大豆E3泛素连接酶基因GmAIRP1的同源克隆及在烟草中的功能鉴定

E3泛素连接酶在植物抵御高盐及干旱等非生物胁迫过程中发挥重要作用。本研究克隆获得大豆E3泛素连接酶基因GmAIRP1,该基因cDNA全长为642 bp,编码213个氨基酸。蛋白结构域分析表明,GmAIRP1具有典型的RING-finger结构域。系统进化树分析表明,GmAIRP1与蒺藜苜蓿MtAIRP1同源性最高,亲缘关系最近。表达分析显示,GmAIRP1可被高盐、干旱和ABA诱导表达,并在胁迫1 h或3 h时表达量达到最大。抗逆表型分析表明,GmAIRP1转基因烟草在高盐和干旱胁迫21 d后,生长状态优于野生型,提高了植株对高盐和干旱胁迫的耐受性。生理指标测定结果显示,在高盐和干旱胁迫下,GmAIRP1转基因烟草的POD和CAT活性提高,整体高于对照,MDA含量始终低于对照。以上研究结果表明,Gm AIRP1能够通过激活抗氧化酶活性、提高渗透调节物质的积累来增强植物抵御高盐和干旱胁迫的能力,在植物响应高盐和干旱胁迫中发挥正调控作用。     

大豆SbPRP3基因表达及转基因烟草非生物胁迫鉴定

为研究大豆(Glycine max L.)细胞壁脯氨酸富集蛋白基因(SbPRP3)在逆境胁迫中的作用,利用实时荧光定量PCR,对SbPRP3在高盐、干旱和低温处理下的表达情况进行检测。结果显示,SbPRP3在高盐处理下表达量升高,在干旱和低温处理下表达量先升高后降低。将SbPRP3构建到植物表达载体pRI101-AN上并转化烟草,获得阳性转基因烟草3株。对转基因烟草植株进行高盐、干旱和低温胁迫处理。结果表明,与对照相比,高盐和低温处理下转基因烟草脯氨酸积累量增加,而丙二醛产生量降低。但干旱处理后转基因烟草脯氨酸和丙二醛的含量与对照相比没有显著差异。由此推测SbPRP3可以提高转基因烟草的耐盐性和耐寒性。     

花烟草NaNHX1基因的克隆及其在非生物胁迫下的表达模式

植物NHX家族基因,在植物的生长发育以及生物与非生物胁迫的应答反应中发挥着十分重要的作用。为了探究花烟草Na+/H+逆向转运蛋白的生理功能,为花烟草耐盐分子机制的研究提供参考。采用同源克隆的方法进行基因克隆,对花烟草进行非生物胁迫,并运用qPCR的方法进行基因表达模式分析。结果表明,从花烟草(Nicotiana alata)中克隆了一个属于Na+/H+逆向转运蛋白家族的基因NaNHX1。该基因的开放阅读框全长为1 599 bp,编码了532个氨基酸残基。生物信息学分析结果表明,该基因编码的蛋白分子量为58.4 kD,等电点为5.66;具有Na+/H+逆向转运蛋白家族典型的保守结构域NhaP2;该蛋白属于疏水性蛋白,包含10个跨膜区。NaNHX1基因主要定位于细胞质膜,并含有多个磷酸化位点。同源性分析的结果显示,NaNHX1基因与美花烟草(Nicotiana sylvestris)、茸毛烟草(Nicotiana tomentosiformis)以及番茄(Solanum lycoperisicum)NHX基因的亲缘关系最近,而与拟南芥的NHX基因同源性最低。NaNHX1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaNHX1的表达呈现3种不同的表达模式。其中,对高盐及低钾胁迫的响应强烈。本研究的结果表明,NaNHX1基因属于Na+/H+逆向转运蛋白家族,可能参与了花烟草高盐和低钾胁迫,以及其它非生物胁迫响应在内的众多生理过程。     

花烟草NaERF1基因的克隆及在非生物胁迫下的表达模式分析

AP2/ERF类转录因子,是植物所特有的最大的一类转录因子家族,在植物的生长发育过程中,扮演着重要的角色。探究花烟草ERF转录因子的生理功能,为花烟草抵御逆境的分子机制研究提供借鉴。采用同源克隆的方法进行基因克隆。通过对花烟草进行非生物胁迫,运用qPCR的方法进行基因表达模式分析。从花烟草(Nicotiana alata)中克隆了一个属于ERF家族的基因NaERF1。该基因的开放阅读框全长为819 bp,编码了272个氨基酸。生物信息学分析结果表明,该基因编码的蛋白分子量为30.7 kD,等电点为6.07;具有AP2/ERF类转录因子家族典型的保守结构域;该基因主要定位于细胞质内,并含有多个磷酸化位点。同源性分析的结果显示,NaERF1基因与茄科植物的ERF同源性较高,并且与普通烟草的ERF亲缘关系最近。NaERF1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。同时,在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaERF1的表达呈现5种模式。其中,对低钾及ABA胁迫的响应强烈。NaERF1基因属于AP2/ERF类转录因子,可能广泛参与了花烟草包括非生物胁迫响应在内的众多生理过程。     

