玉米转录因子 ZmNAC59 调控植物抗盐性

【目的】NAC转录因子家族是植物中研究较多的一类转录因子,在调控植物生长发育和响应非生物胁迫过程中起重要作用。玉米是三大粮食作物之一,其生长过程常会面临各种逆境胁迫,盐害被认为是限制作物生长和生产的主要环境因素之一。因此鉴定玉米抗盐基因,解析其抗盐机制对培育玉米抗逆品种具有重要意义。【方法】研究克隆了玉米转录因子ZmNAC59,并用生物信息学手段分析其保守结构域和系统进化关系,用实时荧光定量PCR方法分析该基因在NaCl和MeJA处理下的表达模式,并用稳定转基因体系将该基因异源表达拟南芥观察表型,同时还用病毒诱导沉默技术将该基因在玉米中沉默后进行盐处理表型观察,并进行酶活性检测。【结果】ZmNAC59能够被NaCl和MeJA诱导上调。病毒诱导沉默ZmNAC59后进行盐胁迫,沉默株系对盐胁迫更敏感,ROS积累更多;而ZmNAC59过表达拟南芥后,过表达株系在盐胁迫处理下存活率更高,活性氧积累更少,Na⁺/K⁺比率低,表明ZmNAC59作为盐胁迫中的正调控因子可以通过调节离子流动提高植物抗盐性。实时荧光定量PCR结果表明拟南芥过表达株系中Na     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

玉米磷转运蛋白基因ZmPht3;1的克隆和功能分析

Pht3(phosphate transporter 3)磷转运子家族属于一类低亲和力磷转运蛋白,在调节植株体内磷素的动态平衡中发挥重要作用。为了初步探讨玉米中Zm Pht3;1基因的结构特征及其磷饥饿的响应机制,利用同源克隆的方法从耐低磷玉米自交系Mo17中分离得到Zm Pht3;1基因,并运用实时荧光定量PCR和亚细胞定位的方法对其进行深入研究。结果表明,Zm Pht3;1的编码区全长1 101 bp,编码366个氨基酸,含有典型的线粒体转运家族(mitochondrial carrier family,MCF)结构特征与6个疏水跨膜结构。荧光定量PCR分析表明,该基因在两个极端材料的根系与叶片中均有表达,而表达模式差异显著,在耐低磷玉米自交系Mo17的根系和叶片中表现为缺磷胁迫前期的一般性反应和后期的特异性反应。转化烟草的亚细胞定位结果显示,Zm Pht3;1主要分布于细胞膜上,可能是一个双亲和转运体,在玉米响应磷饥饿胁迫过程中发挥重要的适应性调节作用。     

玉米中介体亚基ZmMED7基因的克隆及表达分析

为了研究玉米中介体亚基ZmMED7基因的功能,通过荧光定量PCR技术,研究ZmMED7-1和ZmMED7-2在玉米不同组织中的表达及在ABA处理、盐害和渗透胁迫等逆境条件下的应答反应,并将ZmMED7-1和ZmMED7-2转入拟南芥中研究其基因功能。结果显示,ZmMED7在玉米不同发育时期的组织器官中均有表达,ZmMED7-1和ZmMED7-2分别在V1和V5期的叶片中表达量最高。ABA、盐害和渗透胁迫等处理都能够抑制ZmMED7-1和ZmMED7-2基因的表达。过表达ZmMED7-1导致拟南芥在种子萌发时期对盐胁迫的耐受能力减弱。研究结果说明:ZmMED7基因作为负调控因子参与了植物逆境胁迫应答反应。     

玉米逆境胁迫响应基因ZmbZIP71的克隆与表达分析

从玉米抗旱自交系CN165中克隆得到了与逆境胁迫相关的bZIP（Basic Leucine Zipper Protein）基因ZmbZIP71。ZmbZIP71基因的开放阅读框为471 bp,编码156个氨基酸,相对分子量为17.59 kDa,等电点pI为9.24。ZmbZIP71蛋白包含真核生物中高度保守的bZIP结构域。ZmbZIP71基因编码区的基因组序列全长为1050 bp,包括2个外显子和1个内含子。利用实时荧光定量PCR方法分析ZmbZIP71基因在玉米不同组织中的表达差异及其在非生物胁迫下的表达模式,结果表明,该基因在雄穗和雌穗中的表达量较高,并且受干旱、低温和ABA的胁迫诱导上调表达,在盐胁迫下下调表达。     

