烟草ACS基因家族鉴定及二氯喹啉酸胁迫条件下的表达分析

二氯喹啉酸药害给烟叶生产造成较大影响。为明确烟草二氯喹啉酸药害的发生机制,对1-氨基环丙烷-1-羧酸合酶(1-aminocyclopropane-1-carboxylate synthase,ACS)基因家族进行了鉴定,并对二氯喹啉酸胁迫条件下的基因表达模式进行分析。结果表明,普通烟草共有26个ACS基因。染色体定位分析显示,23个ACS基因定位在染色体上,3个定位在scaffold上。亚细胞定位分析显示,ACS主要定位于细胞核或细胞质中。进化分析表明,ACS蛋白进化形成3个类别。序列比对显示,ACS氨基酸序列含有家族保守结构域和活性位点。启动子分析发现,ACS基因启动子区含有脱落酸、茉莉酸、水杨酸、生长素和赤霉素5种激素反应共10种类型的顺式作用元件。表达分析显示,大部分ACS家族成员均受二氯喹啉酸诱导而上调表达。      

巴氏杜氏藻八氢番茄红素合成酶侧翼调控序列的克隆与分析

研究表明八氢番茄红素合成酶( PSY)是巴氏杜氏藻(Dunaliella bardawil)类胡萝卜素代谢途径中的第一个关键调节酶.本实验采用基于LAoPCR的基因组步移法分别设计两组基因特异引物pSP1-3及tSP1-3,克隆巴氏杜氏藻PSY( DbPSY)的启动子和终止子序列,并利用在线启动子分析软件分析其保守调控序列.最后利用生物信息学方法分析预测DbPSY的蛋白质结构.分析结果表明,DbPSY启动子具有两个保守调控序列:BOXLCOREDCPAL和GT1GMSCAM4,其中BOXLCOREDCPAL受紫外光诱导,上调下游基因的表达,GT1GMSCAM4受盐调控,促进下游基因的表达.蛋白结构分析表明,DbPSY的N端在邻近物种间具有较大的多样性.我们推测DbPSY的微调与启动子保守序列及蛋白质N端序列的多样性相关.     

烟草硝酸盐转运蛋白基因NtNRT2.4的克隆及表达分析

植物NRT2基因家族(高亲和硝酸盐转运蛋白基因)在植物吸收和转运硝酸盐过程中发挥重要作用。本研究根据NRT2基因家族的保守序列设计兼并引物,扩增出一条821bp序列,以此序列为信息探针,通过电子克隆和RT-PCR的方法从烟草中克隆获得一个包含1593bp开放阅读框、编码530个氨基酸的c DNA序列,命名为Nt NRT2.4。生物信息学分析表明,该基因编码的蛋白质具有NRT家族的共有结构特征,与烟草已发现的三个本家族基因同源性达到77.54%,与拟南芥NRT2家族基因同源性达到81.63%,其启动子中包含多个与硝酸盐、光照及根系特异表达相关的元件;组织特异表达分析结果显示,烟草Nt NRT2.4基因的组织表达差异较大,在根部的表达量较高,在叶片和茎部的表达量低;不同氮素形态、光照和蔗糖处理下的基因表达分析结果表明,硝态氮、光照和蔗糖处理诱导Nt NRT2.4表达,而铵态氮抑制其表达。     

甘蓝型油菜TFⅢA型锌指蛋白基因鉴别和表达模式

植物TFⅢA型锌指蛋白的锌指区含有保守的QALGGH区,属于C2H2型转录因子,参与调控植物生长发育和逆境胁迫应答.参照拟南芥AtZFP基因的C2H2结构域氨基酸序列,采用生物信息学方法鉴定了46个甘蓝型油菜TFⅢA型锌指蛋白基因,分析了基因结构、生化特性、进化关系和基因表达模式.结果表明：42个基因为单锌指蛋白基因,4个为多锌指蛋白基因;40个基因不含内含子,6个基因含内含子;46个基因的氨基酸序列同源性变幅为9.0% ～99.2%,氨基酸序列保守性差异明显,亚类成员间包含相同的保守基序,亚类间的保守基序不同;PlantCARE分析发现其启动子区富含激素、非生物胁迫及特定生理过程响应的顺式作用元件;RNA-seq分析表明,部分基因在甘蓝型油菜品种中双11号的根、初花期雌蕊和角果中特异表达.     

