植物抗旱和耐重金属基因工程研究进展

干旱和重金属污染严重影响植物的生长发育.植物耐逆相关基因的克隆和功能鉴定研究,为通过基因工程途径提高植物的抗逆性奠定了理论基础.水分亏缺、高盐、低温和重金属胁迫都能诱导LEA(late embryogenesis abundant protein)基因的表达.转基因研究表明,LEA蛋白具有抗旱保护作用、离子结合特性以及抗氧化活性;水孔蛋白存在于细胞膜和液泡膜上,在细胞乃至整个植物体水分吸收和运输过程中发挥重要作用.干旱和盐胁迫促进水孔蛋白基因转录物的积累.过量表达水孔蛋白可增强水分吸收和运输,提高植物的抗旱能力.金属转运蛋白参与重金属离子的吸收、运输和累积等过程.这些蛋白基因在改良草坪草植物的抗旱节水和耐重金属能力等方面具有潜在的应用价值.     

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

胚胎发育晚期丰富蛋白(LEA蛋白)与植物抗逆性研究进展

胚胎发育晚期丰富蛋白(LEA蛋白)在自然条件下主要在种子发育晚期大量积累,植物LEA基因也在多种非生物胁迫下诱导表达。植物LEA蛋白是植物应对失水胁迫(包括干旱、盐碱、冷冻等)逆境的一种广泛存在的亲水性应答蛋白,具有很强的热稳定性。本论文就LEA蛋白的结构、分类、功能及抗逆性分子机制进行了概述与总结,为分离新的LEA蛋白成员,进行功能分析以及进一步发掘其潜在应用价值提供参考。     

Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype

The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant–water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Plant dehydrins and stress tolerance: Versatile proteins for complex mechanisms

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.Key words: abiotic stress, dehydration stress, drought, cold acclimation, freezing tolerance, LEA proteins, dehydrins     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

植物水通道蛋白的干旱应答机制研究进展

干旱胁迫是严重影响全球作物生产的非生物胁迫之一,研究植物耐旱机制已成为一个重要领域。水通道蛋白是一类特异、高效转运水及其它小分子底物的膜通道蛋白,在植物中具有丰富的亚型,参与调节植物的水分吸收和运输。近10年来,水通道蛋白在植物不同生理过程中的作用,一直受到研究人员的关注,特别是在非生物胁迫方面,而研究表明水通道蛋白在干旱胁迫下对植物的耐旱性起着至关重要的作用,能维持细胞水分稳态和调控环境胁迫快速响应。水通道蛋白在植物耐旱过程中的调控机制及功能较复杂,而关于其应答机制和不同亚型功能性研究的报道甚少。该文综述了植物水通道蛋白的分类、结构、表达调控和活性调节,分别从植物水通道蛋白响应干旱表达调控机制、水通道蛋白基因表达的时空特异性、水通道蛋白基因的表达与蛋白丰度,水通道蛋白基因的耐旱转化四个方面阐明干旱胁迫下植物水通道蛋白的表达,重点阐述其参与植物干旱胁迫应答的作用机制,并提出水通道蛋白研究的主要方向。     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice

Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice.     

Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza

Salinity and drought are important abiotic stresses limiting plant growth and development. Late embryogenesis abundant (LEA) proteins are a group of proteins associated with tolerance to water-related stress. We previously cloned an LEA gene, SmLEA, from Salvia miltiorrhiza Bunge. Phylogenetic analysis indicated that SmLEA belongs to Group LEA14, which is involved in the dehydration response. To determine its function in detail, we have now overexpressed SmLEA in Escherichia coli and S. miltiorrhiza. The logarithmic increase in accumulations of SmLEA proteins in E. coli occurred earlier under salinity than under standard conditions. SmLEA-transformed S. miltiorrhiza plants also showed faster root elongation and a lower malondialdehyde concentration than the empty vector control plants did when cultured on MS media supplemented with 60 mM NaCl or 150 mM mannitol. Moreover, SmLEA-overexpressing transgenics experienced a less rapid rate of water loss. Under either salinity or drought, overexpressing plants had greater superoxide dismutase activity and a higher glutathione concentration. These results suggest that SmLEA may be useful in efforts to improve drought and salinity tolerance in S. miltiorrhiza. Our data also provide a good foundation for further studies into the stress resistance mechanism and molecular breeding of this valuable medicinal plant.     

