Cloning of endoplasmic reticulum-located small heat shock protein gene(CaHSP22.5)in Capsicum annuum L. and its expression characters in transgenic tobacco

ObjectiveTo clone and analyze small pepper endoplasmic reticulum-located small molecule heat shock protein(sCaHSPs)gene,and at the same time detect its expression characteristics,to provide a theoretical basis for ex-ploring the regulatory mechanism of CaHSP22.5 in plant stress resistance.MethodThe CaHSP22.5 gene was cloned to construct the pBI121-CaHSP22.5 expression vector,transformed into Agrobacterium LBA4404,and the transformed Agrobacterium was used to transform wild-type tobacco by the leaf disc method to construct a CaHSP22.5 transgenic line. At the same time,a single gene expression pBI121 vector transgenic line was constructed as a control group. Different lines of tobacco were treated with low temperature at 4℃and 6 h,and the effect of CaHSP22.5 gene on the cold resis-tance of the plant was determined according to the survival rate of seedlings. After 10 h treatment at 4℃,the chlorophyll fluorescence of wild-type plants and CaHSP22.5 transgenic lines was measured using a fluorescence-modulated leaf cham-ber fluorometer Li-6400. The effect of CaHSP22.5 expression on plant photosynthesis was determined based on non-pho-tochemical quenching coefficient(NPQ)values. Hydrogen peroxide(H2O2)in tobacco leaves was quantified by the titanium (H2O2)complex method. The effect of CaHSP22.5 on plant photosynthetic systems was confirmed by measuring the ac-cumulation of reactive oxygen species(ROS). The molecular chaperone activity of CaHSP22.5 was determined by the ef-fect of denatured citrate synthetase(CS)renaturation in vitro.ResultAfter low temperature treatment,it was found that the average survival rates of CaHSP22.5 transgenic lines tobacco No. 3,No.24 and No.32 seedlings were 84%,75%and 52%,while the average survival rate of wild type tobacco was 30%,and the survival rate of tobacco seedlings of CaHSP22.5 transgenic lines was higher than that of wild-type seedlings. After 10 h of low temperature treatment,the NPQ of CaHSP22.5 transgenic plants was significantly higher than that of wild-type plants(P<0.05),indicating that the accumulation of CaHSP22.5 in transgenic tobacco was beneficial to protect the photosynthetic system and remove active oxygen under low temperature stress. At the same time,after 10 h of low temperature treatment,it was found that the low temperature stress led to an increase in the production of ROS,and the ROS accumulation level of CaHSP22.5 transgenic plants was lower than that of wild type. Different concentrations of CaHSP22.5 could renature the denatured CS,and their CS activities were higher than those without CaHSP22.5.ConclusionCaHSP22.5 protein enhances the cold tolerance and photosynthesis of plants,reduces the accumulation of ROS in plants,reduces excessive peroxidation,and has molecu-lar chaperones active.