植物Na+/H+逆向转运蛋白与植物耐盐性的研究进展

植物通过外排Na+和液泡区隔化Na+来减少Na+的毒害,这一过程由Na+/H+逆向转运蛋白来完成。通过概述植物Na+/H+逆向转运蛋白的基本特征、与植物耐盐性的关系及其分子生物学研究现状,为阐明植物复杂的盐胁迫机理和利用基因工程手段提高植物耐盐性奠定了基础。     

植物Na~+/H~+逆向转运蛋白研究进展

土壤中过多的Na+是抑制植物生长的主要限制因子之一,也是造成土壤盐渍化的主要离子。而Na+/H+逆向转运蛋白是植物维持Na+稳态,增强植物抗盐性的一条重要途径。通过对Na+/H+逆向转运蛋白的分类、结构、功能,以及和植物耐盐性关系的概述,揭示其耐盐机理,并就其将来的应用和发展前景作了分析。     

NaCl胁迫下拟南芥Na+/H+逆向转运蛋白基因(AtNHX1)的克隆及序列分析

以NaCl胁迫处理的拟南芥幼苗叶片为材料,用TRIzol一步法RNA提取试剂盒抽提总RNA,通过RT-PCR方法和DNA序列测定,证实获得了拟南芥Na /H 逆向转运蛋白基因(AtNHX1)的cDNA克隆.该cDNA全长1 617 bp,包括538个氨基酸编码区和1个终止密码,具有多个物种Na /H 逆向转运蛋白基因的高度保守序列氨氯砒嗪脒(amiloride)的结合位点(LFFIYLLPPI).序列同源性分析结果显示,该cDNA片段与原序列的同源性高达99.75%,与同科芸薹属油菜的同源性为89.00%,但与不同科植物的同源性较低,仅为60%～70%,表明该基因在进化上存在多样性,但它们都具有氨氯砒嗪脒结合位点,对Na 具有高度专一性,对植物的耐盐性起着重要作用.     

盐胁迫对芨芨草种子萌发苗Na+,K+含量与质膜H+-ATPase活性的影响

以芨芨草(Achnatherumsplendens)种子萌发苗为试验材料,分别以不同浓度NaCl和Na₂SO₄进行胁迫,通过对芨芨草叶片和根系中Na⁺,K⁺含量以及质膜H⁺-ATPase活性进行测定,以探讨盐胁迫对芨芨草中Na⁺,K⁺分布以及质膜H⁺-ATPase活性的影响。结果表明:随着NaCl和Na₂SO₄浓度的增加,芨芨草根系和叶片的Na⁺含量增加,K⁺含量下降,K⁺/Na⁺比值下降,根系中质膜H⁺-ATPase活性增加;在NaCl和Na₂SO₄胁迫下,芨芨草叶片中Na⁺含量显著低于根系,K⁺含量显著高于根系,叶片的K⁺/Na⁺比值均大于1并明显高于根系,根系的质膜H⁺-ATPase活性显著高于叶片;与NaCl相比,Na₂SO₄胁迫下,芨芨草根系向叶片的离子选择性运输系数(TS_K,Na)较高,叶片和根系的质膜H⁺-ATPase活性明显高于相同浓度的NaCl胁迫组。与NaCl胁迫相比,芨芨草对Na₂SO₄胁迫的适应性更强。     

超表达桑树MaNHX1和拟南芥AVP1基因增强拟南芥耐盐性的研究

盐胁迫会严重影响作物的生长发育,培育和种植耐盐胁迫的作物品种有利于对盐渍化土地的开发利用。Na+/H+逆向转运蛋白基因(NHX)已被证实与作物的耐盐性有关。将克隆的桑树Ma NHX1基因和拟南芥焦磷酸酶基因AVP1分别转入拟南芥植株,筛选获得转基因株系MN和AV,并通过人工授粉杂交后筛选获得共表达2个基因的转基因拟南芥株系MNV,用于探究Ma NHX1和AVP1共表达增强植株耐盐性的机制,确证Ma NHX1基因在盐胁迫适应性调节中的功能。在200 mmol/L Na Cl胁迫条件下,共表达转基因拟南芥株系MNV与野生型植株相比,叶片中的叶绿素含量高出20.69%,含水率高出5.89%,脯氨酸含量高出28.42%,而丙二醛含量的增幅下降7.73%。研究结果表明:超表达耐盐性基因的转基因株系可以通过维持细胞膜稳定性、降低膜损伤和透性伤害等机制应对盐胁迫环境。     