非生物胁迫下植物脱水素的研究进展

脱水素是LEA蛋白中的一类,广泛存在于植物的各个组织器官及植物胚胎发育后期.脱水素是植物在受低温、干旱和高盐等非生物逆境胁迫时合成的一类高亲水性保护蛋白,具有保护核酸、胞内蛋白和膜结构免受损害的功能.许多研究已经证实在非生物胁迫下,植物脱水素的表达与积累和植物抗逆性之间存在着紧密的联系.对脱水素的结构、亚细胞定位、基因表达模式及非生物胁迫下脱水素作用的最新研究成果进行了综述.     

Identification and functional roles of CaDIN1 in abscisic acid signaling and drought sensitivity

Plants frequently face challenges caused by various abiotic stresses, including drought, and have evolved defense mechanisms to counteract the deleterious effects of these stresses. The phytohormone abscisic acid (ABA) is involved in signal transduction pathways that mediate defense responses of plants to abiotic stress. Here, we report a new function of the CaDIN1 protein in defense responses to abiotic stress. The CaDIN1 gene was strongly induced in pepper leaves exposed to ABA, NaCl, and drought stresses. CaDIN1 proteins share high sequence homology with other known DIN1 proteins and are localized in chloroplasts. We generated CaDIN1-silenced peppers and overexpressing transgenic Arabidopsis plants and evaluated their response to ABA and drought stress. Virus-induced gene silencing of CaDIN1 in pepper plants conferred enhanced tolerance to drought stress, which was accompanied by low levels of lipid peroxidation in dehydrated leaves. CaDIN1-overexpressing transgenic plants exhibited reduced sensitivity to ABA during seed germination and seedling stages. Transgenic plants were more vulnerable to drought than that by the wild-type plants because of decreased expression of ABA responsive stress-related genes and reduced stomatal closure in response to ABA. Together, these results suggest that CaDIN1 modulates drought sensitivity through ABA-mediated cell signaling.     

Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance

Osmotin or osmotin-like proteins have been shown to be induced in several plant species in response to various types of biotic and abiotic challenges. The protein is generally believed to be involved in protecting the plant against these stresses. Although some understanding of the possible mechanism underlying the defense function of osmotin against biotic stresses is beginning to emerge, its role in abiotic stress response is far from clear. We have transformed cotton plants with a tobacco-osmotin gene, lacking the sequence encoding its 20 amino acid-long, C-terminal vacuolar-sorting motif, under the control of CaMV 35S promoter. Apoplastic secretion of the recombinant protein was confirmed and the plants were evaluated for their ability to tolerate drought conditions. Under polyethylene glycol-mediated water stress, the osmotin-expressing seedlings showed better growth performance. The transformants showed a slower rate of wilting during drought and faster recovery following the termination of dry conditions in a greenhouse setting. During drought, the leaves from transgenic plants had higher relative water content and proline levels, while showing reduced H₂O₂ levels, lipid peroxidation and electrolyte leakage. Importantly, following a series of dry periods, the osmotin transformants performed better in terms of most growth and developmental parameters tested. Most relevant, the fiber yield of transgenic plants did not suffer as much as that of their non-transgenic counterparts under drought conditions. The results provide direct support for a protective role of osmotin in cotton plants experiencing water stress and suggest a possible way to achieve tolerance to drought conditions by means of genetic engineering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of differentially expressed genes in abiotic stress response and their role in signal transduction pathways

The study of abiotic stress response of plants is important because they have to cope with environmental changes to survive. The plant genomes have evolved to meet environmental challenges. Salt, temperature, and drought are the main abiotic stresses. The tolerance and response to stress vary differently in plants. The idea was to analyze the genes showing differential expression under abiotic stresses. There are many pathways connecting the perception of external stimuli to cellular responses. In plants, these pathways play an important role in the transduction of abiotic stresses. In the present study, the gene expression data have been analyzed for their involvement in different steps of signaling pathways. The conserved genes were analyzed for their role in each pathway. The functional annotations of these genes and their response under abiotic stresses in other plant species were also studied. The enzymes of signal pathways, showing similarity with conserved genes, were analyzed for their role in different abiotic stresses. Our findings will help to understand the expression of genes in response to various abiotic stresses. These genes may be used to study the response of different abiotic stresses in other plant species and the molecular basis of stress tolerance.     

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.