互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

基于Tail-PCR分析AN2基因上游启动子活性

黑果枸杞(Lycium ruthenicum)富含花青素,AN2基因是调控黑果枸杞花青素合成代谢的主效基因。为解析AN2基因启动子的活性差异,采用Tail-PCR方法分别克隆了黑果枸杞和红果枸杞(L. barbarum) AN2基因起始密码子上游约1 686 bp (LrAN2p)和1 495 bp (LbAN2p)的序列。Plant CARE预测表明,LbAN2p和LrAN2p中分别有133和137个的顺式作用元件, 其中,参与光调控的顺式元件分别有11和15个;参与激素响应相关的顺式元件分别有13和16个。构建AN2启动子植物表达载体pKGWFS7:LbAN2p和pKGWFS7:LrAN2p,利用农杆菌介导的烟草遗传转化体系获得转基因烟草。GUS染色结果表明,LrAN2p能够驱动GUS在烟草中的表达,叶片呈现蓝色,具有较LbAN2p更强的启动活性,qRT-PCR结果表明,LrAN2p转基因烟草中GUS基因具有更高的转录水平,这可能会使AN2基因在黑果枸杞中具有更高的表达,激活黑果枸杞花青素合成代谢通路。这为解析枸杞果色形成及AN2基因的表达调控机制奠定了理论基础。     

The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

新疆野苹果AP2/ERF转录因子家族鉴定与响应腐烂病的表达分析北大核心CSCD

AP2/ERF转录因子家族参与了植物生长发育、抵抗胁迫以及植物激素响应等诸多生物过程,是植物中最重要的转录因子家族之一。该研究基于腐烂病菌侵染后的新疆野苹果(Malus sieversii)全长转录组,使用AP2保守结构域的隐马可夫模型PF00847,鉴定新疆野苹果的AP2/ERF家族成员。利用MEGA6、NCBI CDD-batch、MEME、EXPASY、BUSCA对MsAP2/ERF家族成员进行鉴定、分类和结构分析、理化性质和亚细胞定位分析。通过RNA-seq数据和qRT-PCR实验对差异表达的MsAP2/ERFs基因的表达水平进行分析和验证,旨在鉴定新疆野苹果中潜在具有腐烂病抗性的AP2/ERF家族基因资源。结果显示:(1)在新疆野苹果中共鉴定获得106个AP2/ERF基因,涵盖全部AP2(17个)、ERF(57个)、DREB(25个)、RAV(5个)和Soloist(2个)5个亚家族。(2)进一步的细化分类发现MsERF亚家族包括B1-B6 6个组,而MsDREB亚家族中只有A2、A4、A5、A6共4个组,缺少A1和A3组的基因成员。(3)RNA-seq表达模式分析结果表明,29个MsAP2/ERF基因在腐烂病感染过程中差异表达,其中MsERF亚家族中差异表达基因数量最多(19个)。(4)12个MsAP2/ERF代表基因的qRT-PCR结果表明:8个ERF亚家族基因均受腐烂病病菌诱导显著上调表达,其中B4类ERF成员基因(MsERF40)在腐烂病病菌侵染后5 d表达量上调表达倍数最高;4个MsDREB基因中,3个受到腐烂病病原菌诱导显著上调,1个下调表达;此外,含有TIR保守结构域的MsERF05在腐烂病病菌侵染1 d后上调表达69倍,表明ERF亚家族的MsERF40和MsERF05在新疆野苹果抗腐烂病过程中发挥重要作用。该研究结果为新疆野苹果AP2/ERF基因响应腐烂病的功能和机理研究奠定了基础。     

Late-Pleistocene and early Holocene history of the canid fauna of Europe (Canidae)

In the sub-fossil assemblages of Europe the red fox is clearly the most frequent carnivorous mammalian species with a total of 1553 records. In depositions from the Weichselian Glacial the red fox Vulpes vulpes is, a typical representative of the Holocene fauna, already recorded in 100 assemblages. The Iberian peninsula, Italian peninsula and Balkans were theorised as glacial refugia. Well-founded facts give reason to believe that V. vulpes was also distributed in the Carpathian refuge. Later on, the Crimean peninsula would also appear to be a possible glacial refuge of the red fox.In the last warmer complex of interstadials during the Pleni-Glacial (Hengelo-Denekamp, 38,000–25,000 BC) the red fox was distributed in central Europe. Its distribution during this epoch extended at least in part to southern England. The earliest well-dated records of V. vulpes in central Europe after the Maximum Glaciation lie between 14,000 and 13,500 BC. Already during the early Late-Glacial (13,500 BC) the red fox appeared in typical glacial faunal communities. A separation to glacial refugia was only possible for 10,000 years.During the last warmer Pleni-Glacial complex of interstadials (38,000–25,000 BC) in central Europe a sympatric distribution of the arctic fox (Alopex lagopus) and the red fox probably existed. During the Last Glacial Maximum (22,000–18,000 BC) the arctic fox was exclusively distributed in central Europe, outside of the refuges. The combined distribution of A. lagopus and V. vulpes during the Late-Glacial (15,000–9500 BC) in central Europe, with the probable exception of the Allerød, is precisely documented by sub-fossil assemblages.In the Pleni-Glacial the wolf Canis lupus was distributed in geographic regions that served as glacial refugia of more warm-climate adapted species. Concerning the wolf no drastic decrease of the distribution is assumed. The Holocene presence of C. lupus is probably not caused by recolonisation.

