Analysis Of SlHSP20 Gene Cluster Characteristics And SlHSP17.6A Gene Function Under Cold Stress In Tomato

In plants,small heat shock proteins(s HSPs)are the most abundant and widely distributed heat shock proteins,and their conservative structural characteristics evolved before the differentiation of bacteria and eukaryotes.s HSPs,as molecular partners,help to inhibit a variety of different cell processes,including protein aggregation,cytoskeletal protein dynamics,cell growth,transcription,and differentiation.In this study,the Sl HSP20 gene in tomato was repeatedly arranged in tandem on the same chromosome and connected in the47.54 Mb region and has high homology,but its 5′-UTR has different cis-acting elements.Co-expression analysis showed that the interaction genes of the gene clusters were also different.By analyzing the expression of the Sl HSP20 gene cluster under low temperature stress,it was found that Sl HSP17.6A has an important effect on tomato response to low temperature stress.The above research results lay the foundation for further analysis of the evolutionary origin and function of tomato s HSPs genes.The main findings are as follows:(1)According to the chromosomal location of members of the tomato Sl HSP20 gene cluster,it can be known that there are four tandem repeats(Sl HSP17.6A,Sl HSP17.6B,Sl HSP17.6C,and Sl HSP17.7A)on the sixth chromosome of tomato to form a gene cluster.The conserved ACD domain of the typical β-sheet structure of Sl HSP20 gene cluster is highly homologous and belongs to parallel homologous gene pairs.The members of the Sl HSP20 gene cluster have co-expression differences during tomato fruit development.While the protein products of the interacting genes that they bind to are different,indicating that different amino acids may cause changes in the spatial configuration of the protein and affect its functional differences.(2)Five conserved motifs in the tomato Sl HSP20 gene cluster were detected by MEME software.The motif types and arrangement order are consistent and the motif contains 9conserved β-sheet structures.Cis-acting element functions were predicted that members of the Sl HSP20 gene cluster were found to play a key role in responding to abiotic and resistance stress.(3)The tomato gene database(http://ted.bti.cornell.edu/)was used to analyze the expression of Sl HSP20 gene cluster in different tissues and fruits at different stages of growth and development.Among them,Sl HSP17.6A was more than the other three gene clusters.There were significant differences in expression between members during fruit development and maturity.(4)Significant differences in the expression levels of tandem Sl HSP20 genes by exogenous application of hormones,indicate that they play an important role in related hormone regulation pathways.Effects of exogenous glucose,fructose,and sucrose treatment on the expression of members of the Sl HSP20 gene cluster in tomato green ripe fruit discs,and gene cluster members showed significantly different gene expression levels in response to sugar.Under low temperature induction,the expression of gene clusters changed significantly.Among the members of the Sl HSP20 gene cluster,Sl HSP17.6A,Sl HSP17.6B,and Sl HSP17.6C played key roles in low temperature stress,but Sl HSP17.7A was significantly inhibited in low temperature stress.(5)In order to further verify the tolerance of the Sl HSP20 gene cluster to low temperature,a VIGS gene silencing vector for Sl HSP20 was constructed.Three strains of p TRV2-Sl HSP17.6A,five strains of p TRV2-Sl HSP17.6B,six strains of p TRV2-Sl HSP17.6C,and six strains of p TRV2-Sl HSP17.7A were obtained through expression analysis.A total of20 VIGS gene-positive plants of 4 members of the gene cluster were subjected to low temperature stress,and the results showed that under 4℃ low temperature stress,malondialdehyde(MDA),free proline,superoxide anion content and cold damage index were significantly higher than in the control plants,the soluble sugar content was significantly suppressed,indicating that the Sl HSP20 gene cluster was tolerant to low temperatures.(6)Further research the low temperature sensitive Sl HSP17.6A gene and stable expression of silenced and overexpression transgenic tomatoes.After confirming the transgene selection,select the overexpressing plant OE18 and silenced plant S12 that are most significantly expressed in the Sl HSP17.6A transgenic line.Fruits were subjected to low temperature treatment at 4℃.The results showed that the contents of malondialdehyde(MDA),free proline and superoxide anion were significantly higher than those of control plants,and the soluble sugar content was significantly inhibited compared to control plants,further illustrating the Sl HSP20 gene cluster.Response to low temperature stress in tomato plants.