桑树中拟南芥同源基因mNHX1的克隆及其功能研究

通过RT-PCR法从桑芽的全RNA中逆转录合成了桑芽cDNA库,以此为模板,用PCR技术克隆了拟南芥的同源基因mNHX1,该基因是编码植物细胞液泡膜上Na+/H+逆向运转蛋白的基因。用农杆菌法将mNHX1导入拟南芥,获得了过量表达转基因植株,并进行了功能研究。从51个转基因抗性植株中共筛选到18个纯系。结果表明,转mNHX1基因的拟南芥种子在含有100~200 mmol/L NaC l的MS培养基上,种子的发芽率明显高于野生型。同时,在含有150、250 mmol/L浓度NaC l的基质中,转基因植株的抗盐性能明显高于野生型。根据已报道的At-NHX1同源基因的功能推断,mNHX1所编码的细胞液泡膜上Na+/H+逆向运转蛋白是桑树抗盐性能的一个极其重要的因素。     

逆向转运蛋白基因的克隆及序列分析

根据同源序列区域设计简并引物，利用ＲＴ－ＰＣＲ 及ＲＡＣＥ 方法从盐生植物大叶补血草中分离了Ｎａ^＋／Ｈ^＋逆向运输蛋白基因的ｃＤＮＡ（LgNHX1，ＧｅｎＢａｎｋ登录号ＥＵ７８０４５７）。LgNHX1的ｃＤＮＡ 全长为２３９７ｂｐ，５′端非翻译区长度为５１２ｂｐ，３′端非翻译区长度为２２９ｂｐ，其中包括１２ｂｐ的ｐｏｌｙ（Ａ）尾巴，中间的开放读码框为１６５６ｂｐ，编码１个５５０个氨基酸的多肽，推测分子量为６１ｋＤ。氨基酸同源性分析表明，ＬｇＮＨＸ１与北滨藜、小麦、盐角草和拟南芥液泡Ｎａ^＋／Ｈ^＋ 逆向运输蛋白的一致性分别为８２．６２％，８２．０１％，８０．６４％ 和７５．８６％。预测分析表明，ＬｇＮＨＸ１具有１１～１２个跨膜结构区域。系统发育分析表明该蛋白（ＬｇＮＨＸ１）与液泡型的Ｎａ^＋／Ｈ^＋ 逆向转运蛋白的亲缘关系较近，与质膜型的Ｎａ＋／Ｈ＋ 逆向转运蛋白亲缘关系较远。     

液泡膜Na~+/H~+逆向转运蛋白基因沉默对霸王叶气孔特征的影响

以霸王(Zygophyllum xanthoxylum)野生型植株(WT)、液泡膜Na+/H+逆向转运蛋白基因(Zx NHX)的RNA干扰(Zx NHX-RNAi)株系L2和L7为材料,分析了Zx NHX沉默后霸王叶下表皮气孔特征的变化情况。结果表明,50 mmol·L-1Na Cl处理下,Zx NHX-RNAi株系叶下表皮气孔开度达到峰值的时间比WT延迟了4 h;渗透胁迫下,L7气孔开度的峰值比WT显著下降12%。对照条件下,L7气孔大小显著低于WT,达到峰值的时间比WT延迟6h。无论是对照条件还是盐处理或渗透胁迫下,Zx NHX-RNAi株系叶组织含水量均显著低于WT,尤其是盐处理下,L7比WT低40%。可见,Zx NHX通过调控霸王叶的水分状况,进而影响气孔运动。     

唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析

花青素是植物体内重要的黄酮类次生代谢产物,其强大的抗氧化能力对植物抵抗由各种非生物胁迫带来的氧化损伤发挥着重要的作用。本研究基于转录组数据,从唐古特白刺cDNA中克隆得到一个类黄酮3-O-葡萄糖基转移酶基因,将其命名为NtUFGT。该基因开放阅读框长度为1407 bp,编码468个氨基酸,预测该基因编码的蛋白质相对分子质量为51.37 kDa。多重序列比对分析结果显示,其编码蛋白属于UDP-glycosyltransferases蛋白家族。实时荧光定量PCR(qRT-PCR)分析了该基因在唐古特白刺中的表达模式,发现该基因的表达具有组织特异性,由高到低依次为花>果实>茎>叶>根;同时该基因能被聚乙二醇(PEG)和脱落酸(ABA)等非生物胁迫强烈诱导表达。构建该基因的真核表达载体pPZP221:35S:NtUFGT,使用花序浸染法转化拟南芥并筛选至T₃代,RT-PCR验证表明,NtUFGT基因在转基因拟南芥3个株系中均明显表达。测定干旱胁迫条件下野生型(WT)和转基因拟南芥(OE株系)生长状况和抗逆相关生理生化指标,结果发现转基因拟南芥生长状况明显优于野生型,根长更长,鲜重和叶绿素含量更高,同时OE株系积累了更多的花青素和总黄酮。与表型一致,相较于WT,OE株系具有更高的抗氧化酶活性[超氧化物歧化酶(SOD);过氧化物酶(POD);过氧化氢酶(CAT)],积累了更多的还原性谷胱甘肽(GSH)和脯氨酸,同时其丙二醛(MDA)、过氧化氢(H₂O₂)含量显著低于WT;基因定量分析结果显示,OE株系拟南芥中抗逆相关基因AtCAT1、AtPOD1、AtRD29A及脯氨酸合成基因AtP5CS的表达量明显高于WT。以上结果说明,NtUFGT能有效提高转基因拟南芥中花青素和总黄酮含量,赋予植物更强的活性氧清除能力和渗透调节能力,从而增强了植物对干旱胁迫的耐受性。     

