番茄线粒体和内质网小分子热激蛋白基因的分子克隆

以热激处理的番茄（Lycopersicon esculentum Mill．）花为实验材料,构建了cDNA库,运用ＲＴ－ＰＣＲ方法克隆番茄粒体和内质网小分子热激蛋白cDNA,利用这两个保守区片段为探针,筛选cDNA库,获得线粒体和内质网小分子热激蛋白全序列cDNA。;通过分析线粒体和内质网小分子热激蛋白基因对温度的反应,发现小分子热激蛋白基因在番茄花中的热激应答温度低于它们在叶片中的热激应答温度,并且番茄叶片中的线粒体小分子热激蛋白基因还具有低温应答特性。对线粒体和内质网小分子热激蛋白基因的分子结构特点,小分子热激蛋白基因在番茄花中的特别热激应答温度的调控机理以及线粒体小分子热激蛋白的基因在中片中的低温度应答成因进行了讨论。     

叶绿体小分子量热激蛋白介绍

本文对叶绿体小分子量热激蛋白的研究进行了简要的回顾和总结.叶绿体小分子量热激蛋白是热激蛋白超家族的成员,具有3个特殊的保守区域;当植物遇到热胁迫时,叶绿体小分子量热激蛋白能够保护光合系统Ⅱ和类囊体膜;初步分析了叶绿体小分子量热激蛋白与植物的耐热性和耐冷性关系以及其分子伴侣功能.     

AtsHsp17.6-CⅠ和AtsHsp17.6-CⅡ基因对非生物胁迫的应答研究

小分子热激蛋白是植物受到热胁迫后的主要表达产物之一,与植物细胞耐热有密切关系。该研究发现,拟南芥小分子热激蛋白基因AtsHsp17.6-CⅠ和AtsHsp17.6-CⅡ 除热激之外,重金属离子Ni⁺、Pb²⁺、Cu²⁺、Zn²⁺和Al³⁺均能诱导这2个热激蛋白基因的表达;氧化胁迫和渗透胁迫同样也能诱导它们表达。该研究将由CaMV35S启动子驱动的这2个小分子热激蛋白基因导入拟南芥,RT-PCR分析表明,2个小分子热激蛋白基因在转基因植物中呈现组成型表达。实验结果表明,组成型表达小分子热激蛋白基因AtsHsp17.6-CⅠ的转基因植物表现出对6 μmol·L^-1 Cd²⁺胁迫、0.4% NaCl胁迫的耐受性。研究表明,这2个小分子热激蛋白基因可能参与着多种抗逆途径,推测其能够减轻或抵抗逆境胁迫引起的伤害并对其进行修复。     

过量表达内质网小分子热激蛋白增强番茄的衣霉素抗性

真核细胞内质网腔内未折叠蛋白的过度积累会引起内质网胁迫（ER胁迫）,继而激活未折叠蛋白应答（UPR）信号途径,诱导内质网定位的分子伴侣的大量表达（如BiP和calnexin等）。本工作将CaMV35S启动子驱动的内质网小分子热激蛋白基因（ER-sHSP）导入番茄,发现ER-sHSP的过量表达提高了转基因番茄整株对衣霉素的抗性。衣霉素处理使未转基因番茄中BiP和calnexin基因的表达迅速升高,转基因番茄中这两个基因的表达也有增加,但表达强度明显低于未转基因番茄。说明ER-sHSP能够减轻ER胁迫,并可能参与UPR信号转导途径。     

高温下线粒体小分子热激蛋白对柠檬酸合成酶、线粒体和花粉粒的保护作用

选用pGEX-6P-1表达载体,构建GST与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白.分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效.利用转基因方法,将线粒体小分子热激蛋白基因导入烟草,在CaMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用.此外,转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力.     

