雷公藤AP2/ERF转录因子基因的克隆与分析

该研究以雷公藤发状根为材料,根据雷公藤根转录组数据设计引物,采用RT-PCR方法克隆得到2个雷公藤AP2/ERF转录因子,分别命名为TwAP2/ERF1基因(GenBank登录号:GAVZ01042389.1)和TwAP2/ERF2基因(GenBank登录号:GAVZ01016765.1)。TwAP2/ERF1基因含有一个525bp开放阅读框(ORF),编码186个氨基酸;TwAP2/ERF2基因的ORF为789bp,编码262个氨基酸;2个基因编码的蛋白质均为亲水性蛋白质。系统进化分析表明,TwAP2/ERF1与油桐(Vernicia fordii)AP2/ERF(APQ47444.1)和木油桐(Vernicia montana)AP2/ERF(APQ47365.1)相似性较高,TwAP2/ERF2与毛果杨(Populus trichocarpa)AP2/ERF(XP_002304640.1)和樱桃(Prunus pseudocerasus)AP2/ERF(ALD84477.1)相似性较高。雷公藤发状根经MeJA诱导后,TwAP2/ERF1基因的相对表达量明显提高,并于处理后9h达到最高值,为对照表达量的16.77倍;而MeJA处理对TwAP2/ERF2基因的表达表现出抑制作用,但于处理后48h相对表达量有所提高。研究表明,雷公藤TwAP2/ERF1转录因子响应MeJA早期诱导正调控,推测其可能参与调控雷公藤植物次生代谢产物的生物合成,该研究结果为阐明雷公藤次生代谢物质的生物合成调控与利用现代生物技术提高雷公藤植物细胞中次生代谢物质的含量奠定了基础。     

荔波连蕊茶GA20氧化酶基因的克隆及表达分析

根据植物GA20ox基因编码区的保守序列设计引物,以山茶属荔波连蕊茶幼嫩茎段为材料,提取总RNA,进行RT-PCR。采用RACE技术扩增获得1 567 bp的GA20氧化酶基因全长cDNA序列,命名为ClGA20ox2(GenBank登录号KF823787)。序列分析表明,ClGA20ox2开放阅读框(ORF)为1 146 bp,编码382个氨基酸,5'非编码区115 bp,3'非编码区303 bp。预测的蛋白质分子量为43.56 kD,等电点为7.02,所推导的蛋白氨基酸序列与夹竹桃和杨树GA20ox蛋白的同源性分别为73%和72%。ClGA20ox2与其它植物GA20ox蛋白比较,构建系统进化树,结果显示山茶GA20ox蛋白与夹竹桃和杨树的GA20ox蛋白的亲缘关系最为密切。实时定量PCR结果显示,该基因在荔波连蕊茶的根、茎、叶和种子中均有表达,其表达模式却不同:ClGA20ox2基因在二年生茎段中的表达丰度最高,在顶端分生组织中表达丰度最低,在嫩叶和根中表达丰度较高,成熟叶片和种子表达丰度较低。     

银杏EPSPS基因克隆及表达分析

利用RACE技术,克隆到银杏EPSPS合酶基因(GbEPSPS)的cDNA序列并对其进行生物信息学分析.结果表明:银杏GbEPSPS cDNA长1 403 bp(GenBank登录号GU084139),其包含一个1 035 bp的ORF框,编码344个氨基酸;预测该基因编码蛋白质分子量为36.87 kD,其等电点为5.75.系统进化树分析表明,银杏EPSPS蛋白质序列与其他物种的EPSPS同源性较高.半定量RT-PCR分析结果显示,EPSPS基因在银杏的叶和果实中表达量最高,其次为茎,根中表达水平最低.草甘膦处理能显著诱导银杏EPSPS基因表达量升高;紫外光对银杏EPSPS基因的表达具有诱导作用;ABA诱导GbEPSPS表达量先升后降;GbEPSPS转录水平受到42℃高温显著诱导.     

