Construction Of CDNA Library Of Tomato Fruit And Screening And Expression Analysis Of SIHSP17.7 Interaction Protein

Small heat shock proteins(s HSPs)are a family of proteins consisting of typical C-terminal,N-terminal and ?-crystals,and their molecular weights are generally around 12~42 k Da.In the transcriptome analysis of tomato fruit transformed with Sl CIF gene in the early stage of the research group,the small heat shock protein 17.7(Sl HSP17.7)was highly expressed in the fruit ripening process of tomato(Solanum lycopersicum),but its mechanism of action in tomato fruit development is not clear.In this study,we constructed a c DNA library of mature tomato fruits,and screened the proteins interacting with Sl HSP17.7 to explore the possible pathways of Sl HSP17.7 in fruit development.The c DNA library of tomato fruit was constructed by using the c DNA of mature-green,breaker and red-ripening fruits of Micro-Tom tomato as template.The p GBKT7-Sl HSP17.7 bait vector was used to screen interaction protein.The interaction protein with Sl HSP17.7 was verified by in vivo bimolecular fluorescence complementation(Bi FC)and in vitro GST-pull down analysis.To further elucidate the molecular mechanism of Sl HSP17.7 regulating the low temperature tolerance of tomato fruit.The main experimental results are as follows:1)We constructed a c DNA library of mature tomato fruits.The storage capacity of tomato fruit c DNA library was greater than 2.2×106cfu,the cell density of working fluid was 3.6×107cell/m L,and the degree of normalization was greater than 106,the results were in accordance with the quality control requirements,so we can carry out subsequent experiments.2)The bait vector of p GBKT7-Sl HSP17.7 was non-toxic and self-activating by transforming into Y2 HGold yeast strains,so we can be used for further screening of libraries.3)Furthermore,the calcium cation exchanger Sl CCX1-like protein was obtained by screening c DNA library,sequencing and sequence alignment analysis.The yeast two-hybrid assay was used to verify the interaction relationship between the Sl HSP17.7 and Sl CCX1-like.4)The full length of Sl CCX1-like was cloned by RT-PCR,and the prokaryotic expression vector containing GST tag was constructed and induced to express.Pull-down experiment and Western Blot analysis together with the previously expressed Sl HSP17.7 showed that the two proteins S1HSP17.7 and Sl CCX1-like could interact directly,which proved that Sl HSP17.7 and Sl CCX1-like existed relation in vitro.5)Four functional vectors were constructed to connect Sl HSP17.7 and Sl CCX1-like into p XCGW and p XNGW vectors,respectively,using bimolecular fluorescence complementary system Bi FC.S1HSP17.7-c CFP and Sl CCX1-like-n YFP,Sl HSP17.7-n YFP and Sl CCX1-like-c CFP were transiently infected with tobacco leaves by Agrobacterium-mediated method.The results showed that the fusion expression vector infected tobacco leaves under confocal microscopy within 36 ~ 48 h,clear green fluorescence was observed in the tobacco leaf infection site,and the fluorescence was distributed on the endoplasmic reticulum membrane,indicating the existence of interaction.6)Transgenic silencing of Sl HSP17.7 and overexpression of tomato fruit as material.Real-time quantitative PCR was used to detect the expression of Sl CCX1-Like in transgenic plants.The results showed that Sl HSP17.7 is a negative regulation of the expression of Sl CCX1-Like.