CBL-CIPK信号系统参与小桐子抗冷性形成的生物信息学分析

类钙调磷酸酶B亚基蛋白(calcineurin B-like calcium sensor,CBL)属Ca²⁺结合蛋白,通过与类钙调磷酸酶B亚基互作蛋白激酶(calcineurin B-like calcium sensor interacting protein kinase,CIPK)互作介导Ca²⁺信号转导过程。CBL-CIPK信号系统参与了植物对多种逆境胁迫的响应过程。为深入探讨小桐子的抗冷性机制,该研究基于BLAST序列比对的方法,在全基因组水平对小桐子CBL与CIPK基因家族进行了鉴定,并对其系统进化、基因结构、表达特性及功能互作进行了解析。结果表明:(1)在小桐子基因组中共鉴定到8个CBL基因与18个CIPK基因,CBL与CIPK蛋白长度分别在211～257 aa与422～484 aa之间,等电点分别在4.65～5.08与6.20～9.26之间。(2)另外,CBL基因家族都包含8～10个外显子,而CIPK基因家族分为显著的1～2个外显子(11个基因)和12～15个外显子(7个基因)两类。(3)多序列比对显示,小桐子CBL蛋白都鉴定到1个由14个氨基酸残基组成的非典型EF-hand基序与3个取代程度不同的典型EF-hand基序,而CIPK蛋白都包含N端激酶结构域与C端自抑制FISL/NAF结构域。(4)染色体定位显示,26个小桐子CBL与CIPK基因不均匀地分布于9条染色体上。(5)转录组数据分析表明,大部分CBL与CIPK基因在小桐子叶片、根及种子中都有高水平表达,其中JcCIPK14与JcCIPK18在低温处理时上调表达量达到了极显著水平(P<0.01),参与小桐子的抗冷性过程。综上结果为开展小桐子CBL和CIPK基因的功能鉴定与低温信号转导机制研究提供了借鉴。     

小桐子植物特异性酪蛋白激酶PS-CK1-5基因的克隆及原核表达分析

酪蛋白激酶(casein kinase, CK)作为一类普遍存在的Ser/Thr蛋白激酶，通过调节靶标蛋白的活性与稳定性，在植物整个生理过程及信号转导途径中发挥重要作用。基于同源序列比对，该研究对小桐子(Jatropha curcas)酪蛋白激酶基因家族进行鉴定与表达分析。结果表明，小桐子基因组中共鉴定到7个酪蛋白激酶1基因(CK1)、5个植物特异性酪蛋白激酶1基因(PS-CK1)、3个酪蛋白激酶2α亚基基因(CK2-α)、2个酪蛋白激酶2β亚基基因(CK2-β)，4个亚家族成员在氨基酸长度、等电点及外显子数目等都有其家族特异性。蛋白的氨基酸序列比对表明，小桐子酪蛋白激酶1都包含N端保守激酶结构域，同时其内部都鉴定到典型的激酶活性环基序、ATP结合核心基序、核定位信号肽。qRT-PCR表达分析表明，小桐子JcPS-CK1-5基因在叶片与根中都属于低温诱导基因，可能参与小桐子抗冷性过程。构建其原核表达载体pET-32a-JcPS-CK1-5，并在BL21(DE3)中诱导表达，得到81.6 kD的条带，与理论融合蛋白的分子量一致。这可为小桐子CK基因的功能鉴定及逆境信号转导机制研究提供参考。     