丹参2 酮戊二酸依赖性双加氧酶基因克隆及表达分析

以商洛紫花丹参为材料,对其转录组序列SRA020132进行Blast分析,采用PCR技术克隆得到丹参2-酮戊二酸依赖性双加氧酶基因Sm2-ODD1,GenBank登录号为JN935923。Sm2-ODD1基因全长1 365bp,包含3个外显子和2个内含子;cDNA全长1 189bp,读码框951bp,编码316个氨基酸残基;预测的编码蛋白具有2-酮戊二酸依赖性双加氧酶中结合2-酮戊二酸和亚铁离子的"H-T-D"、"H-X"和"R-Y-S"保守基序以及"果冻状"空间结构。表达分析显示,Sm2-ODD1在丹参各个器官都表达,但表达水平具有组织特异性,在根中表达量最高,在叶中表达量最低;该基因表达明显受到MeJA、GA3和ABA的诱导,可能参与了丹参萜类代谢下游途径。     

甜荞柠檬酸转运蛋白基因FeFRD3的克隆及表达分析

该研究采用同源克隆策略,从甜荞中克隆到1个柠檬酸转运蛋白基因FeFRD3(GenBank登录号为MG462907)。FeFRD3基因含一个1 554bp开放阅读框,编码517个氨基酸,预测蛋白分子量为55.83kD,等电点为8.48。生物信息学分析显示,FeFRD3蛋白含有8个跨膜区,定位于质膜和液泡膜上。蛋白序列分析结果表明,FeFRD3与拟南芥、大豆和水稻的FRD3同源蛋白有较高的序列一致性。系统进化树分析表明,FeFRD3属于具有将铁由根向地上部位长距离转运功能的柠檬酸转运蛋白,且与拟南芥AtFRD3亲缘关系最近。qRT-PCR分析结果表明,FeFRD3基因在甜荞根、茎、叶和种子中均有表达,但在根中的表达量最高,在种子中的表达量最低;缺铁胁迫没有影响FeFRD3基因在根中的表达,但高铁胁迫明显诱导了该基因在根中的表达。研究结果为进一步深入研究FeFRD3基因在甜荞铁长距离转运中的功能奠定了基础。     

牡丹PoSAD基因克隆与表达分析

以凤丹牡丹(Paeonia ostii)叶片为试验材料,采用RACE和RT-PCR方法,克隆得到凤丹牡丹硬脂酰-ACP去饱和酶基因SAD的cDNA全长,命名为PoSAD(GenBank登录号为KY038819)。序列分析表明,该基因cDNA序列全长1 559bp,其中开放阅读框1 197bp,编码398个氨基酸,3′端非编码区长172bp,5′端非编码区长123bp。多序列比对结果表明,凤丹牡丹PoSAD氨基酸序列含有2个保守结构域。系统发育分析结果显示,凤丹牡丹与蓖麻处于同一分支,其亲缘关系最近。TMHMM和TargetP亚细胞定位分析得知,PoSAD蛋白无跨膜区域,可能定位于叶绿体中发挥功能。组织特异性结果分析表明,PoSAD基因在凤丹牡丹的根、茎、叶、花瓣、雌蕊、雄蕊、种子中均有表达,且在花瓣中表达量最高,雌蕊中次之,在根中的表达量最低;不同时期种子中,60d表达量最高,80d次之,10d中表达量最低。     

丹参抗性基因SmORA1的克隆及诱导表达分析

该研究采用PCR方法从丹参中克隆出一条乙烯应答因子结合蛋白(ERF)转录因子编码基因,命名为SmORA1,GenBank登录号为KT359598。经分析发现该基因全长648bp,不包含内含子,编码206个氨基酸残基。编码蛋白SmORA1含有典型的AP2结合结构域。表达分析结果表明,SmORA1主要在丹参根中表达,且该基因的表达明显受到茉莉酸甲酯(MeJA)、脱落酸(ABA)、乙烯(ET)、机械创伤和病原菌等逆境信号的诱导,但低温和脱水情况下SmORA1表达下调。研究表明,SmORA1参与丹参生物胁迫反应,可整合JA、ABA、ET和病原菌等胁迫信号途径。     