烟草HD-ZIP Ⅳ转录因子NtPDF2基因的克隆及表达分析

为了明确NtPDF2在烟草中的生物学功能及其调控腺毛分化发育的作用机制,同源克隆了烟草NtPDF2基因,并对其序列、基因结构、进化关系以及表达模式进行分析。结果表明:NtPDF2 CDS全长为2 199 bp,编码732个氨基酸残基。烟草PDF2蛋白质序列高度保守,尤其是C端序列,均含有3个保守结构域(Homeobox domain,START domain和SAD domain)和1个保守LZ(Leucine zipper)元件。烟草PDF2基因结构高度保守,均由10个外显子和9个内含子组成。进化分析表明,普通烟草NtPDF2基因是由林烟草ML1基因进化而来。表达分析显示,烟草PDF2基因表达具有明显的时空特异性。干旱、黑暗和磷饥饿胁迫可显著下调NtPDF2基因的表达,而盐胁迫对其没有明显影响;NtPDF2基因的表达在赤霉素(GA)、茉莉酸甲酯(Me JA)、脱落酸(ABA)和打顶处理条件下均显著上调,这暗示烟草NtPDF2基因参与了调控多种非生物胁迫和激素应答过程。     

烟草NtGCN2启动子的克隆及功能研究

  目的  探究烟草蛋白激酶NtGCN2启动子驱动GUS基因在ABA、SA、AZA和MeJA四种处理下的表达模式。  方法  克隆烟草NtGCN2上游2000 bp的启动子序列,并采用Plant CARE和PLACE数据库进行顺式作用元件预测。构建NtGCN2启动子驱动β-葡萄糖苷酸酶基因（GUS）的融合表达载体并转化烟草K326。利用获得NtGCN2-Nsy-pro2000:: GUS的转基因植株,探究NtGCN2启动子驱动的GUS报告基因在ABA、SA、AZA和MeJA四种激素处理下的表达模式。  结果  顺式作用元件预测结果发现,在NtGCN2-Nsy-pro2000上存在多种顺式作用元件,如脱落酸应答元件（ABRE）、茉莉酸甲酯应答元件（TGACG-motif）等。ABA、SA和AZA处理下NtGCN2启动子驱动的GUS基因表达和酶活性均显著增加,而MeJA处理下NtGCN2启动子驱动的GUS基因表达及酶活性下降。  结论  NtGCN2上游2000 bp序列具有启动子活性,NtGCN2-Nsy-pro2000驱动GUS基因在不同激素处理下呈现不同的响应模式。ABA、SA和AZA激活NtGCN2-Nsy-pro2000驱动的GUS基因表达,而MeJA抑制NtGCN2-Nsy-pro2000驱动的GUS基因表达。      

褪黑素采前喷施对采后番茄果实抗病性和贮藏品质的影响

为研究褪黑素采前喷施番茄植株对采后果实抗灰葡萄孢菌能力和品质的影响,本实验以'Ailsa Craig'番茄为材料,采用1、50、100、150 μmol/L褪黑素采前喷施番茄植株,测定采后番茄果实品质、相关酶活力及基因表达量。结果表明:与蒸馏水喷施对照组相比,100 μmol/L褪黑素采前喷施处理可以显著降低番茄果实接种灰葡萄孢菌后的发病率、病斑面积(P0.05),诱导提高果实中几丁质酶、β-1,3-葡聚糖酶、多酚氧化酶和苯丙氨酸解氨酶活力,增加抗病相关基因PR1、NPR1、PI Ⅱ和LoxD的相对表达量,提高VC、可滴定酸、可溶性糖和可溶性蛋白含量。综上,100 μmol/L褪黑素采前处理对采后番茄果实抗灰葡萄孢菌的效果最佳,褪黑素可提高番茄果实的抗病相关酶活性和贮藏品质。     

2种山葡萄白藜芦醇合酶基因的克隆与序列分析

目的:白藜芦醇合酶(resveratrol synthetic enzyme,RS)是植物白藜芦醇合成途径中的关键酶,为获得高效表达的白藜芦醇合酶基因,方法:利用已知的葡萄RS基因(AF274281)序列设计并合成一对引物,以2种不同来源的山葡萄叶片基因组DNA为模板,PCR扩增包含RS完整基因在内的一段序列;在此基础上采用悬挂延伸PCR法分别克隆目的基因。结果:测序与序列分析表明,RS全长基因片段大小均为1 536bp,其中包含一个内含子及两个外显子,同源性比较,其内含子碱基变化较大。悬挂延伸PCR法克隆获得的目的基因,开放读码框分别为1 170 bp和1 182bp,编码389/392个氨基酸残基,命名为CNRS1、CNRS3。同源性比较发现,CNRS1、CNRS3与已知葡萄RS基因序列的同源性分别为98%、97.5%。结论:以上结果证实CNRS1、CNRS3属葡萄RS基因家族成员,为今后进一步对该基因的研究与利用打下了基础。     