中间锦鸡儿WRKY75基因对拟南芥耐受盐和ABA能力的影响

该研究从旱生灌木中间锦鸡儿中克隆得到1个CiWRKY75基因。序列分析显示,CiWRKY75开放阅读框长570bp,编码189个氨基酸,含有1个WRKYGQK基序和1个C2H2型锌指结构,属于第二类WRKY转录因子。亚细胞定位显示,CiWRKY75定位于细胞核。实时荧光定量PCR检测表明,CiWRKY75基因的表达受盐胁迫和ABA诱导。在拟南芥中过量表达CiWRKY75后,与野生型拟南芥相比,转基因株系种子的萌发率在盐胁迫下降低,并且对盐胁迫的耐受能力明显减弱;ABA处理下,2个转基因株系的种子萌发率(10.3%、9.6%)较野生型(25.9%)明显降低。研究表明,CiWRKY75是中间锦鸡儿对盐和ABA响应的重要调控因子。     

玉米液泡膜焦磷酸酶基因ZmVPP1的克隆及逆境下的表达分析

采用同源克隆的方法在玉米中得到一个与拟南芥耐盐基因AVP1类似的基因.BLAST分析表明,该基因编码蛋白属于质子泵焦磷酸酶家族(H_PPase superfamily)中的Ⅰ型VPP,将其命名为ZmVPP1.ZmVPP1包舍一个2301bp的开放阅读框,编码766个氨基酸残基.蛋白比对结果表明,该蛋白在不同植物中相当保守.实时荧光定量PCR检测发现,ZmVPP1基因在成熟叶片中高丰度表达,在生殖器官中表达量较少.脱水、高盐、低温等逆境胁迫条件和ABA处理下的表达分析表明,ZmVPP1基因受逆境的诱导表达,属于不依赖于ABA的途径.由此推断,ZmVPP1可能参与玉米对Na+的隔离,从而起到耐盐的作用.     

野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料, 采用同源克隆方法和RT-PCR技术获得一个TIFY 类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明, 与其他物种相比, GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高, 达到56%; 序列分析表明GsTIFY11b蛋白除具有 TIFY保守结构域外, 还具有一个N端保守结构域和一个C端保守的Jas结构域; 实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达; 将GsTIFY11b转化拟南芥来验证其耐盐碱功能, 获得两个转基因纯合体株系, 盐碱胁迫分析结果表明, GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性, 并且与野生型相比, 转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明, GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明, GsTIFY11b定位于细胞核中。上述结果表明, 该基因在细胞核中起着转录调节子的作用, 可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料,采用同源克隆方法和RT-PCR技术获得一个TIFY类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明,与其他物种相比,GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高,达到56%;序列分析表明GsTIFY11b蛋白除具有TIFY保守结构域外,还具有一个N端保守结构域和一个C端保守的Jas结构域;实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达;将GsTIFY11b转化拟南芥来验证其耐盐碱功能,获得两个转基因纯合体株系,盐碱胁迫分析结果表明,GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性,并且与野生型相比,转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明,GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明,GsTIFY11b定位于细胞核中。上述结果表明,该基因在细胞核中起着转录调节子的作用,可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

