盐生植物海马齿盐胁迫全长cDNA文库的构建与分析

为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下:采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN+TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。     

AtNHX5基因过量表达对拟南芥耐盐性的影响

为了研究AtNHX5基因在植物耐盐中的作用,构建了植物过量表达载体pROKⅡ-AtNHX5,并转化拟南芥。结果显示:(1)RT-PCR检测表明,转基因拟南芥中AtNHX5基因的表达大幅提高。(2)对转基因纯合株系进行耐盐性分析显示,AtNHX5过量表达提高了植株在种子萌发和苗期的耐盐性。(3)转基因植株在盐处理下的干重、鲜重以及地上部分Na+、K+含量均高于野生型对照。在200mmol/L NaCl处理下,以转基因株系a1-4为例,其地上部分单株鲜重、单株干重、K+含量分别是野生型的1.27、1.54、1.16倍,较野生型显著升高。研究表明,过量表达AtNHX5基因促进了盐胁迫下转基因植株对K+的吸收,转基因拟南芥的耐盐性明显提高。     

长叶红砂液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特性

为研究长叶红砂(Reaumuria trigyna)离子转运分子机制,利用RT-PCR和RACE技术,克隆到其液泡膜Na+/H+逆向转运蛋白基因(NHX1)的全长cDNA片段,命名为RtNHX1(NCBI序列号为KR919802)。结果表明:RtNHX1的cDNA片段全长2 622bp,开放阅读框1 662bp,5′非编码区509bp,3′非编码区451bp,编码553个氨基酸,推测分子量为60.91kD。该蛋白含有12个跨膜结构域,为疏水蛋白,与其他植物液泡膜Na+/H+逆向转运蛋白NHX1的亲缘关系较近。实时荧光定量PCR对其在NaCl胁迫下的表达检测显示,不同时间和不同浓度NaCl胁迫下,RtNHX1表达量变化均呈先升高后降低趋势,在100mmol/L NaCl胁迫6h和200mmol/L NaCl胁迫后达到最高,表达量分别超过或约是对照的3倍,一定程度反应出RtNHX1参与长叶红砂的盐胁迫应答,是该植物离子转运体系的重要元件。     

绒毛白蜡FvNCED3基因植物表达载体构建及功能分析

[目的]构建绒毛白蜡FvNCED3基因的植物过表达载体并对其基因功能进行分析。[方法]采用双酶切法将绒毛白蜡FvNCED3基因插入pROKⅡ载体中,构建pROKⅡ-FvNCED3植物表达载体;浸花法转化拟南芥,抗性种子经Kan筛选后进行PCR检测;测定不同盐浓度培养基中转基因幼苗和对照植株的鲜重与根长,鉴定其基因功能。[结果]双酶切重组质粒得到1 823 bp的目的片段,表明成功构建pROKⅡ-FvNCED3植物表达载体;转基因种子经50mg/L Kan筛选培养6 d时效果最好,转化率达4.97‰。耐盐性分析表明,盐胁迫下,转基因株系根长显著长于对照,且盐浓度为100 mmol/L、150 mmol/L时,其鲜重分别为对照的1.43倍、2.06倍。[结论]成功构建pROKⅡ-FvNCED3植物表达载体,经PCR证明FvNCED3基因整合到植物基因组中。转基因后代的耐盐性分析初步表明FvNCED3基因具有增强植物耐盐性的功能。     

基于转录组学的蓖麻耐盐基因的挖掘

为获取蓖麻耐盐基因的序列信息,挖掘盐胁迫下差异表达基因及相关代谢途径,本研究以盐胁迫（300 mmol/L NaCl）处理0、12和24 h后通篦5号的幼苗真叶为试验材料,借助高通量测序技术进行转录组测序分析。结果表明,在盐胁迫12 h和24 h分别有4822和3103个差异表达基因。对共有的1872个差异表达基因进行共表达模式聚类分析,发现这些基因共有3种表达模式。KEGG代谢通路分析结果显示,缬氨酸、亮氨酸和异亮氨酸降解（ko00280）、植物昼夜节律调控（ko04712）以及淀粉和蔗糖代谢（ko00500）3个通路在盐胁迫适应过程中显著富集;GO功能富集分析结果显示,多数差异表达基因被富集到生物过程中,其中细胞进程（GO:0009987）和响应非生物胁迫（GO:0009628）过程富集到差异表达基因的数目最多。另外,共有19个转录因子参与蓖麻的盐胁迫响应。植物激素信号转导通路中有42个差异表达基因,其中有97.6%的基因分别在12 h和24 h是上调表达的。此外,还筛选出包括光合作用途径、抗氧化调节、Na+、K+和Ca2+转运相关参与蓖麻幼苗盐胁迫的差异表达基因。qRT-PCR结...     

