柠条锦鸡儿CkLEA1基因克隆及表达分析

植物受到逆境胁迫后,大量逆境响应基因会被诱导表达,LEA蛋白编码基因就是与植物抗旱、抗冷等非生物胁迫密切相关的一类基因.从已构建的柠条锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一条LEA蛋白编码基因并进行了克隆.序列比对与系统进化分析显示该基因属于LEA3基因家族成员,命名为CkLEA1(GenBank登录号是KC309408).克隆得到该基因gDNA长469bp,包含两个外显子和一个内含子;cDNA长357bp,包含300bp的开放阅读框,推导编码99个氨基酸的蛋白质.利用荧光定量PCR技术对CkLEA1基因在各种逆境胁迫条件的表达情况进行初步研究表明,CkLEA1受干旱、ABA、冷、热、盐和碱等处理不同程度地诱导,推测其与柠条锦鸡儿响应逆境胁迫的机制有关.     

植物蛋白激酶与作物非生物胁迫抗性的研究

干旱、盐碱、高温等非生物逆境胁迫严重影响作物生长发育、产量和品质。在遭受非生物逆境的威胁时,植物通过信号受体,可感知、转导胁迫信号,启动一系列抗逆相关基因的表达,最终缓解或抵御非生物逆境胁迫对植物造成的危害。其中,蛋白激酶和蛋白磷酸酯酶的磷酸化/去磷酸化作用在植物感受外界胁迫信号的分子传递过程中起到开关的作用。正常情况下,蛋白激酶磷酸化开启信号转导途径,启动相应的抗逆基因表达反应;当信号消失后,蛋白激酶去磷酸化将信号转导途径关闭,达到调控植物正常生长的目的。因此,蛋白激酶在调控感受胁迫信号、启动各种非生物逆境胁迫响应中起到了极其重要的作用。近年来,对植物蛋白激酶参与非生物胁迫响应的研究倍受关注。本文阐述了不同类型蛋白激酶在改良作物非生物胁迫抗性上的应用,为进一步研究提供资料。     

甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析

MYB转录因子具有多种生物学功能,在植物响应生物和非生物胁迫中发挥重要作用。该文从盐胁迫后的甘薯(Ipomoea batatas)水培苗转录组数据(RNA-seq)中筛选出2个受盐胁迫显著上调表达的MYB基因,分别命名为IbMYB3和IbMYB4。多种非生物胁迫和植物生长物质处理下的基因表达分析显示,IbMYB3受逆境诱导显著上调表达,暗示其可能参与甘薯非生物胁迫响应。生物信息学分析表明,IbMYB3开放阅读框长度为1059 bp,编码353个氨基酸残基,蛋白分子量为39.41 kDa,理论等电点(PI)为5.26,为酸性带负电的亲水性蛋白。亚细胞定位结果表明,IbMYB3蛋白定位于细胞核,具有较强的转录激活活性。上述结果表明,IbMYB3转录因子可能在甘薯非生物胁迫响应过程中发挥重要调控作用,研究结果为进一步探明IbMYB3基因的功能奠定了基础。     

水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应

锌指蛋白在调控植物生长发育和应对逆境过程中发挥着重要作用.为进一步研究锌指类蛋白参与植物非生物胁迫响应的分子机制,对水稻(Oryza sativa)中一个编码含有B-box锌指结构域蛋白的OsBBX25基因进行了功能分析.OsBBX25受盐、干旱和ABA诱导表达.异源表达OsBBX25的转基因拟南芥(Arabidopsis thaliana)与野生型相比对盐和干旱的耐受性增强,且盐胁迫条件下转基因植物中KIN1、RD29A和COR15的表达上调,干旱胁迫下KIN1、RD29A和RD22的表达上调.外源施加ABA时,转基因植物的萌发率与野生型之间没有明显差异.OsBBX25可能作为转录调控的辅助因子调节胁迫应答相关基因的表达,进而参与植物对非生物胁迫的响应.     

一个特异水稻原纤蛋白FBN11的生物信息学和基因表达特性分析

Fibrillin 11(FBN 11)是植物质体中FBN蛋白家族的重要成员之一,比其他成员多300~500个氨基酸残基,说明其可能存在某些特异性结构和功能。本研究在水稻幼苗中扩增获得到了一个受非生物逆境胁迫诱导的OsFBN11基因的全长cDNA,该基因含有12个内含子和13个外显子。其编码蛋白的分子量为72.36 kD,pI值为9.26。生物信息学分析结果表明,该蛋白含有无规则卷曲、α螺旋和β-折叠3种氨基酸二级结构,不含有跨膜结构域,蛋白亲水性强,PSORT软件预测该蛋白可能定位于叶绿体中。进一步对14种植物FBN11蛋白的同源性和5个物种该蛋白的保守结构域分析,发现该蛋白兼有典型的PAP-Fibrillin结构域和蛋白激酶PKc结构域。水稻全生育期芯片分析显示该基因主要在愈伤组织、叶片和根系中高水平表达。RT-PCR检测结果显示,该基因在水稻幼苗中受ABA、NaCl和干旱胁迫处理上调表达。上述结果表明,OsFBN11可能在水稻质体发育和抗非生物胁迫反应中发挥重要作用。     