Zusammenfassung

Die Entwicklung der Canidenfauna Europas im Spätpleistozän und frühen HolozänIn den subfossilen Ablagerungen ist Vulpes vulpes in Europa mit 1553 Nachweisen das mit Abstand am häufigsten nachgewiesene Raubsäugetier. Allein in den Ablagerungen des Weichselglazials konnte der Rotfuchs bereits in 100 Fundkomplexen nachgewiesen werden. Als Glazialrefugium des Rotfuchses wird die Iberische Halbinsel sicher identifiziert. Eine Verbreitung der Art während des Kältemaximums wird außerdem auf der Apenninen-Halbinsel sowie der Balkan- Halbinsel vermutet. Außerdem liegen fundierte Fakten für die Annahme vor, dass V. vulpes im Karpatenrefugium verbreitet war. Auf der Halbinsel Krim scheint ein Glazialrefugium für des Fuchses möglich.Während des letzten wärmeren Interstadial-Komplexes im Hochglazial (Hengelo-Denekamp, ca. 38.000–25.000 v. Chr.) war Mitteleuropa vom Rotfuchs besiedelt. Die nördliche Arealgrenze der Art war innerhalb dieses Klimaabschnitts mindestens zeitweise bis nach Südengland ausgedehnt. Die frühesten, zeitlich relativ gut abgesicherten Nachweise von V. vulpes nach der Weichsel-Maximalvereisung in Mitteleuropa liegen etwa zwischen 14.000–13.500 v. Chr. Schon im frühen Spätglazial (ca. 13.500 v. Chr.) kam der Rotfuchs im nördlichen Mitteleuropa in typischen glazialen Faunengemeinschaften vor. Eine Disjunktion des Areals während der letzten Vereisung kann für höchstens 10.000 Jahre stattgefunden haben. Es wird angenommen, dass während des Hengelo-Deenekamp Interstadials ein sympatrisches Vorkommen von Eisfuchs Alopex lagopus und Rotfuchs V. vulpes in Mitteleuropa existierte. Nur zur Zeit des absoluten Kältemaximums (ca. 22.000–18.000 v. Chr.) war außerhalb der Refugialgebiete in Mitteleuropa ausschließlich A. lagopus verbreitet. Das gemeinsame Vorkommen (von A. lagopus und V. vulpes) während des gesamten Spätglazials in Mitteleuropa, wahrscheinlich mit Ausnahme des Allerød-Interstadials, ist präzise belegt.Während des Hochglazials war Canis lupus auch in geografischen Regionen verbreitet, die für an wärmeres Klima gebundene Tierarten den Charakter von Glazialrefugien hatten. Beim Wolf C. lupus kann keine extreme Arealverringerung während der Weichseleiszeit angenommen werden. Das holozäne Vorkommen von C. lupus in Mitteleuropa dürfte daher nicht generell auf eine Rekolonisation zurückzuführen sein.     

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.     

Association study of the oestrogen signalling pathway genes in relation to age at natural menopause

Genetic factors play a significant role in influencing the variation of age at natural menopause (AANM). Estrogen receptor β (ESR2), is an important factor in the mechanism of action of estrogen, while the aromatase gene (CYP19) and the 17-alphahydroxylase gene (CYP17) are involved in the biosynthesis of estrogen. We tested whether polymorphisms of ESR2, CYP19 and CYP17 genes are associated with AANM in Caucasian females. A total of 52 SNPs (17 for ESR2, 28 for CYP19, and 7 for CYP17) were successfully genotyped for 229 Caucasian women having experienced natural menopause. Comprehensive statistical analyses focusing on the association of these genes with AANM were conducted. The effects of age, height and age at menarche on AANM were adjusted when conducting association analyses. We found that six SNPs (2, 6–7, 9, 13 and 16) within ESR2 were not significantly associated with AANM after Bonferroni correction. However, two blocks of ESR2 were associated with AANM. For CYP19, two SNPs (24 and 27) were nominally associated with AANM. No significant association was observed between CYP17 and AANM. Our results suggest that genetic variation in the ESR2 and CYP19 genes may influence the variation in AANM in Caucasian women.     

