Studies On The Drought And Cold Tolerance Functions Of Tomato Transcription Factor Gene PHYTOCLOCK2(SlPCL2)

Abiotic stresses,including drought,high salinity and extreme temperature,seriously hinder crop production.Tomato(Solanumlycopersicum L.),as a worldwide vegetable crop,has high edible and medicinal value,but the yield of tomato is greatly reduced under adversity.Therefore,understanding the physiological and molecular mechanisms of tomato response and stress tolerance is of great value for improving stress-resistant cultivation techniques,genetic and molecular improvement of stress-resistant varieties.At present,many studies have shown that some transcription factors are extremely important to plant growth and development and response to various stresses.Golden2-like(G2-like)transcription factor family plays a role in chloroplast development,fruit quality,biotic stress,abiotic stress,plant senescence and so on.Previous studies on G2-like transcription factors in tomato mainly focused on chloroplast development,while the research on abiotic stress was insufficient.In recent years,there is more and more evidence that biological clock plays a vital role in plants’ resistance to adverse environment.In this study,the biological clock-related gene PHYTOCLOCK2(PCL2)of tomato G2-like family was analyzed by bioinformatics,and the expression pattern of SlPCL2 under various stresses was analyzed.The phenotypic changes of SlPCL2 in wild-type tomato and Benji tobacco were observed and its function in response to drought and cold stress was verified.The results are as follows:(1)Cloning and bioinformatics analysis of SlPCL2 geneSlPCL2 gene is located on tomato chromosome 6.c DNA full-length 921 bp encodes a polypeptide of 306 amino acids.It is predicted that the molecular weight of the peptide is about 34.09 k Da and the isoelectric point is 6.04.In the 3000 bp region upstream of the promoter region of SlPCL2 gene,a large number of hormone,light response and stress-related response elements were predicted,suggesting that SlPCL2 gene may have some regulation after stress.The phylogenetic tree was constructed by multiple sequence alignment of amino acid sequences of tomato,Arabidopsis thaliana,rice,potato,eggplant,cucumber,cotton,duckweed and so on.The results showed that SlPCL2 gene had the highest homology with Panali tomato,the highest homology with potato outside tomato species,and the highest homology with Columbine outside Solanaceae.(2)SlPCL2 gene was induced by many abiotic stresses in tomatoThe expression pattern of SlPCL2 gene under drought(PEG6000),low temperature(4 ℃)and high temperature(38 ℃)stress was analyzed by qRT-PCR,and the expression level of SlPCL2 in leaves was detected at different time points after treatment.The results showed that the expression level of SlPCL2 gene increased after 6 hours of drought,low temperature and high temperature stress.The results showed that the expression of SlPCL2 gene in tomato leaves was induced by drought,low temperature and high temperature stress.(3)Overexpression of SlPCL2 gene affected the growth and development of TomatoThe overexpression vector of SlPCL2 gene controlled by Ca MV35 S strong promoter was constructed,and 35S::SlPCL2 gene was integrated into tomato genome by Agrobacterium tumefaciens-mediated method.Three overexpression lines of SlPCL2 gene were screened by qRT-PCR.Phenotypic observation showed that there were great differences in morphology between the two tomato lines with high relative expression(173.6 × and 53.9 ×)and wild-type tomato plants,such as significantly shorter plant height,short internodes and weak apical dominance.The phenotype of the lines with higher expression level than that of wild type was basically the same as that of wild type.Spraying GA3 on two dwarfing lines did not recover the phenotype,that is,it was insensitive to external application of GA3.The expression levels of genes related to GA biosynthesis pathway and signal transduction pathway were further detected,and the results showed that some genes related to GA synthesis and Sl GID1,the key gene of GA signal transduction pathway,were inhibited in dwarfing plants.Therefore,it is speculated that the main reason for the dwarfing of tomato plants caused by SlPCL2 overexpression may be due to the blockage of GA signal transduction pathway.(4)Overexpression of SlPCL2 gene enhanced the drought and cold tolerance of transgenic tobaccoAs the overexpression of SlPCL2 gene seriously affected the growth and development of tomato plants,SlPCL2 gene was further overexpressed in tobacco to identify its function of stress tolerance.The results showed that the effect of SlPCL2 on the growth and development of transgenic tobacco plants also had a dose effect,that is,the lines with high expression showed dwarfing phenotype,while the lines with relatively high expression were more normal.The tobacco lines with overexpression of SlPCL2 but not seriously affected plant phenotype were treated with drought and low temperature.the results showed that the transgenic tobacco plants with SlPCL2 gene showed stronger drought and cold tolerance than wild-type tobacco plants.The tobacco seeds transformed with SlPCL2 gene were treated with drought.The results showed that SlPCL2 gene could promote the germination of tobacco seeds under drought.