Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

棉花GhZIP4基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段.结果表明:(1)GhZIP4基因cDNA序列全长1 487 bp,包含1 269 bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域“HRHSHPHG”和“HSHGHGHD”.(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高.(3)GhZIP4 DNA序列编码区全长1 778 bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则.(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用.     

毛白杨PtoGSTF4基因克隆及相关特性鉴定

该研究从毛白杨中克隆到1个Phi类谷胱苷肽S-转移酶（GST）基因（PtoGSTF4）,编码213个氨基酸。表达模式分析发现,PtoGSTF4在正常生长、H₂O₂和莠去津处理后的茎、叶以及茎的韧皮部均表达,属于组成型表达基因。在大肠杆菌中表达并纯化了PtoGSTF4重组蛋白,酶学性质分析表明PtoGSTF4对CDNB、NBD-Cl、NBC和Cum-OOH等4种底物均有活性。动力学分析发现,PtoGSTF4对GSH具有较高的亲和力,而对CDNB的亲和力相对较低。在不同pH及温度条件下对PtoGSTF4蛋白进行活性检测,发现PtoGSTF4在pH 7.5~10.5范围内或30 ℃~60 ℃温度范围内有较高的活性。研究推测,PtoGSTF4可能在毛白杨的抗逆生理中发挥重要作用。     

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

hsc70-4基因促进家蚕核型多角体病毒复制增殖

【目的】热休克应答(heatshockresponse,HSR)是机体细胞应对环境压力的一种重要防御策略,鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能,并揭示其作用的分子机制,为探明宿主与病毒相互作用的分子基础提供理论依据。【方法】通过分子克隆技术对Bmhsc70-4基因进行克隆,并利用BioEdit及GeneDoc对其进行多序列比对分析;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除;利用荧光定量PCR技术检测相应基因的表达量;通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响;通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况,并进一步通过免疫共沉淀验证它们的相互作用。【结果】Bmhsc70-4基因开放阅读框为1950bp,编码649个氨基酸,在昆虫间具有较高的保守性;BmNPV能够诱导Bmhsc70-4基因上调表达,过表达Bmhsc70-4基因能够促进BmNPV的增殖,敲除Bmhsc70-4基因能够抑制BmNPV的增殖,表明Bmhsc70-4基因的表达利于BmNPV的增殖;Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能;荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。【结论】Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能,在BmNPV侵染家蚕细胞过程中,能够与BmIAP相互作用,并促进BmNPV复制增殖。     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer

MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. DPC4/Smad4 is a critical tumor suppressor involved in the progression of pancreatic cancer, but few studies have been conducted to determine its relationship with miRNAs. In this study, we identify miR-421 as a potential regulator of DPC4/Smad4. We find that in human clinical specimens of pancreatic cancer miR-421 is aberrantly upregulated while DPC4/Smad4 is strongly repressed, and their levels of expression are inversely correlated. Moreover, ectopic expression of miR-421 significantly decreases DPC4/Smad4 protein level in pancreatic cancer cell lines and simultaneously promotes cell proliferation and colony formation in vitro. Our findings identify miR-421 as a potent regulator of DPC4/Smad4, which may provide a novel therapeutic strategy for treatment of DPC4/Smad4-driven pancreatic cancer.     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

赤桉ACT1和ACT2基因的克隆与生物信息学分析

为了解赤桉(Eucalyptus camaldulensis)肌动蛋白(Actin)在生长发育过程中的功能,根据赤桉幼苗转录组数据库中的肌动蛋白基因序列,从赤桉嫩叶中克隆了2条Actin基因片段,并利用RACE技术获得Actin基因的全长cDNA,分别命名为ECACT1和EC-ACT2基因。生物信息学分析表明,这两条基因的全长cDNA分别为1533 bp和1387 bp,均含有1个编码377个氨基酸的开放阅读框。经比对分析,赤桉Actin蛋白的氨基酸序列与其他植物Actin蛋白的具有较高的相似性,并且具有Actin蛋白特有的保守序列和相关特征。因此推测这两条基因对桉树的生长发育具有一定的调控作用。     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

黄花棘豆脱水素OoY_2K_4的鉴定及表达分析

该研究以黄花棘豆cDNA为模板,采用同源克隆法,从黄花棘豆转录组数据库中克隆获得1个响应逆境胁迫的胚胎发育晚期丰富蛋白基因,命名为OoY_2K_4;OoY_2K_4基因ORF为786bp,编码261个氨基酸,含有2个保守的Y片段和4个K片段,为典型的Y_2K_4类脱水蛋白亚家族成员;OoY_2K_4蛋白不具有跨膜结构域,不存在信号肽,亲水性极强,含有1个糖基化位点和17个磷酸化位点;亚细胞定位显示,OoY_2K_4蛋白定位于细胞质中。多序列比对发现,OoY_2K_4蛋白与其他物种第二组LEA蛋白(脱水素)序列高度保守;进化树分析显示,该序列与三叶草、蒺藜苜蓿和紫花苜蓿相似度最高,亲缘关系最近。采用qRT-PCR对OoY_2K_4基因在干旱、高盐、低温以及脱落酸、乙烯、赤霉素处理下的表达分析显示,干旱和高盐胁迫可显著诱导OoY_2K_4基因表达,而低温胁迫下基本无变化;激素处理均可诱导OoY_2K_4基因高效表达,其中脱落酸诱导下OoY_2K_4基因表达最显著。研究推测,OoY_2K_4基因可能通过依赖ABA的信号途径参与黄花棘豆对干旱和高盐逆境胁迫的应答反应。