甜椒内质网小分子热激蛋白基因(CaHSP22.5)克隆及其在转基因烟草中的表达分析

目的]克隆甜椒内质网小分子热激蛋白基因(CaHSP22.5),并检测其表达特性,为探究其在植物抗逆中的调控机制提供理论依据.方法]克隆CaHSP22.5基因并构建其表达载体pBI121-CaHSP22.5,转化农杆菌LBA4404后通过叶盘法转化野生型烟草,即获得CaHSP22.5转基因株系;以转pBI121空载体的野生型烟草为对照组.对转基因株系和野生型烟草进行4℃6 h低温处理,根据幼苗成活率测定CaHSP22.5基因表达对植株耐寒性的影响.采用脉冲振幅调制叶室荧光仪Li-6400测量4℃处理10 h后野生型植株与CaHSP22.5转基因株系叶绿素荧光,根据非光化学淬灭系数(NPQ)值测定CaHSP22.5基因表达对在低温下植株光合作用的影响;使用过氧化氢(H2O2)钛络合物法对烟草叶片中H2O2进行定量分析,通过测定活性氧(ROS)积累程度分析CaHSP22.5对植物光合系统的影响;采用检测柠檬酸合成酶(CS)活性的方法,在体外通过对变性CS复性的影响测定CaHSP22.5的分子伴侣活性.结果]经低温处理后发现CaHSP22.5转基因株系13号、24号和32号幼苗的平均存活率分别为84%、75%和52%,而野生型烟草的平均存活率为30%,CaHSP22.5转基因株系烟草幼苗成活率菌高于野生型幼苗.低温处理10 h后发现CaHSP22.5转基因植株NPQ较野生型植株显著升高(P<0.05),说明转基因烟草中CaHSP22.5的积累有利于保护光合系统,在低温胁迫下清除ROS.同时发现低温胁迫导致植株ROS产量增加,CaHSP22.5转基因植系与野生型相比ROS积累水平较低.不同浓度的CaHSP22.5均可使变性的CS复性,且其CS活性均高于未添加CaHSP22.5的CS活性.结论]CaHSP22.5蛋白可增强植株的耐寒性及光合作用,在植物体内可降低ROS积累,减轻脂质过氧化,同时具有分子伴侣活性.

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

Objective]To 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.Method]The 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.Result]After 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.Conclusion]CaHSP22.5 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.