不同浓度β-氨基丁酸处理对葡萄果实抗病性的诱导模式

分析了不同浓度（1～500 mmol/L）β-氨基丁酸（β-aminobutyric acid,BABA）处理对采后葡萄果实抗病反 应的诱导作用并明确相关抗性反应的模式。结果显示,10～500 mmol/L BABA处理可有效抑制果实在20 ℃贮藏期 间灰葡萄孢（Botrytis cinerea）的侵染,从而降低果实发病率。在有效作用浓度中,经高浓度（100、500 mmol/L） BABA处理的模拟接种和B. cinerea接种葡萄果实均出现H2O2迸发、病程相关基因（VvNPR1.1、VvChit4和VvPR2） 表达量提升、抗病相关酶（几丁质酶和β-1,3-葡聚糖酶）活力升高和植保素单体（白藜芦醇和白藜芦醇脱氢二聚 体）积累等一系列典型抗病性反应。10 mmol/L BABA处理不能直接诱导果实上述抗病反应,但经10 mmol/L BABA 处理的果实在接种B. cinerea后则出现最显著的H2O2生成量、PRs转录水平和植保素含量上升现象。同时,低浓度 （10 mmol/L）BABA处理的葡萄果实中可溶性糖含量和甜度指数均显著高于高浓度（100、500 mmol/L）处理果 实。这些结果暗示,高浓度（100 mmol/L或500 mmol/L）BABA处理可诱导葡萄果实的直接抗病反应;10 mmol/L BABA处理则诱导Priming（敏化反应）作用,以使果实在病原菌侵染时展现强烈抗病性;同时,Priming抗性较直 接诱导作用可有效防止底物的过度消耗,以维持果实贮藏品质。     

超声诱导对发芽花生的转录组分析及苯丙烷类合成相关基因的挖掘

为解析超声处理对花生发芽过程中基因及代谢通路的变化差异,本研究以前期筛选出适宜富集白藜芦醇的花生品种为试验原料,以经超声诱导后的发芽花生(CS)为研究对象,并以未经诱导处理的发芽花生(KB)作为对照组开展试验.结果表明:共获得1104个差异表达基因(DEG),其中521个上调表达基因和583个下调基因.在差异表达基因的...     

普通烟草碳酸酐酶家族基因的生物信息学分析

为挖掘普通烟草碳酸酐酶基因的信息并探讨其功能,本研究利用生物信息学方法,在烟草基因组数据库中对普通烟草碳酸酐酶家族成员进行了检索,并对其理化性质、遗传进化、基因结构、蛋白保守基序、顺式作用元件和组织表达模式进行了分析。结果显示,在普通烟草中至少含有9个α和6个β亚家族成员。在进化关系上,不同亚家族成员之间,序列同源性较低;与水稻相比,拟南芥和普通烟草两个亚家族间的亲缘关系较近。亚细胞定位分析结果显示,烟草α亚家族成员在细胞壁、细胞膜、线粒体、叶绿体、细胞质等细胞器中均有分布,而β亚家族成员均存在于叶绿体中。基因结构和保守基序分析说明,各亚家族成员的基因结构和蛋白保守基序呈现较高的一致性。启动子顺式作用元件分析显示,烟草碳酸酐酶基因的启动子中,均含有多个参与光反应、激素响应、非生物胁迫和机械损伤等相关的顺式作用元件。基因组织表达模式分析表明,烟草的碳酸酐酶基因不同成员在不同组织表达水平存在差异,并呈现出时空特异性。本研究结果为烟草碳酸酐酶基因的功能研究奠定了基础。     

Preparation of a resveratrol-enriched grape juice based on ultraviolet C-treated berries

Grapes are rich in bioactive phenolics. However, most of them remain in the by-product after juice processing, and only a minor part pass to the juice. Our aim was to obtain a resveratrol-enriched white grape juice based on UVC-treated berries in combination with different conditions during juice production. Postharvest UVC treatment of berries enabled the further selective stilbenes enrichment of the juice, especially resveratrol. Macerating enzymes did not modify the phenolic profile but increased juice yield up to 30%, keeping its phenolic concentration, and thus reducing the phenolics remaining in the by-product. Maceration with Na₂S₂O₅ was critical to increase the phenolic content in the juice whereas β-cyclodextrin only tended to increase the stilbenes content. Optimum conditions for juice production (maceration for 2 h at 45 °C with 0.2% Na₂S₂O₅) using UVC-treated grape berries significantly increased stilbenes concentration (up to 35-fold over the control), without affecting the sensory properties of the juice. The phenolic (resveratrol)-enriched grape juice obtained here could be an ideal alternative to wine in the search of grape-derived health benefits.Industrial relevanceThe aim of this study is to obtain a resveratrol-enriched grape juice by combining postharvest UV-C treatment of grape berries with several technological approaches in the juice processing. Results obtained indicate that maceration for 2 h at 45 °C with 0.2% Na₂S₂O₅ using UVC-treated grape berries significantly increased phenolic concentration, especially stilbenes (up to 35-fold over the control), without affecting the sensory properties of the juice. The resveratrol-enriched juice has an added-value compared to the conventional ones and could exert more health benefits due to its improved phenolic profile.     

