黑麦草重结晶抑制蛋白基因家族的克隆及其RNAi载体的构建
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摘要
植物冷胁迫相关蛋白是指从耐寒物种中分离得到的参与调节植物抗寒生理生化活动的特殊蛋白质,其中一类能抑制冰晶形成的蛋白质被称为植物抗冻蛋白。植物抗冻蛋白能结合于冰晶表面,阻止冰晶生长,非依数性地降低溶液冰点而不降低熔点,导致熔点与冰点之间出现差值,即表现为热滞(Thermal hysteresis, TH)活性,还可抑制冰晶之间发生重结晶,即重结晶抑制(Recrystallization inhibition, RI)活性。与昆虫和鱼类抗冻蛋白相比较,植物抗冻蛋白的热滞活性极低,却具有极高的重结晶抑制活性,因此,植物抗冻蛋白亦被称为重结晶抑制蛋白(Ice Recrystallization inhibition Protein, IRIP)。不同物种来源的植物重结晶抑制蛋白差异较大,基因序列之间的相似性很低,其作用机理至今未完全解析清楚。多年生黑麦草是耐寒性极强的常绿植物,内含活性较高的IRIP,应用前景较大。为探索黑麦草IRIP的遗传特性,对IRIP基因家族进行了克隆与功能分析研究。本论文的主要研究结果如下：
(1)黑麦草重结晶抑制蛋白基因家族的克隆。根据GenBank公布的黑麦草重结晶抑制蛋白的序列,以黑麦草基因组为模板,分别设计4对特异引物扩增完整基因组DNA序列；将冷诱导黑麦草叶片总RNA逆转录成cDNA第一链,以此为模板,分别设计4对特异引物扩增黑麦草IRIP的cDNA序列。将获得的4条基因组DNA序列和4条cDNA序列分别克隆至T载体,经测序及序列分析,发现IRIP基因家族序列存在着转录后的编辑剪接,令LpIRIPl失去15个碱基的内含子或是5个氨基酸序列,也导致LpIRIP4基因读码框移位,没有终止子。序列比对结果表明,4个成员碱基序列的3’端高度保守,而5’端的保守性很低,序列相似性低。
(2)黑麦草重结晶抑制蛋白基因家族的原核表达。根据所克隆的黑麦草重结晶抑制蛋白的cDNA序列,分别设计4对原核表达引物,扩增出4条目的序列,分别亚克隆至原核表达载体pET-28a和pET-30a,酶切鉴定后转化表达菌株BL32(入DE3),使用终浓度1mM的IPTG进行诱导表达,结果表明,仅第3号成员的表达质粒pET-30a-IRIP3获得成功表达,蛋白质大小为26kDa。
(3)黑麦草重结晶抑制蛋白的双元RNA干扰载体构建。以黑麦草重结晶抑制蛋白基因家族4条序列及黑麦草抗冻蛋白基因LpAFP (AJ277399)的保守区序列作为干扰目的序列,分别以LpAFP, LpIRIP2, LpIRIP3为模板设计引物,扩增得到400bp左右的干扰装载片段,分别以正反向装载至沉默载体中间载体pBlueActin,后亚克隆至双元载体pCAMBIA2301,构建完成黑麦草IRI蛋白基因家族双元沉默载体,为后续通过RNA沉默技术分析黑麦草IRIP基因家族各个成员的功能奠定了基础。
Plant cold-stress associate-proteins are classified as special proteins participate in frost-resistant life events, in which ones in regulation of plant physiological and biochemical activities and against cold environmental stress,it is called antifreeze protein. Antifreeze proteins binding to the surface of ice crystals and prevent ice crystal growth, non-reducing point other than the freezing point,so it creates the gap between the melting point and freezing point, and that is charactered as thermal hysteresis activity.It control the growth of ice crystals and inhibit ice recrystallization, compared with insects and fishes antifreeze proteins, plant antifreeze proteins have lower thermal hysteresis activity, but they have a very high recrystallization inhibition activity, so plant antifreeze proteins also be called recrystallization inhibition proteins in the research.
Recrystallization inhibition activities of plant antifreeze proteins are different among various species, and the homology between different sequences is very low. Although the researchs about ice recrystallization inhibition proteins have done in vitro,and made a number of models to explain its mechanism, but so far still have not been scientifically confirmed. The major works of this paper is about ryegrass because of its special properties and it have heavily attract me,the content are list as follows:
1. The cloning of ryegrass recrystallization inhibition proteins gene family.First,i get the informations about ice recrystallization inhibition Proteins gene family from the Genbank,designed four pairs primers and cloned the special sequences from the DNA templates and cDNAtemplates.According to the analysis of alignment of sequences,we find the LpIRIP sequences may proceeded splicing post of the transcripting,and there maybe exist intron in the inner of IRIP genes.