香鳞毛蕨 1 脱氧 D 木酮糖 5 磷酸合酶基因的克隆及表达分析

1 脱氧 D 木酮糖 5 磷酸合酶(DXS)是甲基 D 赤藓醇 4 磷酸(MEP)途径中控制影响植物萜类化合物合成的第一个限速酶。该研究对香鳞毛蕨(Dryopteris fragrans)DfDXS基因进行序列特征及生物信息学分析,并通过qRT PCR技术分析其在外源激素、干旱、盐胁迫、高温及低温处理下的表达模式,旨在探究DfDXS基因在香鳞毛蕨萜类生物合成及抗逆机制中的作用,为进一步解析香鳞毛蕨抗逆分子机制奠定基础。结果显示：(1) DfDXS1基因全长2 139 bp,编码712个氨基酸,而DfDXS2全长2 160 bp,编码719个氨基酸;结构域分析显示,其具有典型的转酮醇酶保守域,包含焦磷酸硫胺素结合位点和转酮醇酶结构域;DfDXS氨基酸序列与江南卷柏(Selaginella moellendorffii)和银杏(Ginkgo biloba)的DXS等关系较近。(2)水杨酸(SA)处理下,DfDXS基因的相对表达量先升高后降低;脱落酸(ABA)抑制DfDXS的表达;DfDXS1/2在茉莉酸甲酯(MeJA)处理下相对表达水平均显著高于对照;乙烯利(Eth)抑制DfDXS的表达,但DfDXS1处理3 h时表达水平显著高于对照。(3)聚乙二醇(PEG)、高温和低温均诱导DfDXS1上调表达。研究推测,香鳞毛蕨DXS基因在萜类物质合成与逆境胁迫机制中发挥着重要的作用。     

葡萄风信子MaANS基因及启动子的克隆与分析

该研究根据转录组测序结果,在葡萄风信子(Muscari armeniacum) ‘亚美尼亚’中克隆到花青素合酶(ANS)基因的cDNA与DNA序列,该基因命名为MaANS。采用荧光实时定量分析MaANS时空表达模型,同时利用染色体步移法克隆到MaANS上游1 044 bp的一段序列。信息学分析表明：MaANS开放阅读框为1 065 bp,编码355个氨基酸;DNA与cDNA的一致性为89.62%,DNA序列在ATG下游515~594 bp之间插入1个79 bp内含子;启动子序列在-70 bp位置有1个TATA-box,有多个光响应元件及MYB结合位点等。荧光实时定量分析表明,MaANS基因在花中优势表达,并且在完全着色期表达量最高。该研究结果为深入研究MaANS基因功能、分析葡萄风信子着色机理奠定了基础。     

de novo转录组学分析华南地区入侵植物五爪金龙代谢特征

为探究华南地区严重入侵植物五爪金龙(Ipomoea cairica)生物入侵的分子机制,对五爪金龙及其近缘种七爪金龙(I. digitata)和裂叶牵牛(I. nil)进行de novo转录组测序和组装,得到56551条unigenes,其中56522条得到注释,7815条GO注释,15615条COG注释,180201条KEGG数据库注释。转录组分析结果表明,五爪金龙氮代谢通路关键酶基因的表达高于对照。次生代谢关键酶(PAL、4CL、CAD、查耳酮合酶、苯基丙乙烯酮异构酶、槲皮黄3-O-甲基转移酶等)基因在五爪金龙与七爪金龙及裂叶牵牛中均得到协同性的差异表达,而这些代谢通路指导的产物合成对五爪金龙的抗逆境能力、生长、化感作用等均起关键作用。关键基因的RT-qPCR验证结果与转录组结果具有一致性。因此,这从分子生物学层面上对解释五爪金龙在华南地区的入侵机制提供了新的证据。     

广藿香香叶醇合酶基因克隆及表达分析

香叶醇合酶(geraniol synthase,GES)是香叶醇形成过程中非常重要的酶,是萜类代谢途径的限速酶。根据课题组广藿香转录组数据中的GES 转录本序列设计基因全长扩增引物,采用RT PCR方法克隆了广藿香GES基因的全长cDNA序列。对该基因进行了相关的生物信息学分析,并利用荧光实时定量PCR法检测了PcGES1基因在4个广藿香栽培种中不同时期茎、叶中的表达情况。结果显示：广藿香GES基因包含一个完整的ORF框,长1 734 bp,编码577个氨基酸,命名为PcGES1,GenBank登录号为KF926075 ;PcGES1基因编码的氨基酸序列与罗勒GES基因编码的氨基酸序列最为相近。广藿香GES蛋白定位在叶绿体中,无跨膜区域。PcGES1主要在叶中表达,老叶中表达量最高;从不同栽培种来看,PcGES1在石牌广藿香和高要广藿香中表达模式相似,在海南广藿香与印尼广藿香中表达相似,在海南广藿香老叶中表达最高。该研究结果为进一步阐明广藿香萜类代谢途径奠定了基础。     