烟草抗冷相关基因在两种不同育苗方式下的低温应答差异分析

为探究水旱两段式育苗技术抗低温胁迫的分子机制,利用生物信息学方法分析了烟草低温应答关键转录因子CBF（C-repeat binding factor）基因家族的进化关系、基因结构、保守结构域及其启动子顺式作用元件,进一步利用qRT-PCR分析两种育苗方式下（常规漂浮育苗和水旱两段式育苗）NtCBF基因家族、CBF-regulon基因和非CBF-regulon相关基因的低温应答模式。结果表明,NtCBF15~NtCBF21基因与拟南芥CBF5、CBF6基因进化关系相近,NtCBF1~NtCBF14基因与拟南芥CBF1~CBF4基因进化关系相近。NtCBF基因家族成员均具有motif 1/2/4/5保守蛋白结构域,表明该家族在进化过程中较为保守;其中,NtCBF3、NtCBF4、NtCBF5、NtCBF6、NtCBF10、NtCBF13、NtCBF14和NtCBF18等8个基因启动子区具有低温响应元件。低温处理后,NtCBF5、NtCBF12和NtCBF13及CBF-regulon基因中NtRD29A、NtRD29B和NtGOLS3的表达量均显著升高。推测NtCBF5、NtCBF12和NtCBF13及其下游调控基因NtRD29A、NtRD29B和NtGOLS3在水旱两段式育苗抗低温胁迫过程中起重要作用。      

Influence of pressing method on juice stilbene content in muscadine and bunch grapes

ABSTRACT:  Juice stilbene contents of 2 muscadine ( Vitis rotundifolia) cultivars, Noble and Carlos, and 2 bunch grape ( Vitis labrusca) cultivars, Mid South and Miss Blanc were evaluated after crushing and pressing. Four commercial pressing methods were used to produce juice: (1) cold press of fresh berries (CP); (2) hot press of fresh berries (HP); (3) cold press of frozen and thawed berries (FCP); and (4) cold press of pectolytic enzyme treated berries (ECP). Free run juice (FRJ) from the crusher without pressing was also tested. An HPLC with a UV detector was used to verify the presence and quantity of resveratrol, piceid, and their isomers. For the muscadine juices, total stilbenes were greater for HP and FCP than for FRJ and CP treatments. Trans -resveratrol was only detected in HP juices of the muscadines at levels near the detection limits. The bunch grape cultivars behaved similar to the muscadines in the processing treatments. HP juice had the greatest total stilbene levels and was significantly greater than FRJ, CP, and FCP. Total stilbene levels in juices from the 2 bunch grape cultivars, were higher (up to 5 times greater depending on pressing method) than for the muscadine grape cultivars. Total stilbene levels in muscadine juices treated with pectinase before pressing (ECP) were similar to those of the FCP samples. Juice from FCP and ECP treated Noble muscadine had total stilbene levels greater than FRJ and CP but less than HP levels. Juices from HP-, ECP-, and FCP-treated Carlos muscadine had total stilbene levels greater than for FRJ and CP juices.     

Environmental factors affect the activity of biocontrol agents against ochratoxigenic Aspergillus carbonarius on wine grape

The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents-the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2-against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30°C). The three biocontrol agents were able to prevent infections at 60% RH and 20°C. At 60% RH and 25°C only strain AU34-2 achieved some protection on day 5, whereas at 30°C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20°C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25°C, and only strain LS16 at 30°C. After 5days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30°C and 100% of RH for all the three strains, and at 25°C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25°C than at 30°C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25°C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.     

The allantoin and uracil permease gene sequences of Saccharomyces cerevisiae are nearly identical.

We have determined the structure of the allantoin permease (DAL4) gene of Saccharomyces cerevisiae. The gene putatively encodes a hydrophobic protein with a M(r) of 71,755. It possesses the alternating hydrophobic-hydrophilic regions similar to those found in many other integral membrane proteins. The most striking feature of the allantoin permease component encoded by DAL4 is its striking similarity to the uracil permease component encoded by FUR4. Although data available indicate that these proteins do not share any overlap of function, their predicted protein sequences are 68% identical, 81% similar, and their DNA sequences are 70% identical. The upstream regulatory region of DAL4 contains all of the characterized cis-acting elements previously reported for inducible allantoin pathway genes: six sequences homologous to UASNTR, the element responsible for nitrogen catabolite repression-sensitive activation of allantoin pathway gene expression, and two sequences homologous to the cis-acting element responsible for inducer-responsiveness of the allantoin pathway genes, UIS. The finding of these homologous sequences predicted to exist on the basis of DAL4's expression characteristics, supports and strengthens the suggestion that these elements mediate the functions we have previously ascribed to them.