<Emphasis Type="Italic">AVAG2</Emphasis> is a putative D-class gene from an ornamental asparagus

Members of the AGAMOUS (AG) family of MADS-box genes play important roles in regulating the development of reproductive organs in flowering plants. To elucidate the molecular mechanisms of floral development in Asparagus virgatus, we isolated and characterized an Asparagus AG-homologue, AVAG2. AVAG2 contains an open reading frame that encodes a deduced protein with 234 amino acid residues. Phylogenetic analysis indicated that AVAG2 belongs to the D-lineage of the AG gene family. AVAG2 mRNA was detected in the flower, but not in vegetative organs. Moreover, in in situ hybridization experiments, AVAG2 signals were observed in the stamens and carpels during early flower development, and appeared in the ovule only at later developmental stages. This suggests that the AVAG2 gene is involved in ovule formation. Thus, our expression data support the phylogenetic analysis indicating that AVAG2 belongs to the D-class gene family.     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

拟南芥bso-1突变体的基因定位及表型分析

通过EMS化学诱变在拟南芥Columbia(Col-0)野生型突变体库中筛选获得1株器官显著增大的突变体,命名为big size organ1(bso-1)。遗传分析表明,bso-1受单个隐性核基因控制。表型观察发现,突变体植株的幼苗、花、果荚及种子与野生型相比都表现出明显的增大。组织切片结果显示,突变体种子的增大主要由胚细胞个体增大导致胚体积增大而实现,因此突变体种子的重量也较野生型有明显增加。利用图位克隆方法将相关基因初步定位在4号染色体上SSLP标记T5L19与F28M11之间58kb区间内,生物信息学分析显示此区间内未见调控植物器官大小发育相关的已知基因的报道。该研究结果为进一步克隆bso-1突变体相关基因及探讨其在控制植物器官发育尤其是种子发育过程中的作用奠定了基础。     

The Mitochondrial Genome of Xiphinema americanum sensu stricto (Nematoda: Enoplea): Considerable Economization in the Length and Structural Features of Encoded Genes

The complete sequence of the mitochondrial genome of the plant parasitic nematode Xiphinema americanum sensu stricto has been determined. At 12626bp it is the smallest metazoan mitochondrial genome reported to date. Genes are transcribed from both strands. Genes coding for 12 proteins, 2 rRNAs and 17 putative tRNAs (with the tRNA-C, I, N, S1, S2 missing) are predicted from the sequence. The arrangement of genes within the X. americanum mitochondrial genome is unique and includes gene overlaps. Comparisons with the mtDNA of other nematodes show that the small size of the X. americanum mtDNA is due to a combination of factors. The two mitochondrial rRNA genes are considerably smaller than those of other nematodes, with most of the protein encoding and tRNA genes also slightly smaller. In addition, five tRNAs genes are absent, lengthy noncoding regions are not present in the mtDNA, and several gene overlaps are present. [Reviewing Editor: Dr. Yues van de Peer] F. Lamberti: Deceased, 2004     

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1^−/−; Spry2^−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^−/−; Spry2^−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^−/−; Spry2^−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.     

Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension

Cell–cell signaling regulated by retinoic acid (RA), Wnt/β-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/β-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2^−/− mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2^−/− mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/β-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/β-catenin signaling as the body axis extends.     

Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n = 200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n = 195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n = 5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2–TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.     

The Bt gene <Emphasis Type="Italic">cry2Aa2</Emphasis> driven by a tissue specific ST-LS1 promoter from potato effectively controls <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R₁ transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

旱麦草EtAP2基因的克隆及其对干旱胁迫的响应表达

以旱麦草(Eremopyrum triticeum)为实验材料,利用RT-PCR技术从旱麦草叶片中克隆了1个AP2/ERF家族基因,命名为EtAP2(GenBank登录号KX622583)。EtAP2基因含有1 128bp开放阅读框,编码375个氨基酸,相对分子质量40.87kD,等电点为5.36。多序列比对和进化树分析表明,该基因编码蛋白具有2个AP2保守结构域,与小麦AP2/ERF家族蛋白具有较近的亲缘关系。实时荧光定量PCR分析表明,15%PEG 6000模拟干旱胁迫可诱导EtAP2基因在根和叶中表达,且在根中对干旱胁迫的响应大于叶片。研究表明,EtAP2可能参与旱麦草对干旱逆境胁迫应答的调节。