Studies On Function Of Small Heat Shock Protein CI-HSP17.7 In Tomato Under Low Temperature Stress

Tomato(Solanum lycopersicum)is one of the most important economic crops in the world.It is popular of the multi-layered taste and rich nutrients.However,as a thermophilic solanaceous vegetable,when tomato is suffering from low temperature stress,the stress will decrease its quality and production,then it will bring growers an immeasurable economic loss.Small heat shock proteins(s HSP)are a group of the proteins which can protect plants when they are subjected to biotic or abiotic stresses such as high temperature,low temperature,saline,and mechanical damage.s HSPs not only have the molecular chaperone property of protecting cells under the stress of heat stress,but also play an important role in fruit development during cold storage and recoverying after cold stress,but the exact role of s HSPs in fruit development and the molecular mechanism of cold tolerance are still not clear.In order to clarify the function of CI-HSP17.7 gene under low temperature stress,this study is aimed to analyze the expression characteristics of CI-HSP17.7 in the mature stage of low temper ature resistant tomato Chmeilewskil and low temperature sensitive tomato Moneymaker.And further construct the RNA interference vector and overexpression vector of the CI-HSP17.7gene for genetic transformation and phenotypic identification of the transgenic plants.The main findings are as follows:1)Real-time quantitative PCR is used to analyze the different expression of Cl-HSP17.7 in two sugar-accumulating tomatoes.The results show that the expression level is the highest in the chmielewskii tomato green ripening period and Moneymaker tomato coloring period2)The PCR product is ligated with the intermediate vector p ENTR/D-TOPO using Gateway technology to form p ENTR/D-TOPO-CI-HSP17.7 vector.After the detection,the p ENTR/D-TOPO-CI-HSP17.7 vector is reacted with the final vector p B7GWIWG2(I)for LR reaction.The detection is successful in E.coli and Agrobacterium transformation.After sequencing,the CI-HSP17.7 silencing vector is obtained.A silent Agrobacterium recombin-ation genetic transformation system is established by Agrobacterium-mediated leaf disc-transformation,and 70 positive transgenic lines are obtained.The overexpression vector p CAMBIA3301-CI-HSP17.7 isconstructed using restriction endonucleases Bg III and Bst EII,followed by Agrobacterium transformation.A cotton Agrobacterium tomato over-expression genetic transformation system is established by Agrobacterium-mediated leaf disctransformation method,and 55 positive transgenic plants are obtained.3)Thirty transgenic T0 wild-type kemelus green ripening fruits and normal plant green ripe fruits are taken,and the transverse diameter and longitudinal diameter of the fruit aremeasured.The transverse diameter,longitudinal diameter and weight of the transgenic fruits are compared with the control group.There is a significant decrease,while the fruit of the transgenic Moneymaker tomato red ripening period increased slightly compared with the control group in terms of transverse diameter,longitudinal diameter,and weight.4)By analyzing the expression of CI-HSP17.7 gene in the two groups under low temperature treatment,it can be seen that when the normal fruit encounters low temperature stress,the content of CI-HSP17.7 gene in the fruit will increase and there will be an accumulation trend with the increase of treatment time.The expression of CI-HSP17.7 gene in the fruit of L.Chmeilewskil tomato was not only lower than that of normal fruit,but also the expression level of small amount changed little when encountering low temperature,while the expression level of CI-HSP17.7 gene in overexpressed Moneymake tomato fruit was higher than that of normal fruit.The trend of expression accumulation is more obvious after encountering low temperature treatment.This indicates that low temperature can induce the accumulation of the CI-HSP17.7 gene.5)After low temperature treatment,the content of three soluble sugars in normal fruits changed significantly,and the content of sucrose increased significantly,while the content of fructose decreased significantly,indicating that Kremuzki tomato resists low temperature stress by accumulating sucrose.The content of three soluble sugars in transgenic silenced lines was significantly lower than that in normal fruits.After transgenic treatment,the content of three soluble sugars was significantly lower than that of normal fruits,and the change trend was not obvious.There was no accumulation process of sucrose.This indicates that the transgenic silenced tomato has a reduced cold tolerance.The content of soluble sugar in over-expressed transgenic tomato was higher than that in normal tomato in control group.Under low temperature treatment,the content of fructose and glucose in normal tomato fruit increased and the content of sucrose decreased,which indicated that Moneymaker tomato resisted low temperature stress by accumulating fructose and glucose.For the transgenic tomato,the three soluble sugars contained more than normal fruits,and the accumulation of fructose and glucose was more obvious,which indicated that the transgenic Monermaker tomato had increased cold tolerance.By analyzing the changes of sugar content in two groups of transgenic tomato under low temperature stress,it can be explained that CI-HSP17.7 gene has a response to low temperature stress,and CI-HSP17.7 gene can improve the cold tolerance of fruit.