中华卵索线虫β-actin基因的克隆与分析

利用RACE技术克隆中华卵索线虫Ovomermis sinensisβ-actin基因,RT-PCR方法检测该基因在中华卵索线虫不同发育时期的表达情况。结果首次获得了中华卵索线虫β-actin基因全长cDNA序列。该基因cDNA全长1636bp,其中ORF长1131bp,编码376个氨基酸;5′UTR长137bp;3′UTR长367bp。由该cDNA推导的蛋白质序列与秀丽隐杆线虫Caenorhabditis elegans、人类等物种的β-actin蛋白序列相似性均在95%以上。系统进化树显示其系统发育关系与传统的分类关系基本一致。β-actin基因在不同发育时期的线虫中持续恒量表达。由此推断中华卵索线虫β-actin基因具有高度保守性,可以作为生物物种进化的分子标志,且能较好反映物种间的系统发生关系,是量化实验的理想内标参照。     

水稻条纹病毒胁迫对灰飞虱mucin基因表达量的影响

为分析灰飞虱mucin基因在水稻条纹病毒(Rice stripe virus, RSV)侵染过程中的作用,采用荧光定量PCR的方法分析了RSV 胁迫条件下灰飞虱mucin基因mRNA表达量的变化。饲喂RSV病叶1天和2天时灰飞虱mucin基因的表达量没有明显变化,而在3天和7天时灰飞虱mucin基因的表达量增加了3.60倍和1.97倍。饲喂健康水稻叶片1、2、3和7天后,灰飞虱mucin基因的表达量分别为饲喂前的1.07、1.12、0.78和0.34倍,而饲喂病叶1、2、3和7天后,mucin基因的表达量分别为饲喂前的1.15、1.19、3.19和1.01倍。结果表明,病毒胁迫使灰飞虱体内mucin基因的表达量增加,暗示mucin基因在RSV与灰飞虱互作过程中起着重要作用。     

小麦小GTP结合蛋白基因TaRop3克隆及其表达分析

利用同源克隆法从小麦抗条锈病基因Yr5近等基因系（Taichung29*6/Yr5）克隆到编码Rop蛋白基因的全长cDNA序列,并将基因命名为TaRop3（Triticum aestivum Rop3）,聚类分析其所在Rop蛋白家族中的亚组,并利用半定量RT-PCR方法分析其组织表达特异性和不同诱导条件下表达动态。序列分析表明,TaRop3开放阅读框为639 bp,预测编码含213个氨基酸残基Ⅱ型Rop蛋白,C末端具有膜定位基序,与大麦HvRop4和水稻OsRac4聚类在同一亚组。TaRop3基因在小麦幼苗和成株根、茎、叶片和茎节、柱头及花药中均有表达,其中在幼苗及成株茎部表达水平较高。非亲和条锈菌小种CYR17侵染和水杨酸处理诱导TaRop3表达增强,而干旱、高温胁迫以及脱落酸、乙烯利和茉莉酸甲酯处理均降低该基因表达水平。     

马铃薯甲虫热激蛋白基因Ld-hsp90的克隆及温度胁迫下的表达

马铃薯甲虫Leptinotarsa decemlineata是一种抗逆性极强的世界性检疫害虫,对温度胁迫具有很强的适应性,为进一步明确其对温度胁迫适应性的分子生态机制,采用RT-PCR及RACE技术克隆得到了1个hsp90基因的cDNA全长序列,命名为Ld-hsp90,并利用实时荧光定量PCR技术检测其在温度胁迫后相对表达量的变化。Ld-hsp90全长为2 489 bp,开放阅读框(ORF)长度为2 160 bp,编码719个氨基酸,理论等电点为4.98,分子量约为82.09 kD,5'端与3'端非编码区长度分别为113 bp和216 bp。氨基酸序列中含有HSP90家族的5个签名序列及胞质特征序列MEEVD。实时荧光定量PCR检测结果显示:38℃和44℃高温热激1 h,马铃薯甲虫成虫体内的Ld-hsp90的表达量均显著高于对照组,而0℃与-10℃低温处理1 h后的表达量与对照组均无显著差异。研究表明Ld-hsp90的上调表达在马铃薯甲虫抗高温中起着重要的作用。     

中华卵索线虫tra-1基因cDNA片段的克隆及实时定量表达分析

差异(P>0.05).线虫在宿主体内不同的感染强度(40/1与10/1)对tra-1基因转录活性无显著影响(P>0.05).表明tra-1基因参与调控中华卵索线虫雌性生殖系统的发育、卵的形成以及胚胎的发育.     

大豆花叶病毒CP基因RNAi载体的构建及大豆遗传转化

为获得含有大豆花叶病毒（Soybean mosaic virus,SMV）CP基因干扰片段的转基因大豆新材料,对11个SMV流行株系的CP基因进行了核苷酸序列比对,采用GATEWAY技术构建了RNA干扰（RNA interference,RNAi）载体pB7GWIWG2（Ⅱ）-CPi,对重组载体测序比对,通过酶切以及PCR方法鉴定载体,并利用农杆菌介导的转基因体系进行大豆遗传转化。克隆出264 bp高度保守的CPi干扰片段,通过测序证实其与目标序列的匹配度达到100%;重组质粒经过XbaⅠ单酶切后出现783 bp和10 818 bp两条带,经过EcoRⅠ、Hind Ⅲ双酶切后出现2 256 bp和9 346 bp两条带,说明2个ccdB结合位点均被CPi取代;PCR反应得到474 bp和460 bp两条带,说明CPi以反向重复形式与pB7GWIWG2（Ⅱ）组合。共获得10棵T₀代转基因幼苗,经除草剂涂抹、PCR、PAT/bar转基因检测试纸检测后,6株为阳性,表明该方法可筛选出对大豆花叶病毒具有抗性的转基因大豆新种质,为SMV抗病育种工程提供良好的亲本材料。     