桑树Ca~(2+)/H~+反向转运体基因MCAX-1的克隆及序列与表达分析

植物体内的Ca2+/H+反向转运体在Ca2+介导的营养和信号转导中发挥着重要作用。选择桑树幼叶cDNA文库中功能注释为Ca2+/H+反向转运体基因(CAX)的一条EST序列设计引物,通过cDNA末端快速扩增(RACE)与反转录PCR(RT-PCR)在桑品种育71-1的幼叶中克隆获得该基因的全长cDNA序列,命名为MCAX-1(GenBank登录号:JN716318)。序列分析显示:MCAX-1全长1 784 bp,包括197 bp的5'端非翻译序列和243 bp的3'端非翻译序列,含有一个1 344 bp的完整ORF,编码447个氨基酸残基,预测蛋白质分子质量为47.93 kD,等电点为5.67。构建的桑树和其它植物基于CAX蛋白氨基酸序列的系统进化树显示:桑树与盐地碱蓬(Suaeda salsa)、毛果杨(Populus trichocarpa)、蓖麻(Ricinus communis)等有较近的亲缘关系。半定量RT-PCR分析表明:MCAX-1在桑树幼芽、嫩叶、根和幼花中的表达量较高,在韧皮部、木质部和成熟果实中的表达量较低;MCAX-1在低温胁迫下的表达量减少,-1℃时几乎无表达,在干旱胁迫下的表达量随着胁迫时间延长逐渐达到最大值之后又迅速降低,在盐分胁迫初期的表达量无变化,但胁迫18 d时表达量明显降低。初步推测MCAX-1与桑树的抗逆性能有一定关联。     

Increases in the expression of Na+/H+ exchanger 1 and 3 are associated with insulin signalling in the ruminal epithelium

Na⁺/H⁺ exchanger (NHE), which catalyses the exchange of extracellular Na⁺ for intracellular H⁺, is of importance in the maintenance of Na⁺ and pH homoeostasis for rumen epithelial cells. Studies in ruminants showed that high concentrate diets could increase the expression of NHE in ruminal epithelium. Results of recent studies further indicated that insulin, as an important hormone closely related to dietary concentrate, could enhance the expression of NHE. In this study, we have investigated the mechanisms of insulin regulating the expression of NHE in rumen epithelial cells and its potential role in dietary modulation of NHE expression in ruminal epithelium of cows. In primary culture, insulin increased phosphorylation of ERK 1/2 and AKT in rumen epithelial cells. However, this promotion was diminished by insulin receptor inhibitor. Insulin also stimulated NHE1 and NHE3 expression. But this increase was suppressed by insulin receptor inhibitor, ERK inhibitor and AKT inhibitor. In the present animal experiment, NHE1 and NHE3 expression increased in rumen epithelium of cows ingesting a high concentrate diet (HC, 60% concentrate), accompanied by increased insulin concentration in plasma, compared to those feeding a low concentrate diet (LC, 20% concentrate). Furthermore, the phosphorylation of ERK1/2 and AKT was higher in the rumen epithelium of the HC group than those in the LC group. Collectively, these results indicate that diet‐dependent change of NHE1 and NHE3 abundance was mediated, at least in part, by plasma insulin through the ERK and AKT pathway.     