青杄PwNAC42基因的克隆及表达模式分析

NAC转录因子是植物中最大的转录因子家族之一,广泛参与了植物的生长发育过程,并在植物响应盐害、干旱、冷害和脱落酸等多种非生物胁迫的过程中发挥重要作用。通过青杄转录组数据获得PwNAC42基因的cDNA序列,对其蛋白序列进行生物信息学预测,通过实时荧光定量PCR检测PwNAC42基因在青杄各个组织及非生物逆境胁迫下的表达水平。生物信息学分析结果显示:PwNAC42基因cDNA全长共1 749 bp,其中编码区1 140 bp,共编码379个氨基酸;蛋白理论分子质量为42.92 k D,理论等电点为6.53,有丝氨酸、苏氨酸和酪氨酸磷酸化位点,不存在信号肽结构域,为非跨膜的亲水蛋白。组织特异性表达结果显示,PwNAC42在青杄球果中表达量最高。逆境胁迫实验表明PwNAC42的表达量在NaCl、干旱、4℃以及脱落酸处理下产生显著的变化。因此推测PwNAC42在青杄球果发育及青杄对逆境胁迫的响应过程中发挥作用。     

二穗短柄草根部木栓质调控基因BdMYB92的克隆及功能研究

为了探究二穗短柄草(Brachypodium distachyon)根部木栓质合成的调控机制,该研究以二穗短柄草Bd21为试验材料,利用生物信息学分析方法克隆了二穗短柄草根部木栓质合成的调控转录因子基因BdMYB92(GenBank登录号为OP497966);采用荧光定量PCR方法,分析BdMYB92基因在二穗短柄草不同组织中的表达模式以及对6种非生物胁迫处理(空气中干旱、20%PEG-6000模拟干旱、4℃中冷处理、200 mmol/L NaCl、100μmol/L ABA和机械损伤)的响应表达特征;利用双荧光素酶和酵母单杂交验证BdMYB92蛋白和BdFAR4基因启动子的互作关系。结果表明：(1)二穗短柄草BdMYB92基因cDNA全长为1 343 bp,开放阅读框为990 bp,编码329个氨基酸,蛋白分子量为36.4 kD,理论等电点为5.54。(2)亚细胞定位结果显示,BdMYB92定位在细胞核。(3)荧光定量PCR分析表明,BdMYB92在二穗短柄草根中的表达量显著高于叶鞘、节、穗、节间等其他组织;6种非生物胁迫处理均能诱导BdMYB92的上调表达,且对干旱胁迫的响应最为迅...     

甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析

MYB转录因子具有多种生物学功能,在植物响应生物和非生物胁迫中发挥重要作用。该文从盐胁迫后的甘薯(Ipomoeabatatas)水培苗转录组数据(RNA-seq)中筛选出2个受盐胁迫显著上调表达的MYB基因,分别命名为IbMYB3和IbMYB4。多种非生物胁迫和植物生长物质处理下的基因表达分析显示, IbMYB3受逆境诱导显著上调表达,暗示其可能参与甘薯非生物胁迫响应。生物信息学分析表明,IbMYB3开放阅读框长度为1059bp,编码353个氨基酸残基,蛋白分子量为39.41kDa,理论等电点(PI)为5.26,为酸性带负电的亲水性蛋白。亚细胞定位结果表明,IbMYB3蛋白定位于细胞核,具有较强的转录激活活性。上述结果表明, IbMYB3转录因子可能在甘薯非生物胁迫响应过程中发挥重要调控作用,研究结果为进一步探明IbMYB3基因的功能奠定了基础。     

