【作者】 王艳；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2008， 博士

【摘要】 抗冻蛋白(AFP)是广泛存于各种生命体中的一种高效抗冻活性物质,它能以非依数的形式(冰晶的熔点温度与其生长点温度不具有相同的变化)降低水溶液的冰点而对熔点影响甚微,因而保护多种有机体免受结冰引起的伤害,其抗冻活性通常定义为热滞值,大小用热滞系数表示。比较来源不同的多种抗冻蛋白的热滞活性,昆虫抗冻蛋白的热滞值最高为3～6℃,因而昆虫抗冻蛋白基因目前越来越多地做为提高植物抗寒性转化的首选基因。本实验室从新疆极地荒漠昆虫准噶尔小胸鳖甲中克隆了抗冻蛋白基因MpAFP149 (含信号肽),它编码120个氨基酸残基,分子量大小为12.7 kDa。去除信号肽的成熟肽含有98个氨基酸,理论分子量大小为10.2 kDa。在成熟的蛋白质序列中,一级结构由l2个氨基酸TCTxSxxCxxAx (X表示其它的任意氨基酸残基)的TXT重复序列组成,每隔6个氨基酸残基,就有一个半胱氨酸重复,除了第一和第二个重复序列,其他重复单位中的每两个半胱氨酸形成一个二硫键。本试验中我们构建了含有MpAFP149基因的不同植物表达载体,采用不同的转化方式转化模式植物烟草,试探讨新疆荒漠昆虫抗冻蛋白基因在植物体内的抗寒功能及提高昆虫抗冻蛋白在植物体内表达和热滞活性的几种途径和方法。以高效的烟草核转化体系为平台,构建了含有新疆准噶尔小胸鳖甲抗冻蛋白基因MpAFP149的核单价植物表达载体,通过叶圆盘法转化模式植物烟草,获得了2株转录水平较高的T1代转基因植株T1-5和T1-39。在-1℃处理1 d、2 d和3 d的抗寒实验中,转基因烟草的抗寒能力明显优于野生型烟草。当低温处理1 d时,转基因烟草和对照野生型烟草的离子外渗率都有一定程度的上升但差异不大。当低温处理延长至2 d时,野生型烟草的电导率达到65%,而转基因烟草T1-5和T1-39的相对电导率仅分别为28%和27%。当处理第三天时,相对电导率的增长趋势与第二天相似,野生型烟草、T1-5和T1-39的相对电导率分别为71%,28%和36%。丙二醛是反应植物细胞膜脂过氧化程度的一个指标,它含量的变化可间接反应相对电导率的变化。冷处理前,野生型和转基因烟草的丙二醛差异不大,但随着低温处理时间的延长,野生型烟草的丙二醛含量是转基因烟草丙二醛含量的3倍。结果表明野生型烟草遭受了比转基因烟草更为严重的膜脂氧化伤害。同时丙二醛和相对电导率的含量都随着低温处理的延长而增加,二者之间存在明显的相关性,相关系数为R2=0.9132,这表明相对电导率的提高是由于低温引起的膜脂过氧化造成的。T1代单价转基因烟草(pCAMBIA1302-MpAFP149)在低温下的表型及生理生化结果进一步揭示了准噶尔小胸鳖甲昆虫抗冻蛋白MpAFP149能够赋予植物一定程度的耐寒性。国外学者在体外试验中发现,小分子物质柠檬酸可提高昆虫抗冻蛋白的热滞活性,柠檬酸是抗冻蛋白的增强剂。为了进一步提高抗冻蛋白在植物体内的热滞活性,利用RT-PCR技术扩增了烟草柠檬酸合成酶基因tacs,使其构建至单价植物表达载体pCAMBIA1302-MpAFP149中,最终构建成一个同时含有MpAFP149和烟草柠檬酸合成酶基因tacs的植物双价表达载体pCAMBIA1302-MpAFP149-tacs,转化至农杆菌EHA105中。利用叶圆盘法转化烟草,获得了T1代双价转基因烟草。对T1代单价和双价转基因烟草进行了体外-1℃低温试验,转基因烟草的表型明显优于野生型烟草,但单双价转基因烟草之间的表型差异不大。室温恢复中,转基因烟草可恢复生长并仍能存活,而野生型烟草已造成了不可逆的低温伤害。试验结果表明含有昆虫抗冻蛋白基因MpAFP149的单双价转基因烟草均表现出比野生型烟草较强的抗寒能力,但柠檬酸在植物体内未能协同抗冻蛋白进一步提高双价转基因烟草的抗寒性。由于叶绿体基因工程能有效解决当前转基因植物中外源基因表达量过低的问题,因而构建了抗冻蛋白基因MpAFP149的叶绿体表达载体,转化植物,旨在提高异源蛋白在植物中的表达量,从而进一步提高植物的耐寒性。根据已构建的大豆叶绿体表达载体,设计特异性引物,将昆虫抗冻蛋白基因MpAFP149插入此载体中,利用基因枪轰击法,转化烟草,经500 mg/L壮观霉素的反复多轮筛选获得四株转基因株系。在-1℃抗寒表型实验中,叶绿体型转基因烟草的耐寒性优于野生型烟草,与核转化的T1代单价转基因烟草抗寒性差别不大,推测可能是由于异源基因MpAFP149在烟草叶绿体基因组中同质化程度较低所造成。通过激光扫描共聚焦和免疫胶体金技术显示异源抗冻蛋白MpAFP149主要表达在转基因植物质外体空间的细胞壁上。透射电镜对-1℃连续三天低温处理的细胞器形态学变化进行比较,转基因烟草和野生型烟草的超微结构存在差异,尤其表现在细胞膜、叶绿体和线粒体的膜上。这种超微结构差异可能是由于异源表达的抗冻蛋白与细胞壁间发生相互作用,同时降低了植物细胞的冰点,因而抑制了质外体中冰晶的生长稳定了膜系结构,避免了细胞的冻害,最终提高了转基因烟草细胞的冷耐受性。总之本试验获得的转基因烟草在-1℃抗寒实验中均比野生型烟草表现一定的抗寒能力,说明此昆虫抗冻蛋白基因MpAFP149无需进行密码子优化就可有效地在植物体内进行正确表达,可以做为今后转化作物的抗寒首选基因。该结果为减轻新疆冷敏感经济作物棉花在春季遭受冻害及葡萄冬季掩埋等问题提供了理论依据和应用基础。更多还原

【Abstract】 Antifreeze proteins (AFPs) have been isolated from a variety of organisms and play an important role. AFP causes the freezing point of a solution to be lowered without influence on the melting point in a noncolligative manner by adsorption to the ice surface. The difference between the melting point and the nonequilibrium freezing point can be determined and is termed thermal hysteresis activity (THA). THA is widely used as an indicator of AFPs activity, so AFPs are often referred to as thermal hysteresis proteins (THPs). The THAs among species are quite different and insect AFPs are comparatively high (3–6℃), so AFP genes from insects were widely used for improving the cold tolerance of crops.MpAFP149 gene was isolated from Microdera puntipennis dzungarica, a local beetle in Xinjiang desert region. The