百合LbCAT和LbGPX基因的克隆与表达分析

以切花百合(Lilium brownii var. viridulum)‘卡瓦纳’cDNA为模板,克隆了过氧化氢酶(LbCAT)和谷胱甘肽过氧化物酶(LbGPX)基因。序列分析表明,这2个基因分别包含1 479 bp和519 bp的开放阅读框(ORF),编码492个和172个氨基酸。进化分析结果表明,LbCAT蛋白与岷江百合CAT蛋白的氨基酸序列相似性最高(99.19%),且亲缘关系最近;LbGPX蛋白与油棕GPX蛋白的氨基酸序列相似性最高(78.61%),亲缘关系最近。qRT PCR结果显示,LbCAT和LbGPX在百合根、鳞茎、叶和花中都有表达。LbCAT在叶中表达量最高,LbGPX在花中表达量最高。这2个基因在百合花蕾的生长发育过程中均有表达,且表达量逐渐增加;在PEG处理后2个基因的转录水平升高,但独角金内酯(SLs)处理却显著降低了这2个基因的转录水平;该结果为百合抗逆性机理研究以及抗逆育种奠定了基础。     

黄毛草莓FnMYB24转录因子基因和启动子的克隆及表达分析

该研究以黄毛草莓(Fragaria nilgerrensis Schltdl.)为材料,采用RT PCR技术克隆了黄毛草莓FnMYB24基因的cDNA和启动子序列。生物信息学分析表明,FnMYB24的cDNA序列长为1 033 bp(GenBank登录号为MN879283),其开放阅读框(ORF)长为609 bp,编码202个氨基酸,含有1个保守的MYB_DNA binding结构域。同源分析结果显示,黄毛草莓FnMYB24基因编码的氨基酸序列与森林草莓(Fragaria vesca)编码的氨基酸相似性较高;同时进一步克隆了该基因编码起始位点上游长度为718 bp启动子序列(GenBank登录号为MN879285),预测该序列包含激素响应元件、光调控元件等多个顺式作用元件。通过构建pFnMYB24∷GUS表达载体进行烟草瞬时转化,发现pFnMYB24启动子具有转录活性且能够驱动FnMYB24基因表达。实时荧光定量PCR结果显示：抗病品种黄毛草莓和易感病栽培品种‘妙香3号’的叶片接种胶孢炭疽菌(Colletotrichum gloeosporioides)后MYB24基因表达量均有上调,但‘妙香3号’的MYB24表达量始终低于黄毛草莓的表达量;SA处理后2个草莓品种的MYB24表达量均高于对照组,表明MYB24基因受水杨酸(SA)的诱导表达。研究表明,草莓MYB24基因可能参与调控抗炭疽病,为进一步研究MYB24基因在草莓抗炭疽病中的功能奠定了基础。     

陆地棉GhSAD2基因克隆与表达特征研究

Δ9硬脂酰 ACP 脱氢酶基因(GhSAD2)是脂肪酸合成代谢过程中关键的去饱和酶基因,为明确该基因在棉花脂肪酸合成代谢中的功能,该研究克隆了陆地棉GhSAD2基因,并对该基因的序列特征、进化关系及表达特性进行分析。序列分析显示,GhSAD2基因（GenBank登录号为KX197920）cDNA全长1 188 bp,编码396个氨基酸,具有脂肪酸去饱和酶家族2个高度保守的组氨酸簇EENRHG和DEKRH,分别位于氨基酸的185和271位。系统进化分析显示,GhSAD2基因与可可树的同源基因进化关系非常接近。qPCR分析显示,GhSAD2基因在叶中的表达量高于茎和根,且在花后25 d的种子中表达量达到最高值。低温胁迫诱导结果表明,GhSAD2基因在不同程度低温处理下均有上调表达,6 h表达量最大,之后逐渐下调。研究表明,GhSAD2基因可能对棉子油不饱和脂肪酸的合成具有重要作用,同时在棉花抗寒方面也起一定的生理作用。     

棉花抗枯萎病相关ERF-B3亚组转录因子的克隆与表达

以拟南芥ERF-B3亚组转录因子的AP2/ERF结构域为探针,利用NCBI中的棉花(Gossypium hirsutum)EST数据库,通过电子克隆结合RT-PCR方法,从枯萎病菌诱导后的高抗枯萎病品种‘中棉所12’克隆到1个与抗枯萎病相关的ERF-B3亚组转录因子基因GhB301。序列分析结果显示,GhB301基因cDNA全长593 bp,开放阅读框384 bp,编码127个氨基酸,含有一个保守的AP2/ERF结构域。实时荧光定量PCR检测该基因的表达显示,枯萎病菌诱导后,GhB301基因在棉花根中、抗病品种中优势表达;在乙烯、水杨酸、干旱、低温及高盐的诱导下表达量均发生变化。研究结果表明,GhB301基因可能参与了棉花对病原菌、激素及非生物胁迫的应答反应。     