LEA蛋白及其在植物抗逆改良中的应用

干旱、盐碱、高温和寒冷等逆境制约着植物的生长发育。植物中含有一组亲水性极强的蛋白,称为胚胎发育晚期丰富蛋白(late embryogenesis abundant,LEA蛋白),在自然条件下这种蛋白质一般在种子发育晚期积累,其对多种非生物胁迫具有很强的抵抗能力,并能响应干旱、寒冷、高盐和ABA等信号。LEA蛋白通过保持细胞渗透压、保护细胞膜结构、作为分子伴侣保护其他蛋白等方式维持植物正常的代谢反应。就LEA蛋白的分类、结构、抗逆机制以及在植物抗逆改良中的应用进行了简要综述。     

高度耐盐双价转基因烟草的研究

随着全球性人口的增长和土地退化的加剧,开发利用广阔盐碱地和干旱土地的需要日益迫切。植物生物技术的日臻完善,为培育高效耐盐植物迎来了一丝曙光。在高渗条件下,耐盐的微生物或植物细胞通过增加胞内一些相溶性溶质的浓度来维持渗透压的平衡。这些可溶性溶质包括无机离子、糖类、多元醇、氨基酸和生物碱等。通过基因工程手段,使细胞内积累脯氮酸⑴、甜菜碱⑵、甘露醇⑶、海藻糖⑷,能够不同程度地提高转基因烟草的耐盐性。多元醇含有多个羟基,亲水性能强,能有效维持细胞内水活度。山梨醇、甘露醇等己糖分子结构、理化性质和生理功能相近。故此．我们认为：不同糖醇在转基因烟草中的积累．可能具有协同(或累加)效应,有希望更大地提高植物耐盐性。我们在获得大肠杆菌mtlD基因(编码l-磷酸甘露醇脱氢酶)和gutD基因(编码6-磷酸山梨醇脱氢酶)克隆⑸的基础上,获得了分别表达mtlD和gutD基因的单价转基因烟草,并首次证实了gucD基因的表达,能显著地提高转基因烟草的耐盐性⑹。本文工作进一步报道同时表达大肠杆菌mtlD和gutD基因双价转基因烟草的高效高度耐盐性。     

沙棘HrTCP转录因子家族鉴定及其干旱胁迫下的表达分析

TCP家族是植物特有的响应高盐、干旱等非生物胁迫的重要转录因子。该研究基于沙棘转录组数据,利用生物信息学与qRT-PCR对HrTCP转录因子家族进行鉴定,预测其家族成员的结构和功能,为解析TCP转录因子调控沙棘抵御干旱胁迫的作用机制奠定基础。结果表明:(1)获得了11个HrTCP转录因子成员,并命名为HrTCP2/4/7/8/11/13/15/17/18/19/20,编码氨基酸序列长度在218～590之间,蛋白质相对分子量为23.44～61.78 kD;亚细胞定位预测发现,除HrTCP13/17/18蛋白定位于细胞质,其余8个蛋白均定位于细胞核。(2)在干旱(15%PEG-6000)和高盐(200 mmol/L NaCl)胁迫后HrTCP4/7/19/20基因表达量呈不同程度上升趋势,其中HrTCP20表达量极显著高于对照,分别是对照的24倍与23倍。(3)外源激素脱落酸(0.1 mmol/L ABA)和茉莉酸甲酯(0.1 mmol/L MeJA)处理后,HrTCP7/19/20基因表达量也均呈上升趋势,其中,ABA诱导下HrTCP19基因表达量达到最高,是对照的16倍,而MeJA诱导下HrTCP20基因表达量上升最高,是对照的5倍。研究发现,HrTCPs转录因子家族成员可受干旱、高盐和激素诱导表达,进而调控沙棘对干旱胁迫的响应。     

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.     

