Functional Analysis Of Small Heat Shock Protein SlHSP17.7 In Tomato Fruits Responses To Low Temperature Stress

Tomato(Solanum lycopersicum)has been widely cultivated in the open fields and the greenhouses in the northern part of China.Tomato is popular with its sweet and juicy taste and the nutritional value.During the ripening process,the quality of tomato fruits is easily affected by many factors,especially the low temperature stress.In this process,many complex physiological and biochemical changes and gene expression are involved.By exploring the expression of stress-responsive genes,important imformation for cultivation of tomato with low temperature tolerance will be provided.As a cell wall invertase inhibitor gene,SlCIF1 can regulate cell wall invertase activity at the post-translational level,thereby accumulating sucrose in tomato fruits.In the present study,the mature-green,red-ripening transgenic-SlCIF1 and Micro-Tom tomatoes were used as the experimental materials.The expression of a small heat shock protein 17.7(SlHSP17.7)gene was upregulated and was screened in the transgenic mature-green fruits based on the microarray expression profile.As a chaperone,small heat shock proteins(s HSPs)gene family play important roles in plant growth and in response to abiotic stress.In this study,the bioinformatics analysis of small heat shock protein gene family members,localization of SlHSP17.7 gene and the response of SlHSP17.7 gene to low temperature stress in two different low temperature tolerance tomatoes were investigated.It will lay a solid foundation for further study of SlHSP17.7 gene function.The main results were shown as follows:1.Three T3 transgenic lines of transgenic-SlCIF1 were identified in tomato based on hygromycin resistance genes.After analysis of the three lines of tomato fruits during mature-green and red-ripening stages,the expression level of SlCIF1 gene increased about 6-fold in transgenic line 31 tomato red-ripening fruits according to quantitative real-time PCR analysis.The cell wall invertase CWINs gene expression,activity,and sugar content were determined in transgenic line 31.The expression of CWINs genes Lin5,Lin6,Lin7 and Lin8 was no significant changed,the CWIN activity of TG and TR decreased by approximately 8.17% and 10.5%,respectively,sucrose content increased,while,fructose and glucose contents decreased in transgenic-SlCIF1 tomato.The result showed that the expression level of SlCIF1 gene could inhibit CWIN activities at post-translational and affect sugar content in tomato fruit.2.The mature-green and red-ripening fruits of transgenic tomato were used for microarray analysis.The result showed that 622 and 883 differentially expressed genes was changed in the mature-green and red-ripening fruits of transgenic-SlCIF1 gene,respectively.In addition,a small heat shock protein 17.7(SlHSP17.7)gene was screened in transgenic mature-green fruits which expression quantity was increased by thousands of times.GO and KEGG analysis showed that 5 ethylene synthesis related genes and 2 small heat shock proteins were correlated with tomato fruit ripening compared with non-transgenic tomato fruit.In addition,the expression of 5 WRKY transcription factors,3 b HLH transcription factors and 5 plant hormone response transcriptions were up-regulated.In TR-CR,the glycometabolism related differentially expressed genes Sl SUT2 and Sl SWEET4 were up-regulated by approximately 2.5-fold and 5.6-fold,respectively.3.Based on the tomato gene database and bioinformatics analysis,at least 43 members of the tomato s HSPs gene family were identified which were distributed unevenly on 12 chromosomes of tomato.The s HSPs of tomato,pepper and potato had homologous relationship.Sequence analysis of s HSPs promoter sequence revealed a number of cis-acting elements involved in plant glycometabolism and stress,including SURE and W-box.Based on the analysis of two tomatoes(Heinz1706 and pimpinellifolium)in the RNA-Seq database,it was found that most of the members of the family were highly expressed during fruit development and relatively low in other tissues.These results provided a scientific basis for the identification of the evolutionary origin and function of tomato s HSPs gene.4.SlHSP17.7 gene mainly located in midrib pulp,pericarp,pectinic placenta and dissepiment in tomato fruits by histochemical localization.Subcellular localization analysis showed that SlHSP17.7 gene was mainly located in the cytoplasm and nucleus of onion epidermal cells and same results were found in tobacco leaves.However,SlHSP17.7 gene was found only localized in the cytoplasm of root epidermal cells of Arabidopsis thaliana.5.The low temperature tolerance-chmielewskii and sensitive-moneymaker tomato fruits were treaeted under low temperature of 4 ?.The expression of SlHSP17.7 was determined at transcription and translation level.The results showed that the expression of SlHSP17.7 had significant changes in the process of response to low temperature,meanwhile,the soluble sugar content also showed significant changes.The results of Trypan blue and reactive oxygen species staining of tomato leaves overexpressing SlHSP17.7 gene revealed that three overexpressing lines exhibited a small number of blue spots compared to non-transgenic lines.It was indicated that overexpression of SlHSP17.7 gene can alleviate the damage of tomato leaves at low temperature.Above results demonstrated that SlHSP17.7 may regulate post-harvest tomato fruit soluble sugar content changes,maintain homeostasis of reactive oxygen species and inhibit programmed cell death.Then,it can further enhance the cold tolerance of tomato fruit which provides theoretical basis for cultivation of low temperature tolerance tomato fruits.