cDNA of MpAFP149 encoded a polypeptide of 120 amino acid residues with calculated molecular mass of 12.7 kDa. MpAFP149 polypeptide (minus the signal peptide) was 98 residues with calculated molecular mass of 10.2 kDa and comprised of tandem repeats of 12-aa sequence (TCTxSxxCxxAx) with regularly spaced Cys. The two Cys within each repeat form a disulfide bond with the exception of repeats 1 and 2, which are linked by an additional disulfide bond. In the experiments, different plant expression vectors carrying MpAFP149 gene were constructed and transformed the model plant tobacco by using different transformation methods for researching the function of MpAFP149 responded to cold in plants and also looking for the best ways to improve the AFPs expression and thermal hysteresis in plants.Basing on the effective system of nuclear transformation to tobacco, the construct of MpAFP149 gene with the signal peptide sequence under control of a cauliflower mosiaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens–mediated transformation. Two T1 generation transgenic tobacco lines T1-5 and T1-39 were obtained which indicated the high transcripts in mRNA level. The cold tolerance of transgenic tobacco was obviously better than wild-type tobacco with cold treatment for 1 d, 2 d and 3 d at -1℃. At an initial stage of cold treatment, ion outflow was all little increased and no difference between transgenic and wild-type tobaccos on one day. There was a clear difference in ion leakage rate after two days exposure to -1℃: the electrolyte leakage reached 65% for wild-type tobacco, 28% for T1-5 and 27% for T1-39. On day three, the ion outflow was increased in similar tendency as day two, with 71%, 28%, 36% for wild-type tobacco plants, T1-5 and T1-39 lines, respectively. The increase of relative conductivity was regarded as the cause of the rise in MDA. MDA concentration in wild-type and transgenic tobacco plants did not vary much before the cold treatment, but it increased more than three times for wild-type tobaccos and two times for T1 transgenic plants after 2-3 days cold treatment. These result suggested that the wild-type plants were suffered severer oxidative lipid injury than transgenic tobacco ones. The amount of both ion leakage and MDA clearly increased with prolonged times of cold treatment. A significant and high correlation (R2=0.9132) was observed between MDA content and permeability of cell membranes to ions which suggested that permeability of cold damaged cell membrane increased with the increasing peroxidation of fatty acids. In terms of phenotype experiments, ion leakage and MDA content, a relationship between the expression of MpAFP149 and an improvement in cold resistance in transgenic plants was inferred.Citrate was found as the enhancer of antifreeze proteins and can improve the thermal hysterisis activity of AFP in vitro. Citrate synthase gene (tacs) was amplified from Nicotiana tabacum L. by RT-PCR according to sequence published on Genebank. Another recombinant expression vector pCAMBIA1302-MpAFP149-tacs was constructed and then transformed the tobacco by Agrobacterium