高温下线粒体小分子热激蛋白对柠檬酸合成酶、线粒体和花粉粒的保护作用

选用pGEX-6P-1表达载体,构建GSP与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白,分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效,利用转基因方法,将线粒体小分子热激蛋白基因导入烟覃,地．aMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用,此外,。转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力。     

叶绿体小分子量热激蛋白介绍

本文对叶绿体小分子量热激蛋白的研究进行了简要的回顾和总结。叶绿体小分子量热激蛋白是热激蛋白超家族的成员,具有3个特殊的保守区域;当植物遇到热胁迫时,叶绿体小分子量热激蛋白能够保护光合系统Ⅱ和类囊体膜;初步分析了叶绿体小分子量热激蛋白与植物的耐热性和耐冷性关系以及其分子伴侣功能。     

过量表达内质网小分子热激蛋白增强转基因番茄果实的耐冷藏性

利用番茄内质网小分子热激蛋白(ERsHSP)特异性抗体,对番茄果实蛋白进行Western分析,以测定低温冷藏下番茄果实中ERsHSP的表达量,并测定果实硬度、腐烂度和失重率等指标,以比较中蔬4号转ERsHSP基因番茄和未转基因番茄果实在4℃低温下的耐冷藏性.结果表明:在4℃冷藏30 d期间,转基因番茄果实具有较高的ERsHSP表达水平,而未转基因番茄果实中没有ERsHSP的表达;相对于未转基因番茄果实,转基因番茄果实冷害症状轻,并具有较高的果实硬度(平均值为2.84 kg/cm2)、较低的果实腐烂度(平均值为21.03%)和失重率(平均值为6.33%).     

番茄LeHsp110/ClpB基因的分子克隆及其对植物耐热性的影响

HSP100ClpB是Clp蛋白家族的一员,具有分子伴侣功能,与细胞“获得耐热性(acquiredthermotolerance)”相关。从番茄cDNA文库中筛选到长度达3144bp的cDNA,依据最长的开放读码框推导出的多肽含980个氨基酸残基,分子进化分析结果表明该蛋白属于HSP100ClpB家族,因其计算分子量为110kD,所以命名为LeHSP110ClpB。实验证明,LeHsp110ClpB在番茄叶片中没有组成型表达,为热诱导型基因,其编码蛋白定位于叶绿体基质。利用农杆菌介导法,将CaMV35S驱动的反义LeHsp110ClpBcDNA片段导入番茄,高温下转反义基因的番茄株系中LeHsp110ClpBmRNA水平明显低于对照,转基因株系的PSⅡ对高温胁迫更加敏感,说明HSP110ClpB在植物耐热性方面起重要作用。     

番茄LeHsp110/ClpB基因的分子克隆及其对植物耐热性的影响

HSP100/ClpB是Clp蛋白家族的一员,具有分子伴侣功能,与细胞“获得耐热性(acquired thermotolerance)"相关。从番茄cDNA文库中筛选到长度达3144 bp的cDNA,依据最长的开放读码框推导出的多肽含980个氨基酸残基,分子进化分析结果表明该蛋白属于HSP100/ClpB家族,因其计算分子量为110 kD,所以命名为LeHSP110/ClpB。实验证明,LeHsp110/ClpB在番茄叶片中没有组成型表达,为热诱导型基因,其编码蛋白定位于叶绿体基质。利用农杆菌介导法,将CaMV35S驱动的反义LeHsp110/ClpB cDNA片段导入番茄,高温下转反义基因的番茄株系中LeHsp110/ClpB mRNA水平明显低于对照,转基因株系的PSⅡ对高温胁迫更加敏感,说明HSP110/ClpB在植物耐热性方面起重要作用。     

组成型表达的ClpB基因提高番茄植株的耐冷性

用农杆菌介导法将CaMV35S启动子驱动的ClpB cDNA导入番茄,并比较了转基因和未转基因番茄的抗冷能力。当受冷胁迫后,转基因番茄比未转基因番茄表现出较轻的冷胁迫症状,并维持较高的PSII水平。     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.