盐胁迫诱导盐穗木Rev1、Rev3基因的表达分析

根据盐穗木盐胁迫下响应的转录组测序结果,参考盐穗木HcRev1、HcRev3基因的ESTs序列设计荧光定量PCR特异性引物,建立检测盐穗木Revs基因相对表达量的荧光定量PCR方法,分析Rev1和Rev3基因在盐穗木不同浓度盐胁迫处理不同时间的转录水平。结果表明,HcRev1、HcRev3基因具有相似的表达模式,在100 mmol·L^-1 NaCl低盐胁迫下表达稳定,在300、500、700 mmol·L^-1 NaCl胁迫下,随胁迫浓度增高、胁迫时间延长,表达量升高。其中HcRev1在700 mmol·L^-1 NaCl胁迫14 d后达到峰值,是对照组的4.63倍。HcRev3基因在300 mmol·L^-1 NaCl胁迫14 d时,表达量迅速升高,是对照组的15.55倍,表达差异极显著。研究结果说明HcRev1、HcRev3基因都受盐胁迫诱导表达,提示HcRev1、HcRev3基因虽然表达量存在差异,但在盐胁迫过程中参与了DNA损伤修复。研究有助于阐明Rev1、Rev3基因在DNA损伤修复和植物耐盐性间的调控功能作用。     

产毒和非产毒霍乱弧菌在甘露醇 和Luria-Bertani培养液中的基因表达差异

摘要：【目的】分析霍乱弧菌产毒株和非产毒株在甘露醇发酵液和LB (Luria-Bertani) 培养液中生长的基因表达谱和代谢差异特征。【方法】提取甘露醇发酵液和LB培养液中霍乱弧菌甘露醇慢发酵株（产毒株）N16961和快发酵株（非产毒株）93097生长第一小时的总RNA,应用霍乱弧菌N16961基因组芯片分析各菌株在不同培养液中的表达差异基因。【结果】 筛选出产毒株N16961在甘露醇发酵液和LB中表达差异基因142个,非产毒株93097有418个,这些表达差异基因主要分属于6个不同的功能类群,主要是转运结合、能量代谢以及蛋白质合成代谢功能。【结论】甘露醇发酵液和LB中产毒株和非产毒株的许多功能基因的转录水平有显著差异,这些表达差异基因可能与霍乱弧菌在甘露醇发酵液中代谢产酸有关,这为进一步分析霍乱弧菌代谢甘露醇的机制、以及分析产毒株与非产毒株的甘露醇发酵快慢机制提供了基础。     

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis -regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.     

Calcium signaling-mediated and differential induction of calmodulin gene expression by stress in Oryza sativa L

Ca(2+)/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of Ca(2+)-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic Ca(2+)-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stressinduced expression of OsCam1 gene requires the [Ca(2+)]cyt elevation that is known to occur in response to these stimuli.     

Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.Keyword: Abiotic stress, Cinnamoyl CoA reductase, Developing seedlings, Leucaena leucocephala     

Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.     

Transgenic peanut overexpressing <Emphasis Type="Italic">mtlD</Emphasis> gene confers enhanced salinity stress tolerance via mannitol accumulation and differential antioxidative responses

Globally, peanut is an important oilseed crop, which is cultivated under different agro-climatic zones. Soil salinity is one of the major constraints in peanut cultivation. Therefore, to understand the physio-biochemical mechanisms imparting salinity stress, four transgenic peanut lines (cv. GG20) already developed and confirmed by our lab, having bacterial mannitol dehydrogenase gene (mtlD), were subjected to different levels of salinity stresses (1, 2 and 3 dS m^?1) in pots under containment facility. Further, these lines were also characterized for various physio-biochemical parameters at flowering, pegging and pod formation stages. All the transgenic lines recorded significantly higher mannitol dehydrogenase (MTD) activity and mannitol accumulation than the wild type (WT). Under salinity stress, significantly higher levels of superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase activities, while significantly lower levels of H₂O₂ and malondialdehyde contents, were recorded in the transgenics compared to WT. Similarly, significantly higher ascorbic acid and relative water content (RWC) were recorded in transgenic lines. The MTD activity showed positive correlation with various antioxidant enzymes, growth parameters and RWC, while negative correlation was recorded with H₂O₂ and malondialdehyde content at most of the plant growth stages. The mtlD transgenic peanut lines under pot conditions were found maintaining lower oxidative injuries, indicating amelioration of salinity-induced oxidative stress by enhanced protection mechanisms via mannitol accumulation and antioxidative responses. The best lines identified (MTD1 and MTD4) may be used further as pre-breeding source for imparting salinity stress tolerance in peanut. Besides, these lines may also be tested under open-field trials for release as salt-tolerant variety.