烟草NtCBL1基因的克隆、表达载体构建及表达分析

CBL是一类Ca²⁺感受蛋白,在植物适应或抵制逆境胁迫的过程中发挥重要的作用。从烟草品种K326中克隆到了一个CBL1的同源基因,该序列包含了一个642 bp的开放阅读框,编码一个由213个氨基酸残基组成的蛋白,预测分子量为24.5 kDa,等电点为5.03。同源性分析结果显示,该基因与林烟草CBL1、甜辣椒CBL1等具有较高的同源性,故命名为NtCBL1。生物信息学分析表明,NtCBL1具有CBL家族保守的EF-hand钙结合结构域。组织表达分析发现该基因在成熟期的根、茎、叶、花中均有表达,在根中的表达量最高。逆境胁迫实验表明,该基因表达受低钾、高盐、干旱、ABA和低温诱导调控,参与烟草生物与非生物逆境胁迫的响应。并成功构建了NtCBL1-pBI121过表达载体。研究结果为解析NtCBL1在响应逆境胁迫的功能奠定一定理论基础。     

海蓬子中高亲和钾离子转运体SbHKT1基因的克隆、表达及生物信息学分析

本研究利用RACE技术从真盐生植物海蓬子中获得了高亲和钾离子转运体SbHKT1基因1647bp完整的ORF框。序列分析结果表明,该基因编码548个氨基酸,分子量为62.10kD,理论等电点为9.33;氨基酸序列中第1个～第35个属信号肽序列,第197个～第537个属离子转运体(TrkH)家族特征序列;该基因编码的蛋白具有10个跨膜结构,N端跨膜区及中部膜上呈现明显的疏水性,C端及中部多个跨膜区呈现强疏水性,符合载体类运输蛋白的特点,因此推测SbHKT1蛋白为跨膜运输蛋白。Blast分析显示该蛋白与碱蓬SsHKT1氨基酸同源性高达77%,与冰叶日中花、赤桉和小麦HKT类蛋白的同源性分别为63%、52%和46%。SbHkt1基因表达存在组织特异性:正常生长条件在根、茎中表达较低,在叶片中几乎看不到表达;在高盐低钾的环境下,各组织表达明显升高,高盐低钾胁迫处理8h,其根部表达处于高峰期;经100μmol/L脱落酸处理4h,根部表达达到最高;干旱胁迫(20%PEG6000)处理2h,根部表达量明显上升。由此推断,该基因参与了植物在高盐低钾、渗透、干旱等非生物胁迫下的生理调控。由于目前已克隆的HKT类蛋白基因多来自非盐生植物,对盐生植物内源HKT基因的研究相对较少,因此,海蓬子内源HKT1基因的全长的获得有助于我们进一步研究该基因在高盐钾饥饿环境下运输钾离子,调节植物体内Na+/K+平衡的功能,对于揭示真盐生植物的耐盐机制,将其运用于非盐生植物,培育新的耐盐品种具有一定的意义。     

Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity

Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T(2) plants transformed with (+mtlD) or without (-mtlD) mtlD. Calli were exposed to -1.0 MPa of polyethylene glycol 8,000 or 100 mM NaCl. T(2) plants were stressed by withholding water or by adding 150 mM NaCl to the nutrient medium. Fresh weight of -mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In -mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in -mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 micromol g(-1) fresh weight in the callus and 0.6-2.0 micromol g(-1) fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.     

Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli

The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined. Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon. Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence. Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites. Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified. Probable terminator sequences were not found in any of these three regulatory regions. Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein. Computer techniques were used to analyse the mtlD gene and its product.     

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.     

Corrected sequence of the mannitol (mtl) operon in Escherichia coli

The previously published sequences of the operator-promoter region of the mannitol operon of Escherichia coli and of the mtlD gene have been found to contain a number of errors. The major conclusions reported previously were correct, but additionally it is now clear that a C-terminal portion of mannitol-1-phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino-terminal region of human liver fructose-6-phosphate-2-kinase:fructose-2,6-bisphosphatase.     

Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata

The mtl operon of Klebsiella pneumoniae KAY2026 (formerly Aerobacter aerogenes 1033-5P14) was shown to contain as the promoter-proximal gene mtlA, encoding a D-mannitol-specific enzyme II transporter (IICBA(Mtl)). This gene is followed by mtlD, coding for a mannitol-1-phosphate dehydrogenase (MtlD, 382 amino acid residues), and mtlR (MtlR, 195 amino acid residues) coding for a putative repressor, gene mtlR overlaps the termination codon of mtlD. The DNA and protein sequences are highly similar to the corresponding genes (81% identical bp) and proteins (79-85% identical amino acids) of Escherichia coli K-12. A truncated form of MtlD lacking the 162 C-terminal amino acid residues still shows 10% dehydrogenase activity which may explain the controversy in the literature concerning the properties of mannitol-phosphate and other medium-length dehydrogenases.     

Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication

The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scatted point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.     

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.