大麦Dhn6基因的克隆、蛋白质结构预测与干旱胁迫表达模式

脱水素(Dehydrins,DHNs)是高等植物胚胎发育晚期产生的一类特异多肽,其表达累积程度与植物的发育阶段、低温、ABA和脱水信号调节等因素密切相关。为了解脱水素的结构与干旱胁迫表达累积反应,文章从六棱大麦分离到序列全长为1 767 bp的Dhn6基因,序列分析结果表明,该基因含一个92 bp内含子,90~1 759 bp为一个开放阅读框,与裸大麦Dhn6基因(GenBank登录号:AF043091)的同源性最高,达93.18%,编码523个氨基酸残基的多肽,预测蛋白质的分子量为49.68 kDa,理论等电点为8.04。结构分析发现,蛋白质具有3个螺旋区,无规则卷曲构成二级结构的主要组分,亲水氨基酸比例超过83%;三维结构预测发现,多肽链自身反向平行排列成松散的亲水索链,K-片段参与兼性?-螺旋结构域的形成,意味着该脱水素具有束缚自由水、稳定细胞膜相结构的功能。实时定量RT-PCR检测结果表明,Dhn6基因的相对表达水平在干旱处理8 h快速累积,推测DHN6在大麦对干旱胁迫的早期响应中发挥重要功能。     

LEA蛋白与植物抗逆性

植物受到逆境胁迫后,LEA蛋白大量表达,可以减轻逆境引起的伤害。本文对LEA蛋白的种类、特性和功能,LEA蛋白基因结构及其表达调控,以及LEA基因表达和LEA蛋白积累与植物抗逆性的关系等方面的研究进展作了简要综述。     

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation-tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11-mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti-aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

Black Point is associated with reduced levels of stress, disease- and defence-related proteins in wheat grain

Black Point in wheat is a dark discoloration at the embryo end of the grain, which causes substantial financial losses to wheat growers due to down-grading of otherwise high-grade wheat. There does not appear to be a single cause for Black Point, although evidence suggests that fungal infection is the main link to Black Point symptoms. We sought to identify grain proteins from Black Point-affected and Black Point-free wheat cultivar SUN239V, which is known to be very susceptible to Black Point. The proteomes of both the germ and endosperm-bran components of Black Point-affected and Black Point-free grain were compared using two-dimensional gel electrophoresis (2-DE) with six replicate gels run for each protein sample. Approximately 1478 discrete protein spots were found in 2-DE gels from the germ fraction of the grain, of which 354 were identified by mass spectrometry (MS). Similarly, 1360 discrete protein spots were found from the endosperm-bran fraction, of which 303 were identified by MS. No proteins of fungal or bacterial origin were positively identified, suggesting that, at least in some cases, Black Point is not associated with microbial activity. Of the germ proteins, 252 were differentially expressed in Black Point-affected tissue, with 67 of these proteins identified by MS. Of the endosperm-bran proteins, 317 were differentially expressed in Black Point-affected tissue, with 86 identified. The largest of 12 functional classes to which the differentially abundant proteins were assigned was the 'stress' class, i.e. products of genes associated with stress, disease and defence. Higher levels of these proteins were found in Black Point-free grain, suggesting that protection from the disease might be afforded by increased levels of the 'stress' proteins.     

Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.     

Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA‐I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine‐containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

小麦TaLEA1基因的表达特征及抗逆功能验证

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,因此耐盐基因挖掘对作物耐盐育种非常重要。许多研究表明胚胎发育晚期丰富蛋白(LEA)在植物应对非生物胁迫中发挥积极作用。本研究以小麦TaLEA1基因为研究对象,分析了其表达蛋白的理化性质及基因表达模式,并通过在拟南芥中过表达,分析Ta LEA1基因的抗逆功能。结果表明,TaLEA1基因的表达蛋白属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。Ta LEA1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TaLEA1基因,显著提高了盐胁迫下转基因拟南芥的种子萌发率、根长及盐和旱胁迫下的叶绿素含量。本研究结果为LEA基因抗逆机理的研究和耐盐基因的挖掘提供了重要信息。     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.     

Mapping of quantitative trait loci associated with protein expression variation in barley grains

Barley (Hordeum vulgare) is an important cereal crop grown for both the feed and malting industries. Hence, there is great interest to gain deeper insight into the determinants of grain nutritional quality in order to improve the assessment of new traits. Two-dimensional gel electrophoresis was employed for the characterization of the grain proteome of doubled-haploid introgression lines (IL) representing a wild barley genome (Hordeum spontaneum Hs213) within a modern cultivar background (H. vulgare cv. Brenda). Proteome maps were subjected to differential cluster analysis and revealed ILs with similar or different protein expression patterns compared to the Brenda parent. A total of 51 quantitative trait loci for protein expression (pQTL) were detected, and proteins underlying these pQTL were further examined by mass spectrometry. Identification was successful for 49 of the segregating spots and functional annotation of proteins revealed that most proteins are involved in metabolism and disease/defence-related processes. Among those, multigene families of glyceraldehyde-3-phosphate dehydrogenases, heat shock proteins, peroxidases, and serpins were identified. Overall, eight pQTL signals were discovered in two independently grown sets of plants. The mapped spots included protein disulfide isomerase, α-amylase inhibitor BDAI, NADP malic enzyme, adenosine kinase and peroxidase BP1. Specific marker information of proteins involved in developmental events and protein storage as well as in disease- and defence-related processes now allows for targeted breeding approaches to improve the grain quality in barley.     

植物耐盐性的分子生物学研究进展

研究证明了植物的耐盐机制十分复杂,安与植物的小分子物质的积累,离子摄入和区域化,以及基因表达和大分子蛋白质的合成有关,如调渗蛋白、通道蛋白、晚期胚胎发生富集蛋白。同时,利用克隆技术分离到了一些盐诱导基因。现将这部分工作做一综述。     

Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins

To study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system. Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 promoter, and the gene products were detected using specific antisera. The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions. The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as compared to a control strain, while the cells expressing le4 did not show improved growth. Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1. No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1. Increased freezing tolerance was observed in both lea-expressing cells, while no effect on heat tolerance was observed. These results support the hypothesis that different LEA proteins play a distinctive role in the protection against cellular dehydration.     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.