小桐子JcCIPK2基因的克隆与表达分析

钙调磷酸酶B类似蛋白互作蛋白激酶(CBL-interacting protein kinase, CIPK)是一类植物中特有的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,参与多种生物和非生物胁迫响应过程。该实验通过RT-PCR克隆了小桐子(Jatropha curcas L.)JcCIPK2基因cDNA全长序列,采用荧光定量qRT-PCR分析JcCIPK2基因在不同组织及不同处理(12℃和1℃低温、42℃高温、30%PEG、250 mmol/L NaCl、150μmol/L ABA)下的表达模式。结果显示:(1)小桐子JcCIPK2基因开放阅读框全长1 398 bp,编码465个氨基酸,相对分子量为52.95 kD,等电点为8.89。(2)蛋白质结构分析表明,JcCIPK2的N端含有位于第11～265个氨基酸之间的丝氨酸/苏氨酸激酶_蔗糖非发酵-1型相关蛋白激酶3催化域STKc_SnRK3,在激酶结构域内还具有激活环(Activation Loop);C端含有位于第316～430个氨基酸之间的CIPK蛋白激酶调控域CIPK_C,其调控域中含有CIPK家族典型的能与CBL特异性结合的NAF结构域,位于第314～369个氨基酸之间。(3)系统进化分析显示,小桐子JcCIPK2蛋白与同属于大戟科的木薯(Manihot esculenta Crantz.)同源关系最近,序列一致性达87%。(4)qRT-PCR分析表明,JcCIPK2基因在小桐子根、茎、叶中均有表达,经12℃和1℃低温处理后,叶片中JcCIPK2基因的表达都呈现先上调后下调表达的趋势,且都在低温处理24 h的表达量最高,与对照相比分别上调了6.0倍和16.72倍;小桐子JcCIPK2基因在42℃高温、30%PEG、150μmol/L ABA、250 mmol/L NaCl处理下也受到不同程度的诱导表达。研究推测,JcCIPK2基因在小桐子对逆境的响应与适应中起重要作用。     

非生物逆境胁迫下ZmCIPK10基因表达分析

干旱、高盐、低温、高温等非生物逆境严重影响植物的生长发育,研究参与逆境胁迫应答基因具有重要的理论意义和应用价值。从玉米中克隆了一个CIPK蛋白激酶基因,暂时命名为ZmCIPK10。该基因全长2 730 bp,转录长度2 100 bp,编码438个氨基酸。顺式元件分析发现在基因启动子区域存在ABRE、HSE、TC-rich repeats等推测的逆境顺势元件。荧光实时定量PCR结果表明ZmCIPK10在干旱、低温、盐胁迫下表达量上调,在ABA、高温胁迫下表达量下调。研究结果初步证实ZmCIPK10基因响应非生物逆境胁迫,为ZmCIPK10参与植物逆境信号途径及其功能研究提供理论依据。     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

水稻中水杨酸和茉莉酸特异诱导蛋白激酶基因OsSJMK1的分离和鉴定

裂原激活蛋白激酶（MAPK）级联系统负责把接受自胞外或胞内的信号进一步传递和放大而最终作用于特异的转录因子,从而启动或调控基因的表达。MAPK信号级联系统在细胞分裂、分化、生物胁迫和非生物胁迫等多种信号传递途径中起着十分重要的作用。该文以一个茉莉酸（JA）诱导的表达序列为基础,在水稻中分离到了一个裂原激活蛋白激酶基因OsSJMK1的全长cDNA。序列比较分析表明该基因编码498个氨基酸,蛋白质等电点为8．43,包括完整的MAPK家族的蛋白激酶结构域。OsSJMK1与所有物种的MAPK一样包括蛋白激酶的全部11个次级结构域,在Ⅶ和Ⅷ次级结构域间一个双磷酸化位点;该位点苏氨酸（T）和酪氨酸（Y）残基之间为天冬氨酸（D）,而不是其他MAPK中常见的谷氨酸（E）、脯氨酸（P）或甘氨酸（G）。除了典型的MAPK激酶功能域外,在羧基端还有一段长约150个氨基酸残基的可能参与蛋白互作的结构域。以上这些结构特征表明OsSJMK1属于植物中第v类MAPK家族成员。蛋白激酶结构域序列比较表明OsSJMK1与报道的稻瘟病菌和机械伤害诱导的BWMK1的序列相似性高达81％,而且基因内含子和外显子的组成也非常相似,属于同一亚类,但在蛋白质序列的C端差异却很大。与BWMK1不同,OsSJMK1的表达不受伤害诱导,而受稻瘟病轻微诱导,但在JA和SA（水杨酸）处理早期表达量却迅速升高。在JA处理后1h,OsSJMK1转录水平升高到最大,而12h后回落到处理前的本底水平;在SA处理后30min转录水平就开始上升,2h达到最高值,而随后开始下降到处理前的本底水平。SA类似物BTH也能诱导OsSJMK1的表达。其他一些激素处理（如ABA）和非生物胁迫（如干旱、盐胁迫）都不对基因的表达产生任何影响,而且在植物大部分组织中的表达量都非常低。这些结果说明OsSJMK1可能特异性的参与JA和SA介导的防卫反应。     

Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes from plant growth and development to biotic and abiotic stress responses. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), play crucial roles in MAPK cascades to mediate a variety of stress responses in plants. However, few MAPKKs have been functionally characterized in cotton (Gossypium hirsutum). In this study, a novel gene, GhMKK5, from cotton belonging to the group C MAPKKs was isolated and characterized. The expression of GhMKK5 can be induced by pathogen infection, abiotic stresses, and multiple defence-related signal molecules. The overexpression of GhMKK5 in Nicotiana benthamiana enhanced the plants' resistance to the bacterial pathogen Ralstonia solanacearum by elevating the expression of pathogen resistance (PR) genes, including PR1a, PR2, PR4, PR5, and NPR1, but increased the plants' sensitivity to the oomycete pathogen Phytophthora parasitica var. nicotianae Tucker. Importantly, GhMKK5-overexpressing plants displayed markedly elevated expression of reactive oxygen species-related and cell death marker genes, such as NtRbohA and NtCDM, and resulted in hypersensitive response (HR)-like cell death characterized by the accumulation of H(2)O(2). Furthermore, it was demonstrated that GhMKK5 overexpression in plants reduced their tolerance to salt and drought stresses, as determined by statistical analysis of seed germination, root length, leaf water loss, and survival rate. Drought obviously accelerated the cell death phenomenon in GhMKK5-overexpressing plants. These results suggest that GhMKK5 may play an important role in pathogen infection and the regulation of the salt and drought stress responses in plants.     

褪黑素改善认知作用研究进展

对褪黑素改善认知作用的研究进展进行综述,为褪黑素的进一步开发利用提供科学依据。     

A cotton group C MAP kinase gene, GhMPK2, positively regulates salt and drought tolerance in tobacco

Mitogen-activated protein kinase (MAPK) cascades play important roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Compared with well-studied MAPKs in groups A and B, little is known about group C. In this study, we functionally characterised a stress-responsive group C MAPK gene (GhMPK2) from cotton (Gossypium hirsutum). Northern blot analysis indicated that GhMPK2 was induced by abscisic acid (ABA) and abiotic stresses, such as NaCl, PEG, and dehydration. Subcellular localization analysis suggested that GhMPK2 may activate its specific targets in the nucleus. Constitutive overexpression of GhMPK2 in tobacco (Nicotiana tabacum) conferred reduced sensitivity to ABA during both seed germination and vegetative growth. Interestingly, transgenic plants had a decreased rate of water loss and exhibited enhanced drought and salt tolerance. Additionally, transgenic plants showed improved osmotic adjustment capacity, elevated proline accumulation and up-regulated expression of several stress-related genes, including DIN1, Osmotin and NtLEA5. β-glucuronidase (GUS) expression driven by the GhMPK2 promoter was clearly enhanced by treatment with NaCl, PEG, and ABA. These results strongly suggest that GhMPK2 positively regulates salt and drought tolerance in transgenic plants.     

Systematic analysis of <Emphasis Type="Italic">NPK1</Emphasis>-like genes in rice reveals a stress-inducible gene cluster co-localized with a quantitative trait locus of drought resistance

Phosphorylation by protein kinase is a ubiquitous key mechanism in translating external stimuli such as drought stress. NPK1 is a mitogen-activated protein kinase kinase kinase identified in Nicotiana tabacum and plays important roles in cytokinesis and auxin signaling transduction and responses to multiple stresses. Here we report the evolution, structure, and comprehensive expression profile of 21 NPK1-like genes in rice (Oryza sativa L.). Phylogenetic analysis of NPK1-like sequences in rice (OsNPKL), Arabidopsis, and other plants reveals that NPK1-like genes could be classified into three subgroups. Three OsNPKL gene clusters, located on chromosome 1 (OsNPKL1, 2, 3, and 4), 5 (OsNPKL14 and 15), and 10 (OsNPKL19 and 20), respectively, were identified in the rice genome. These clustered genes, which most likely evolved by tandem gene duplication, belong to the same phylogenetic subgroup, with similar genomic structures and conserved motifs in the kinase domain, which is unique to this subgroup. Expression analysis of OsNPKL genes under abiotic stresses suggests that the stress-responsive genes are mainly from the same subgroup. Especially interesting is that all the clustered genes are induced by drought, salt, or cold stress, and a few members are very strongly induced by drought. Some of the clustered genes are also induced by abscisic acid. The gene cluster on chromosome 1 is co-located with a quantitative trait locus (QTL) related to drought resistance. Although the drought-induced expression levels of the four genes in the cluster show no difference between the two parents used for QTL mapping, sequence variation in coding regions of the genes between the parents has provided some clues for further functional characterization of this gene cluster in abiotic stress tolerance in rice.