小桐子糖原合成激酶基因JcGSK的克隆与表达分析

以小桐子(Jatropha curcas L.)cDNA为模版,克隆了JcGSK基因的CDS序列。序列分析表明,JcGSK基因包含1 230bp完全阅读框(ORF),编码409个氨基酸。预测其编码蛋白质的相对分子量为46.33kD,理论等电点为8.58。Blast搜索结果及进化分析结果表明,JcGSK蛋白与巴西橡胶树GSK蛋白的氨基酸序列一致性最高(94%)且亲缘关系最近;JcGSK基因编码的蛋白具有一个蛋白激酶特有的结构域。组织表达结果显示,JcGSK基因在小桐子根、茎、叶、花、果皮和种子中都有表达,且在根中表达量最高。小桐子幼苗在NaCl、ABA、PEG、低温和机械损伤处理后JcGSK基因表达量有不同程度的上调,推测其参与小桐子非生物胁迫响应和信号传导过程。JcGSK基因在种子中也有较高表达,在种子发育过程中表达量的变化与种子生长发育趋势基本一致,推测JcGSK基因也参与调控小桐子种子的生长发育。     

Nested Inverse Polymerase Chain Reactions: An Effective Method for Cloning of Full-Length Sequences of Chalcone Synthase

Chalcone synthase is the key enzyme in biosynthesis of flavonoids, which play roles in pigmentation of flowers and protection against ultraviolet and pathogens. Inverse polymerase chain reaction (IPCR) is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. In this study, IPCR united with nested PCR was successfully applied in cloning full-length sequences of three Phalaenopsis chalcone synthase genes (phchs3, phchs4, and phchs5, respectively). Firstly, routine PCR with homologous primers were performed, and gene fragments of phchs3 (1 kb), phchs4 (1.2 kb), and phchs5 (800 bp) were obtained and then sequenced. Then, inverse PCR were carried out for cloning full-length sequence of each gene. Because products were not unique in single round inverse PCR, nested PCR were performed, and the specificity was much enhanced. At last, full-length sequences of 2,499 bp for phchs3, 2,502 bp for phchs4, and 1,855 bp for phchs5 were obtained. This study proved that IPCR could be more efficient if being united with nested PCR.     

Enzymatic control of anthocyanin expression in the flowers of pea (Pisum sativum) mutants

Using enzymological and immunological methods we have investigated the relationship between chalcone synthase and the A locus, a major gene involved in the control of anthocyanin expression in pea (Pisum sativum L.) flowers. Pea plants containing the dominant allele A usually synthesize anthocyanins in the petal tissue, whereas plants homozygous for the a allele do not produce anthocyanins. We sought to determine whether or not the A locus also controlled the presence or absence of chalcone synthase, the first enzyme of the flavonoid pathway in the flowers of three genetic lines (A, purple-violet flowers; A,am, white flowers with sometimes pink edges; and a, white flowers). Chalcone synthase was found to be present in all three genetic lines by enzyme activity measurement, indirect enzyme-linked immunosorbent assay (ELISA), and Western blotting. Spectroscopic investigations showed that only the genetic lines A and A,am contained anthocyanins and flavonol glycosides, respectively, in the flowers; line a accumulated p-coumaric acid or its derivatives. These data suggest that the A locus in Pisum is not the structural gene for chalcone synthase and it does not appear to regulate the expression of this enzyme.This work was supported by a grant from the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation and a consortium of industries.     

Stress-induced curcin-L promoter in leaves of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and characterization in transgenic tobacco

Ribosome-inactivating proteins (RIPs) represent a type of protein that universally inactivates the ribosome thus inhibiting protein biosynthesis. Curcin-L was a type I RIP found in Jatropha curcas L.. Its expression could be activated in leaves by treatments with abscisic acid, salicylic acid, polyethylene glycol, temperature 4, 45°C and ultraviolet light. A 654 bp fragment of a 5′ flanking region preceding the curcin-L gene, designated CP2, was cloned from the J. curcas genome and its expression pattern was studied via the expression of the β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the CP2 was leaf specific, and was able to drive the expression of the reporter gene under stress-induction conditions. Analysis of a series of 5′-deletions of the CP2 suggested that several promoter motifs were necessary to respond to environmental stresses.     