松材线虫fem-1基因的克隆与表达特性研究

 松材线虫病是世界性的检疫病害,严重威胁松林等森林资源和生态安全。有研究表明,Feminization(fem)基因家族在秀丽隐杆线虫中参与性别决定和分化。本研究克隆了松材线虫fem-1基因,并对其表达特征和调控功能进行研究。结果表明,该基因cDNA全长为856 bp,包含681 bp的开放阅读框,编码226个氨基酸,进化分析显示与秀丽隐杆线虫位于同一进化分支。荧光定量PCR以及原位杂交结果显示, fem-1基因在松材线虫各个发育阶段均有表达,其中在2龄虫期表达水平最高;在卵期至3龄虫期阶段于虫体中后部表达,而发育后期,多位于皮下细胞中表达,肠道、性腺等部位极少出现杂交反应。利用RNA干扰技术沉默fem-1基因后,线虫种群雌雄比例出现下降,说明fem-1基因可能参与松材线虫性别分化和决定过程。研究结果有助于了解松材线虫性别决定相关基因表达特性与功能,为深入研究松材线虫发育生物学提供一定的理论依据。     

植物寄生线虫分支酸变位酶基因的研究进展

 植物寄生线虫食道腺中表达的寄生基因编码的分泌蛋白在线虫侵入寄主植物、建立取食位点和抑制寄主的防御反应过程中起重要作用。利用植物寄生线虫食道腺削减cDNA文库及基于同源克隆等方法,鉴定了这些过程中起作用的分支酸变位酶(CM)基因。带有氨基酸末端信号肽的根结线虫CM、孢囊线虫CM与细菌CM的蛋白质序列非常相似。mRNA原位杂交表明,CM基因专门存在于植物寄生线虫的亚腹食道腺中。RT-PCR分析表明,它们的转录丰度在线虫寄生的早期丰度较高,在后期较低或者难以检测到。Southern杂交表明,这些CM基因为多基因家族。CM的蛋白质在专性内寄生线虫中广泛存在,表明这种多功能的酶在控制线虫侵染植物的过程中起重要作用。     

斜纹夜蛾幼虫被中华卵索线虫寄生后的食量分析

斜纹夜蛾 (Spodopterlitura)原属间歇性害虫 ,2 0世纪 90年代该虫发生频繁 ,已发展成常发性害虫。该虫具有较强的隐蔽性、突然暴发性、夏秋高温干旱的多发性及食性杂而又有偏嗜性等特点[1] 。中华卵索线虫 (Ovomermissinensis)寄生前期幼虫能主动寻找宿主。为了明确斜纹夜蛾被中华卵索线虫寄生后的食量和病理变化 ,以便于更进一步保护和利用该线虫控制害虫对蔬菜等的危害 ,我们在室内测定了斜纹夜蛾幼虫被中华卵索线虫寄生前期幼虫人工感染后的食量变化 ,现将结果报道如下。1　材料和方法1 .1　供试虫　中华卵索线虫采自河南上蔡县麦田 ,室…     

短小芽胞杆菌抑菌相关基因的高效克隆

为有效挖掘生防短小芽胞杆菌Bacillus pumilus的抑菌相关基因,对DX01菌株进行了Solexa基因组扫描,并选取其中2个代表性基因TasA和Surf2,以DX01基因组DNA为模板,采用PCR方法对其进行克隆、序列比对及系统进化分析。结果表明,DX01菌株共得到4 267个基因,有3 145个具有明确生物学功能的蛋白。其中,与短小芽胞杆菌抑菌活性相关的基因（簇）有84个,包括2个细菌防御酶基因、2个细菌生物膜形成相关基因、1个信号转导基因、27个细胞壁降解或水解酶基因、15个抗生素合成或转运基因、4个抑菌蛋白基因、1个次生代谢物合成基因簇及32个细菌鞭毛生物合成与调控相关基因。TasA基因在芽胞杆菌属细菌中具有高度的保守性,各同源基因的序列相似性在92%以上;而Surf2基因的同源基因在该属细菌中具有一定的变异性,有的序列相似性低至73%。     

A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH-Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5 kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框（GenBank登录号为JQ309841）,编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。     

Cloning of a rice tau class GST isozyme and characterization of its substrate specificity

The GST cDNA was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU5 (GenBank Accession No. AF309377) sequence. The cDNA was composed of a 687-bp open reading frame encoding for 228 amino acids. The deduced amino acid sequence of this gene shared over 60% sequence identity with the sequences of the tau class ZmGSTU6 and ZmGSTU19. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTU5 formed a homo-dimer composed of 25 kDa subunit and its pI value was approximately 7.5. The OsGSTU5 displays high activities towards 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. The activity of the OsGSTU5 was significantly inhibited by hematin and ethacrynic acid. The OsGSTU5 shows the highest activity towards chloro-s-triazine and acetanilide herbicides.     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

南方根结线虫Col基因分子克隆及其VIGS效应分析

为了明确线虫角质层胶原蛋白基因(Col)与根结线虫病害的关系,利用从线虫基因组中预测的Col设计特异引物,克隆了南方根结线虫Meloidogyne incognitacol基因MiCol.该基因完整编码区长903bp,编码300个氨基酸.克隆的MiCol与预测的MiCol基因序列一致性高达99.67％,氨基酸序列一致性高达100％.利用病毒介导的基因沉默技术,将其导入番茄植株并接种南方根结线虫,60 d后,沉默载体pTV-MiColi处理番茄植株的根结数比空载体对照及清水对照分别减少49.4％和49.2％,表明MiCol基因沉默显著降低了南方根结线虫的侵染数量.