德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 Blattella germanica TPS基因,研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列,利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因,分别命名为 BgTPS1 (GenBank登录号：KR050213) 和 BgTPS2 (GenBank登录号：KR050214)。其中,BgTPS1基因cDNA序列全长2 987 bp,开放阅读框 (ORF) 2 502 bp,编码833个氨基酸;BgTPS2基因cDNA序列全长3 212 bp,开放阅读框2 469 bp,编码822个氨基酸。BgTPS1和BgTPS2基因都主要在5龄幼虫脂肪体中表达,且BgTPS2基因的表达量为BgTPS1基因表达量的3.9倍。在两种不同极端温度诱导下,BgTPS1和BgTPS2基因mRNA均上调表达。其中,BgTPS2 的表达量始终显著高于 BgTPS1。在0℃时,BgTPS1和BgTPS2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达,且BgTPS2基因的表达量显著高于BgTPS1基因;低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。     

甜瓜乙烯受体基因Cm-ETR1 cDNA的克隆及表达特性分析

乙烯感知和信号转导的初始成分是乙烯受体,为探明甜瓜乙烯受体基因Cm-ETR1在甜瓜果实成熟过程中的作用,以甜瓜品种河套蜜瓜为材料,根据GenBank中登录的甜瓜乙烯受体基因Cm-ETR1的cDNA序列(登录号为AF054806),设计合成特异性引物,采用RT-PCR技术克隆得到Cm-ETR1基因全长cDNA序列,提交到GenBank中(登录号为EF495185)。序列分析表明,序列长度为2 256 bp,编码区为2 223 bp,编码740个氨基酸,与已报道的cantalupenis甜瓜ETR1基因的cDNA序列完全一致。Cm-ETR1蛋白的系统进化树分析结果表明,该乙烯受体蛋白在各物种间高度保守,与黄瓜乙烯受体蛋白相似性最高,一致性为99%,与龙眼乙烯受体蛋白相似性最低,一致性为86%。定量PCR分析结果显示,随着甜瓜果实内源乙烯合成量和成熟程度的增加,Cm-ETR1基因的表达量同步增加,在果实乙烯跃变期,Cm-ETR1的表达量也达到最高值,内源乙烯合成量与Cm-ETR1基因表达量间呈显著正相关,表明Cm-ETR1基因在甜瓜果实成熟过程中可能具有重要的作用。     

甘蓝型油菜赤霉素受体基因的克隆与表达

采用同源克隆技术从甘蓝型油菜中克隆到1个赤霉素受体基因,命名为BnGID1B(GenBank登录号为HQ589349).该基因含有1个内含子和2个外显子,其cDNA序列全长1 077 bp,编码358个氨基酸,推测蛋白质的相对分子量是40 203.4 Da,理论等电点为6.26.序列比对结果显示,BnGID1B基因与拟南芥GID1B基因核苷酸序列的相似性为86.3%,氨基酸序列的相似性为91.64%.实时荧光定量PCR分析表明,BnGID1B基因在苗期不同组织以及不同激素处理下的表达量有所不同,主要在根中表达,下胚轴中的表达量明显低于根,可见该基因具有一定程度的组织表达特异性.     