小桐子JcACBP基因克隆和序列分析

以小桐子(Jatropha curcas)cDNA为模板,克隆了酰基辅酶A结合蛋白(Acyl-CoA-binding Protein)基因(JcACBP)的CDS序列,对其序列进行了生物信息学分析,并采用实时荧光定量PCR方法,研究了JcACBP基因在小桐子不同器官和果实生长发育阶段的表达模式。结果显示:JcACBP基因完全阅读框(coding sequence,CDS)全长279bp,编码92个氨基酸。预测其编码蛋白质的分子量为10.30kD,具有ACBP家族典型的结构域。JcACBP基因推测氨基酸与油桐(Vernicia fordii,AFZ62125)的亲缘关系最近(96%)。JcACBP基因在小桐子根、茎、叶、花、发育中的胚及果实等组织中都有表达,其中在花后40d的种子中表达最高,其次是果皮,而在根中表达较少;在果实发育过程中的表达与果实油脂积累的变化趋势基本一致。     

A chalcone synthase/stilbene synthase DNA probe for conifers

A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them.     

Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members

Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.     

Genetic divergence and phylogenetic analysis of genus <Emphasis Type="Italic">Jatropha</Emphasis> based on nuclear ribosomal DNA ITS sequence

The genus Jatropha belongs to the family Euphorbiaceae having significant economic importance. The present investigation was undertaken with an aim to understand phylogenetic relationships among seven species (J. curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis.) which are widely distributed in India, using nuclear ribosomal DNA ITS sequence (nrDNA ITS) and to compare the results with multilocus marker analysis systems reported earlier for the same genus. The size variation obtained among sequenced nrDNA ITS regions was narrow and ranged from 647 to 654 bp. The overall mean genetic distance (GD) of genus Jatropha was found to be 0.385. Highest interspecific GD (0.419) was found between J. glandulifera and J. multifida. The least interspecific GD (0.085) was found between J. gossypifolia and J. tanjorensis. The highest intraspecific GD was observed in J. podagrica (0.011) and least in J. gossypifolia (0.002). The phylogram obtained using nrDNA ITS sequence showed congruence with the phylograms obtained using multilocus markers system reported earlier with minor variations. The present study also strongly supports high phylogentic closeness of J. curcas and J. integerrima. The only exception found was J. podagrica which clustered with J. multifida in earlier based on multilocus marker analysis, was clustered with J. curcas in the present analysis. The sequence data generated in the present investigation will help for further studies in intraspecies population, and their phylogentic analysis, biogeographical, molecular evolution studies and also pave way for future phylogetic and/or evolution studies among the other groups belongs to the family Euphorbiaceae.     

小桐子YABBY全基因组家族成员的鉴定、表达和可变剪接分析

为发掘能源植物小桐子(Jatropha curcas)的YABBY转录因子,以最新公布的小桐子基因组序列为参考,在全基因组层面鉴定出5个亚家族的7个YABBY基因,同一亚家族的成员具有相似的氨基酸序列、基因结构和保守基序组成。YAB2和FIL/YAB3亚家族的2个旁系同源基因对(JcYAB2A/JcYAB2B、JcYAB1/JcYAB3)具有良好的共线性关系,表明片段复制或全基因组复制是小桐子YABBY家族扩张的主要方式。纯化选择是进化的主要动力,而YAB2亚家族成员可能在进化中经历了更明显的功能分化。基因表达模式和蛋白互作预测分析表明JcYAB2B和JcYAB3可能在种子的发育过程中起到重要的调控作用;同时,细胞分裂素、干旱或高盐胁迫处理抑制了大多数JcYABs成员的基因表达。此外,转录组测序结合q RT-PCR分析表明,低温处理有效诱导JcYAB2A和JcYAB2B的基因表达模式发生变化,并伴随着新的、截短的可变剪接转录本的动态积累。因此,推测JcYABs可能通过剪接体的功能竞争或功能互补参与低温响应的调节,这些结果有助于更好地了解YABBY家族成员的功能分化并阐明可变剪接如何调控...