tumefacines. At -1℃, transgenic tobacco showed the better cold phenotype than that of wild-type tobacco, but there was no difference between transgenic tobacco containing only MpAFP149 gene and binary transgenic tobacco containing MpAFP149 and tacs gene. When returned to room temperature, MpAFP149 expressed plant overcame dehydration and got recovery completely. However, the wild-type tobaccos displayed severe chlorosis and wilting and suffered inreversible damage to some extent. The result showed that both transgenic tobaccos either containing MpAFP149 or MpAFP149-tacs gene displayed better cold tolerance than wild-type tobacco, but there was no cooperated relationship between MpAFP149 and citrate to further improve the cold tolerance in transgenic tobacco.In order to resolve the low expression of heterologous gene in transgenic plants, MpAFP149 gene was constructed into bean chloroplast vector by designing the specific primers and transformed tobacco by gene-gun. We expected increasing the MpAFP149 expression to further improve the plant cold tolerance finally. Four transgenic tobacco lines were obtained by repetitive screen using 500 mg/L spectinomycin. At -1℃, chloroplast transgenic tobaccos displayed the better cold tolerance than wild-type tobacco, but there was no difference with transgenic tobacco containing MpAFP149 gene by nuclear transformation. This result was not consisted with our expected aim and it was possible that homologous degree of interested gene MpAFP149 was relatively low.The result of laser scaning microscope and immunogold localization showed that MpAFP149 protein uniformly accumulated in the outer layers of cell wall of transgenic tobacco.The ultrastructure difference at -1℃between transgenic and wild-type plants for consecutive three days was compared by the observation of organelles morphology changes. The organelle morphological alterations in leaves of transgenic and wild-type plants, before and after cold treatment at -1℃, were examined by TEM. The observations revealed that there were altered appearance inside the cells after the cold treatment and the changes were prominent in membrane of cellular organelles, especially in cell membrane, chloroplasts and mitochondria. This protection might be attributed to an interaction between the antifreeze peptides and cell wall, which resulted in the prevention of the ice growth in the apoplast, so that the whole cell could avoid freezing. These results suggested that the expression of the heterologous protein MpAFP149 have significant effects on the morphological changes in organelles, mitigate the freezing-induced structural damage in the membrane system and eventually lead to enhancement of freezing tolerance in transgenic tobacco cells.According to above experiment results, we can draw a conclusion that transgenic tobaccos containing MpAFP149 display the better cold tolerance than wild-type tobaccos. It suggested that the MpAFP149 gene did not be optimized according to tobacco codon bias and can be used as the candidate gene for the improvement of frost resistance of commercially important crops.更多还原