北美海蓬子SbNHX1基因耐盐性及功能结构域分析

对从北美海蓬子中分离的Na+/H+逆向转运蛋白基因SbNHX1进行了耐盐性及功能结构域分析.利用套叠PCR技术去除SbNHX1基因C末端162个核苷酸,得到SbNHX1-C基因,然后将SbNHX1、SbNHX1-C和拟南芥Na+/H+ 逆向转运蛋白基因AtNHX1分别插入pET22b(+)表达载体,转化大肠杆菌B菌株,进行各种金属盐离子胁迫分析.结果表明,北美海蓬子Na+/H+ 逆向转运蛋白基因SbNHX1只对Na+ 、K+离子有抗性,且耐盐性强于拟南芥Na+/H+ 逆向转运蛋白基因AtNHX1.缺失C末端的SbNHX1-C基因对Na+、K+离子胁迫无抗性,说明北美海蓬子Na+/H+ 逆向转运蛋白基因SbNHX1的耐盐作用与该基因C末端1 353 bp至1 514 bp的序列密切相关.     

K+营养对NaCl胁迫下盐地碱蓬生长及叶片液泡膜V-H+-ATPase和V-H+-PPase活性的影响

对溶液培养的盐地碱蓬(Suaeda salsa L.)幼苗进行不同浓度NaCl胁迫并改变培养液中K+浓度,以了解K+营养对NaCl胁迫下盐地碱蓬幼苗生长及叶片液泡膜V-H+-ATPase、V-H+-PPase活性的影响.提高培养液K+浓度可明显增加盐胁迫下碱蓬植株的鲜重、干重,促进盐地碱蓬叶片及根部组织K+积累.盐地碱蓬叶片液泡膜V-H+-ATPase至少由A、B、C、D、E及c亚基组成,其表达量在缺K+处理(12 μmol/L K+)下随盐胁迫浓度的增加而减小,而在正常K+(6 mmol/L)培养下则随盐胁迫浓度的增加而增加;盐地碱蓬叶片液泡膜V-H+-PPase分子量为72 kD,在缺K+和正常K+供应情况下,V-H+-PPase均有较高表达.V-H+-ATPase及V-H+-PPase活性变化与其亚基表达量变化基本成正相关.结果表明: K+对盐生植物碱蓬的耐盐性有重要作用,盐胁迫下,K+可能参与了V-H+-ATPase和V-H+-PPase活性调控.     

盐生植物海马齿耐盐的生理特性

以盐生植物海马齿为研究材料,分别用淡水、1/4海水、1/2海水、全海水浇灌15 d和30 d,研究盐生植物耐盐的生理特性和机理。海马齿植物在低于1/2的海水浇灌时,植物生长旺盛,主要表现为叶片增大和变厚,地上部分生物量增加;而全海水抑制了植物的生长。在盐胁迫下,海马齿植物中Na+的含量叶中最高,茎中含量次之,根中含量最低。长时间盐胁迫时,海马齿植物根、茎、叶中的相对含水量与淡水浇灌相比,变化不大,叶中略有增加;而脯氨酸含量显著增加,且可溶性糖的含量也比淡水浇灌的高。由此推测:海马齿植物主要以有机小分子作为渗透调节物质来维持细胞渗透压,在其耐盐中起着重要的作用。土壤中Na+的毒害,并没有减少土壤中可被植物利用的可交换K+,反而使其增加,说明海马齿植物根部对Na+的吸收能力和Na+/K+交换能力非常强。海马齿植物耐盐性强,还表现为能阻止盐胁迫对植物细胞原生质膜的氧化损伤,不破坏植物叶片内叶绿素的合成,能基本维持植物茎、叶中K+和根、茎中Mg2+的相对稳定。     

