato growing to the fifth leaf stage,and the roots and leaves of the tomatoes were collected at 0h,3h,6h,9h,and 12h after the treatment,respectively.q RT-PCR showed that the SlSGR2 gene responded to different concentrations of cadmium.Among them,tomato roots and leaves responded significantly to 50μM CdCl₂,indicating that the SlSGR2gene responded to cadmium stress.（5）Construction of overexpression vector p CAMBIA2300 of the SlSGR2 gene,infection of wild-type Arabidopsis after transformation with Agrobacterium,and identification by PCR and q RT-PCR,T₃ generation transgenic lines OE4-1 and OE4-2were obtained.Eight sgr2 mutant lines were identified by the three primer method.Under Cd stress,the growth of overexpressed lines was significantly better than that of WT and sgr2;the WT and sgr2 died or the leaves yellowed and whitened.The physiological indicators of Arabidopsis after stress showed that the chlorophyll content of the overexpressed lines was higher than that of WT and sgr2,while the MDA content was on the contrary side.This indicates that SGR2 gene responds to stress-induced plant senescence and plays negative role in chlorophyll degradation.