大豆GmABI4基因克隆及表达分析

该研究基于大豆基因组数据库,根据拟南芥ABI4蛋白的氨基酸序列,经比对分析,获得了大豆中的2个GmABI4基因,分别命名为GmABI4-1(GenBank登录号为XM_014766551.1)和GmABI4-2(GenBank登录号为NM_001249003)。TMHMM软件和系统进化转录分析表明,这2个基因编码的蛋白均不具有信号肽,二级结构主要以无规则卷曲和延伸链为主;进化树分析表明,大豆GmABI4和野生大豆亲缘关系较近。荧光定量PCR分析表明,GmABI4-1与GmABI4-2基因在大豆种子与豆荚中的表达量均高于根、茎、叶、花等其他组织,推测可能与调控大豆种子生命活动相关。     

Molecular characterization of flavonoid biosynthetic genes and accumulation of baicalin,baicalein, and wogonin in plant and hairy root of Scutellaria lateriflora

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora.     

黄秋葵八氢番茄红素脱氢酶基因的克隆与分析

该研究根据黄秋葵(Hibiscus esculentus L.)转录组测序获得的八氢番茄红素脱氢酶基因(HePDS)序列(GenBank登录号为MG372370)设计引物,克隆验证得到1条HePDS基因全长为2 020 bp cDNA,开放阅读框(ORF)包含1 686个碱基;预测其编码561个氨基酸,理论分子量为62.62 kD,等电点为8.155;编码的蛋白与海岛棉(Gossypium barbadense)、雷蒙德氏棉(Gossypium raimondii)、陆地棉(Gossypium hirsutum)同源蛋白的相似性均在93%以上,均含有1个保守的二核苷酸结合域和1个类胡萝卜素结合域,显示其高度的保守性。荧光定量PCR 分析表明,HePDS基因在黄秋葵根、茎、叶、花和果荚中均有表达;叶发育过程以嫩叶中表达最高,果实发育中以花后2 d表达量最高。类胡萝卜素含量随着叶、果实发育逐渐升高,成熟叶的含量最高,果实以花后4 d含量最高,且HePDS基因的表达与类胡萝卜素含量存在密切的相关性。该研究结果为进一步探讨HePDS基因的功能和调控机制,以及采用VIGS和CRISPR/Cas9技术开展黄秋葵基因功能的反向遗传学研究奠定了基础。     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.     

地黄RgMYB10基因的克隆与表达分析

MYB转录因子是植物最大的转录因子家族之一,广泛参与植物的生长发育、逆境胁迫和次生代谢产物积累.该研究通过同源比对和功能注释,在地黄(Rehmannia glutinosa)转录组中筛选出MYB的转录本,设计特异性引物对MYB基因的cDNA序列进行PCR扩增,用水杨酸(SA)、Ag+、茉莉酸甲酯(MeJA)和腐胺(Pu...     

Gene ontology study of methyl jasmonate-treated and non-treated hairy roots of <Emphasis Type="Italic">Panax ginseng</Emphasis> to identify genes involved in secondary metabolic pathway

The roots of Panax ginseng C.A. Meyer, known as Korean ginseng have been a valuable and important folk medicine in East Asian countries. It mainly used to maintain the homeostasis of the human body, with the presence of ginsenosides and non-saponin compounds like phenol compounds, acidic polysaccharides and polyethylene compounds. Functional genomics aid to annotate EST sequences based on gene ontology. In this study, we focused, genes which involve in secondary metabolic pathways and to visualize temporal changes of gene expression in ginseng hairy roots with methyl ester methyl jasmonate (MeJA) along with non-treated hairy roots. A 5.774 EST clones were clustered and assembled into 501 contigs and 2.955 singletons. Annotations categorized with molecular functions, biological processes, cellular compounds of gene ontological terms and biochemical functions, enzyme commission number, and metabolic pathways are assigned through Kyoto Encyclopedia of Genes and Genomes database. Comparatively, EST sequences are assigned to cellular process, metabolic process, biotic and abiotic stress stimuli, developmental and biological regulations and transports are up-regulated 2–3 fold in MeJA treated hairy roots. 46 different sub groups of enzymes found in the MeJA treated plants. These annotated ESTs represents a significant proportion of the P. ginseng and provides molecular resource for develop microarray to study genes expressions to development, metabolism and reproduction.