2. The research about expression of IRIP genes in E.coli. First,we succeed in construction of the four IRIP genes prokaryotic expression vector,subclone the genes coding sequences into pET-28a and pET-30a vectors.Next,after the identification of restriction enzyme digestion, transformed the vectors into the expression cell BL32 (λDE3). At the concentration of 1mM IPTG induced,the recombination pET-30a-IRIP3 successfully express the target protein,it is about 26kDa.
3. The construction of Ryegrass binary silence vector. First, we design special primers according to conserved region by the result of alignment among four IRIP genes sequences and antifreeze protein LpAFP(AJ277399). Next,we clone the slience target sequences, and load the fragments into the RNA interfere intermediate vector pBlueActin, and then transform them into binary vector pCAMBIA2301,made the plant slience binary vectors.And after that works,transfer the binary silencing vector into agrobacterium LBA4404.
(1)黑麦草重结晶抑制蛋白基因家族的克隆。根据GenBank公布的黑麦草重结晶抑制蛋白的序列,以黑麦草基因组为模板,分别设计4对特异引物扩增完整基因组DNA序列；将冷诱导黑麦草叶片总RNA逆转录成cDNA第一链,以此为模板,分别设计4对特异引物扩增黑麦草IRIP的cDNA序列。将获得的4条基因组DNA序列和4条cDNA序列分别克隆至T载体,经测序及序列分析,发现IRIP基因家族序列存在着转录后的编辑剪接,令LpIRIPl失去15个碱基的内含子或是5个氨基酸序列,也导致LpIRIP4基因读码框移位,没有终止子。序列比对结果表明,4个成员碱基序列的3’端高度保守,而5’端的保守性很低,序列相似性低。
(2)黑麦草重结晶抑制蛋白基因家族的原核表达。根据所克隆的黑麦草重结晶抑制蛋白的cDNA序列,分别设计4对原核表达引物,扩增出4条目的序列,分别亚克隆至原核表达载体pET-28a和pET-30a,酶切鉴定后转化表达菌株BL32(入DE3),使用终浓度1mM的IPTG进行诱导表达,结果表明,仅第3号成员的表达质粒pET-30a-IRIP3获得成功表达,蛋白质大小为26kDa。
(3)黑麦草重结晶抑制蛋白的双元RNA干扰载体构建。以黑麦草重结晶抑制蛋白基因家族4条序列及黑麦草抗冻蛋白基因LpAFP (AJ277399)的保守区序列作为干扰目的序列,分别以LpAFP, LpIRIP2, LpIRIP3为模板设计引物,扩增得到400bp左右的干扰装载片段,分别以正反向装载至沉默载体中间载体pBlueActin,后亚克隆至双元载体pCAMBIA2301,构建完成黑麦草IRI蛋白基因家族双元沉默载体,为后续通过RNA沉默技术分析黑麦草IRIP基因家族各个成员的功能奠定了基础。
Plant cold-stress associate-proteins are classified as special proteins participate in frost-resistant life events, in which ones in regulation of plant physiological and biochemical activities and against cold environmental stress,it is called antifreeze protein. Antifreeze proteins binding to the surface of ice crystals and prevent ice crystal growth, non-reducing point other than the freezing point,so it creates the gap between the melting point and freezing point, and that is charactered as thermal hysteresis activity.It control the growth of ice crystals and inhibit ice recrystallization, compared with insects and fishes antifreeze proteins, plant antifreeze proteins have lower thermal hysteresis activity, but they have a very high recrystallization inhibition activity, so plant antifreeze proteins also be called recrystallization inhibition proteins in the research.
Recrystallization inhibition activities of plant antifreeze proteins are different among various species, and the homology between different sequences is very low. Although the researchs about ice recrystallization inhibition proteins have done in vitro,and made a number of models to explain its mechanism, but so far still have not been scientifically confirmed. The major works of this paper is about ryegrass because of its special properties and it have heavily attract me,the content are list as follows:
1. The cloning of ryegrass recrystallization inhibition proteins gene family.First,i get the informations about ice recrystallization inhibition Proteins gene family from the Genbank,designed four pairs primers and cloned the special sequences from the DNA templates and cDNAtemplates.According to the analysis of alignment of sequences,we find the LpIRIP sequences may proceeded splicing post of the transcripting,and there maybe exist intron in the inner of IRIP genes.
2. The research about expression of IRIP genes in E.coli. First,we succeed in construction of the four IRIP genes prokaryotic expression vector,subclone the genes coding sequences into pET-28a and pET-30a vectors.Next,after the identification of restriction enzyme digestion, transformed the vectors into the expression cell BL32 (λDE3). At the concentration of 1mM IPTG induced,the recombination pET-30a-IRIP3 successfully express the target protein,it is about 26kDa.
3. The construction of Ryegrass binary silence vector. First, we design special primers according to conserved region by the result of alignment among four IRIP genes sequences and antifreeze protein LpAFP(AJ277399). Next,we clone the slience target sequences, and load the fragments into the RNA interfere intermediate vector pBlueActin, and then transform them into binary vector pCAMBIA2301,made the plant slience binary vectors.And after that works,transfer the binary silencing vector into agrobacterium LBA4404.
引文
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