酸和铁胁迫对紫花苜蓿根系质膜H＋-ATPase活性的影响

以紫花苜蓿品种WL-525为材料，研究了在pH4．5和pH6．0的环境下，缺Fe^2＋和富Fe^2＋胁迫对紫花苜蓿根系质膜H^＋-ATPase活性的影响。实验结果表明：紫花苜蓿根系质膜H＋-ATPase酶的活性在强酸胁迫、缺Fe^2＋和富Fe^2＋胁迫，以及酸、铁共同胁迫下均发生明显的变化，呈现出短时间胁迫，质膜H^＋-ATPase活性升高，然后随着处理时间的增加，酶活性呈现不同程度的下降趋势。研究结果说明，在强酸和Fe^2＋胁迫下，苜蓿根系质膜H＋-ATPase酶的活性会发生变化，以减轻苜蓿根系受到的伤害。     

高谷物日粮促进山羊瘤胃上皮单羧酸转运蛋白1及钠钾ATP酶mRNA的表达

本试验旨在研究高谷物日粮对山羊瘤胃上皮形态结构及单羧酸转运蛋白(monocarboxylate transporter, MCT)和钠钾ATP酶mRNA表达的影响。将10头装有永久性瘤胃瘘管的健康阉割公山羊随机分为饲喂全粗料日粮的对照组(Hay,0%谷物,n=5)和饲喂高谷物日粮的处理组(HG,65%谷物,n=5),试验期为7周。试验开始后,于每周晨饲后的0、2、3、4、6、8和12 h连续采集瘤胃液监测瘤胃pH值的变化,收集其中第0、3、6和12 h的瘤胃液待测挥发性脂肪酸(volatile fatty acid, VFA)浓度。试验的第50天,屠宰采集瘤胃上皮用于形态学及基因定量分析。研究结果显示:与全粗料组山羊相比,高谷物组山羊瘤胃pH值、乙酸浓度及乙丙比都显著下降(P<0.001),而瘤胃丙酸浓度、丁酸浓度及其他VFA浓度都显著升高(P<0.001);高谷物日粮组的瘤胃乳头长度显著高于对照组(P=0.001),瘤胃乳头宽度显著低于对照组(P<0.001),但是两组间的瘤胃乳头表面积并无显著差异;透射电镜结果显示,长期饲喂高谷物日粮导致瘤胃上皮细胞线粒体发生降解;实时定量PCR结果表明,与对照组相比,高谷物日粮显著升高了MCT1(P<0.001)和钠钾ATP酶(P=0.001)的mRNA表达量,显著降低了MCT4的mRNA表达量(P=0.041),但对MCT2的表达没有显著影响(P=0.305);进一步分析这些基因的mRNA表达量与pH值和VFA浓度之间的相关性,结果显示,MCT1和钠钾ATP酶的mRNA表达量与瘤胃pH值和乙酸浓度呈显著负相关,与总VFA、丙酸、丁酸的含量呈显著正相关,而MCT4的mRNA表达量与pH值呈显著正相关,与总VFA、丙酸、丁酸的含量呈显著负相关。以上结果提示:高精料引起的瘤胃pH值下降和VFA的变化可能与瘤胃上皮MCT和钠钾ATP酶表达量的变化相关。研究结果对深入认识高谷物饲喂引发的瘤胃功能紊乱具有重要意义。     

Ontogeny of the stomach in yellow catfish (Pelteobagrus fulvidraco): detection and quantifictation of pepsinogen and H+/K+‐ATPase gene expression

Yellow catfish (Pelteobagrus fulvidraco) is an important commercial species with high aquaculture potential in China. To better understand the process of digestive functioning of gastric gland development during the larval from 1 dph (day post‐hatching) to 30 dph, real‐time PCR was used to detect and quantify the pepsinogen and H⁺/K⁺‐ATPase gene expression in P. fulvidraco. These data were also compared with the adult situation. The results showed that the expression of pepsinogen and H⁺/K⁺‐ATPase genes in P. fulvidraco larvae both started at 1 dph, though the expression level was very low until 3 dph. The quantification of pepsinogen gene expression increased significantly from 4 to 8 dph, increased fluctuantly from 8 to 23 dph and rose sharply from 23 to 30 dph. In comparison with adult fish, there were no significant differences with larvae at 5 and 23 dph. However, data of 10 and 30 dph larvae were obviously higher than those of adult group. H⁺/K⁺‐ATPase gene expression increased linearly from 1 to 30 dph. However, it was significantly lower than that of adult. The results show that P. fulvidraco larvae have an earlier functional stomach, though the function of the stomach is still not perfect. There is a gradual acidification environment within the stomach during the P. fulvidraco larvae development. Based on these results, we suggest that the weaning time for P. fulvidraco larvae would be much better after 23 dph.     

Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ ATPase isoform in sturgeon sperm cryopreservation

Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane‐impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca²⁺ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S₁₅T₁₅) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L₁₅T₁₅) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L₁₅T₁₅ in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight‐line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.