盐胁迫对弗吉尼亚栎生长及矿质离子吸收、运输和分配的影响

以低浓度(50 mmol.L-1)和高浓度(150 mmol.L-1)NaC l处理弗吉尼亚栎(Quercus virginiana)2年生扦插苗,研究了弗吉尼亚栎生长和根系形态学参数变化以及Na+、K+、Ca2+、Mg2+、NO3-等矿质离子在不同器官的吸收、运输和分配。结果表明,盐胁迫不同程度促进了地上部和根系生长,地上部和根系干重、根长、表面积和体积在低浓度盐胁迫下明显增加(P0.05),而在高浓度盐胁迫下变化不大。随着根系对Na+和C l-吸收的增加,K+、Ca2+、Mg2+在根部和茎部的积累明显降低,矿质离子由根部向茎部运输的能力在低浓度盐胁迫增加而高浓度下受到抑制。叶片在低浓度和高浓度盐胁迫下对K+、NO3-具有很强的选择吸收能力,这对于维持叶片离子平衡和正常的光合作用及代谢过程具有重要意义。Na+和C l-在根部的浓度远远大于地上部,说明弗吉尼亚栎根系对盐离子具有较高的耐受性,而减少盐离子在地上部的积累,对于维持地上部的正常生长具有重要意义,这也是弗吉尼亚栎对盐胁迫的适应机制之一。     

GhCyt b5基因的克隆及其蛋白参与电子传递功能的分析

从陆地棉品种中棉所35盐胁迫EST文库中筛选到与其它植物高度同源的细胞色素b5蛋白(Cyt b5)基因片段,利用RACE技术,获得Cyt b5基因的cDNA序列,命名为GhCyt b5,基因全长810bp,最大阅读框402 bp,编码由134个氨基酸组成的蛋白质,分子量约为17kDa.将该基因的编码序列插入到原核表达载体pET32a中,构建重组质粒为pET32a-Cyt b5,对不同条件下进行蛋白诱导表达分析,发现在28℃和1 mmol/L IPFG条件下能够获得可溶性的GhCyt b5蛋白.利用Ni2+柱亲和层析纯化和SDS_PAGE鉴定分析表明获得重组蛋白为目的蛋白.通过提取棉花细胞物质为反应介质,进行体外电子传递功能分析,发现GhCyt b5能够从还原态变为氧化态,参与电子的传递功能.     

Molecular phylogeny of siboglinid annelids (a.k.a. pogonophorans): a review

Siboglinid, or pogonophoran, annelids are tubicolous worms that rely on chemoautotrophic endosymbionts for nutrition. Three clades within the siboglinids are recognized: Frenulata, Vestimentifera, and Monilifera. As a group, these worms have received considerable attention from molecular phylogenetists. Most studies have focused either on the evolutionary origins of the group or on the relationships within vestimentiferans, which live at hydrocarbon seeps and hydrothermal vents. Here I review the literature to date on siboglinid molecular phylogeny and summarize the clade’s evolution. The vestimentiferans have been well studied, especially in the eastern Pacific. The seep taxon Lamellibrachia is basal in the clade with vent species being more derived. Recent studies of seeps are finding new species and suggest that habitat depth can be correlated with species boundaries. In contrast to the vestimentiferans, frenulate evolution has been poorly studied. Despite their greater apparent diversity, frenulate specimens have not been sampled so extensively, and thus little is known about their evolution. Sclerolinum, also referred to as Monilifera, is a recognized genus of siboglinids that forms the sister group to Vestimentifera. Like the frenulates, little is known about the history of this group. Our present understanding of siboglinid phylogeny has, in large part, been dictated by insufficient sampling effort.     

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.     

Topographic anatomical data on the a.testicularis, a.ductus deferentis and a.cremasterica in ram

130 Ram testes have been used in the study, and the following methods were applied to fill and study the testical arteries: r?ntgenography, stereor?ntgenography, corosive method, Indian ink and gel injections. Variabilities occur as to the site where the a.testicularis arises in rams. Individual differences can also be observed in the arrangement of the epididymal arteries, in most cases, however, they arise from the first loops of the convolution. Both epididymal arteries are considerably thinner than the testicular artery, forming 2 independent vessel convolutions, located at both sides of the convolution of the a.testicularis, and not interfering with the loops of the latter. A.accessoria testis has been found in one ram only. 2 to 3 large waved loops can be observed in the pars marginalis and the a.testicularis of the ram. Bifurcation mostly occurs in the second third of the margo epididymidis. Individual variations have been found at further ramification of the rr.testiculares. The shape of the lobulus testis is indicated by the centripetal branch with its centrifugal twigs. In rams the a.ductus deferentis forms anastomoses both with the branches running from the a.epididymidis caudalis and the a.cremasterica.