An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

亚洲玉米螟6-磷酸葡萄糖异构酶基因(Pgi)全序测定及分析

为进一步从核酸水平上研究亚洲玉米螟Pgi基因相关特性,采用反转录PCR及RACE等技术对该基因编码序列及DNA全序列进行了测定,与Gen Bank中其它昆虫Pgi基因相关信息进行比较分析,并构建了系统发育树。结果表明:亚洲玉米螟Pgi基因编码区序列长为1 671 bp,共编码556个氨基酸;其DNA序列全长为10 078 bp(短序列为9 311 bp),由12个外显子与11个内含子镶嵌而成;各外显子长度与鳞翅目大部分昆虫相同,介于95~188 bp之间,内含子序列总长度为8 407 bp(短序列为7 640 bp),各内含子长度介于418~1 547 bp之间,且在内含子3、4、5、11上均发现杂合现象。系统发育结果显示,大部分昆虫Pgi基因c DNA严格按物种聚类,除了双翅目的家蝇Musca domestica与黑森瘿蚊Mayetiola destructor出现一定的交叉现象外,其余没有出现交叉现象。     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.     

Cloning, Sequencing and Expression of a Xylanase Gene from the Maize Pathogen Helminthosporium Turcicum

A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and 62 bp, encoded a protein of 227 amino acids showing up to 95% amino acid homology with other fungal xylanases. The precise splicing site of the introns was identified by sequencing the corresponding cDNA. A northern blot showed that the gene is expressed when the fungus is grown in a medium containing xylan as a sole carbon source. The cloned xylanase gene was expressed in maize plants during infection.     

A rapid qualitative molecular method for the identification of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> and <Emphasis Type="Italic">C. gloeosporioides</Emphasis>

Identification of the causal agent for anthracnose caused by C. acutatum and C. gloeosporioides based on morphological and cultural criteria is problematic as both are morphologically and genetically diverse. To evaluate a qualitative molecular method to readily distinguish between these two species, Restriction fragment length polymorphisms (RFLP) of a 1-kb intron of the glutamine synthetase (GS) gene was evaluated utilizing representative isolates from a world-wide collection. Unique band patterns of the 1-kb GS intron were obtained for C. acutatum (two fragments with 600 and 350 bp) and C. gloeosporioides (four fragments with 238–340, 252–254, 204, and 108–116 bp) based on PstI enzyme digestion of the amplified PCR product. These data were also confirmed by PstI digestion of the intron DNA sequences using BioEdit software. The identification based on RFLPs of the 1-kb GS intron was consistent with the identification based on previously evaluated species-specific primers (CaInt2 and CgInt). In addition, both species can be differentiated by multiplex PCR. CaInt2, CgInt and ITS4 in one PCR will distinguish between C. acutatum and C. gloeosporioides by differences in PCR product fragment size: 490 bp and 470 bp, respectively. Also, a rapid DNA extraction method was developed, which reduced the time for DNA extraction from two hours to five minutes. In summary, RFLP of the 1-kb GS intron is a reliable technique for identification and differentiation between both species, does not require a sequencing step, and may be useful to diagnostic clinics in helping to make disease management recommendations.     

禾谷缢管蚜水通道蛋白基因RpAQP1的克隆、分子特性和表达分析

为探究禾谷缢管蚜Rhopalosiphum padi (Linnaeus)水通道蛋白基因RpAQP1的序列特征及其在不同龄期的表达,利用RT-PCR和RACE技术克隆了RpAQP1基因的cDNA全序列,采用生物信息学软件分析了RpAQP1编码蛋白的特性,利用qRT-PCR分析了RpAQP1在不同龄期的表达量。结果显示:禾谷缢管蚜水通道蛋白基因RpAQP1的cDNA全长为1 216 bp,其750 bp的开放阅读框编码250个氨基酸,蛋白分子量为27.36 kD。RpAQP1属于水通道蛋白亚家族DRIP(果蝇内嵌蛋白Drosophila integral protein)的一员,具有6个跨膜区,2个保守的NPA结构单元和1个压缩区域Ar/R;qRT-PCR结果显示,RpAQP1在整个发育历期均有表达,其在2龄若蚜中表达水平最高,是成蚜表达量的1.432倍;在4龄若蚜中表达量最低,为成蚜表达量的0.444倍,显著低于其它龄期,而其余各龄期RpAQP1表达量无显著差异。     

烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。     

以Ypt1基因序列为靶标的辣椒疫病菌快速分子检测

PCR primers were designed based on the sequence of Ras-related protein gene (Ypt1) of P. capsici. According to the multiple sequence alignment, Ypt1 has the sufficiently polymorphic intron region for the development of P. capsici-specific primers (PcYpt1F/PcYpt1R). One primer pair was developed which can amplify one P. capsici-specific fragment of 156 bp. Using the primer pair, the P. capsici infected plants and soils were detected. Additionally, Ypt1 has an appropriate region for the development of Phytophthora genus-specific primers (Ypt1F/Ypt1R), which can amplify a fragment of about 540 bp from 14 different Phytophthora specices and a fragment of about 350 bp in Pythium species, with no amplification from fungal species. By PCR optimization using P. capsici genomic DNA, the detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (PcYpt1F/PcYpt1R) and nested PCR (Ypt1F/Ypt1R and PcYpt1F/PcYpt1R), respectively. The developed primers were proved to be efficient in detection of Phytophthora pathogens from diseased plant tissues and residues in soils.     

两种金小蜂体内Wolbachia的wsp基因分子检测及序列分析

采用Wolbachia的通用引物及A和B大组特异性引物对蝶蛹金小蜂Pteromalus puparum和丽蝇蛹集金小蜂Nasonia vitripennis体内Wolbachia的wsp基因进行PCR扩增及序列测定，并对测定的序列进行了同源性比较和基因特征分析。结果表明：蝶蛹金小蜂和丽蝇蛹集金小蜂均被A和B两个大组的Wolbachia复合感染；同种寄生蜂的雌蜂和雄蜂的wsp基因片段序列完全一致。采用通用引物从蝶蛹金小蜂中扩增出Wolbachia的wsp基因片段序列的长度为540bp，属于B大组中Pip组，而从丽蝇蛹集金小蜂中扩增出Wolbachia的wsp基因片段序列的长度为558bp，属于A大组中Uni组。用A-Wolbachia引物从蝶蛹金小蜂和丽蝇蛹集金小蜂中扩增出的wsp基因片段序列长度为548bp，同源性达99．8％；而用B-Wolbachia引物从两者中扩增的两条wsp基因片段序列长度分别为424bp和439bp同源性达87．5％．     

烟粉虱MED隐种蜕皮激素受体基因cDNA克隆、转录与组织表达

蜕皮激素受体(ecdysone receptor,Ec R)是调控昆虫蜕皮、变态和生殖等过程中基因表达的重要调控因子。为研究Ec R在烟粉虱Bemisia tabaci生长发育中的作用,利用RT-PCR和RACE等技术扩增了烟粉虱MED隐种Ec R基因的c DNA全长序列,并利用实时荧光定量PCR技术检测其在烟粉虱不同发育时期相对表达量的变化。Bt Ec R c DNA序列全长2 844 bp,含有一个1 518 bp的开放阅读框,共编码505个氨基酸,预测蛋白分子量为56 k D,等电点为6.43,含有特殊结构功能域:DNA结合域(DBD_Ec R)和配体结合域(LBD_Ec R),该序列编码的蛋白质序列与其它已报道的昆虫Ec R蛋白序列具有很高的相似性,命名为Bt Ec R(Gen Bank登录号:KR534774)。Bt Ec R在MED隐种若虫、雌成虫各发育时期均有表达,若虫期在伪蛹前期达到最高值,成虫期呈现先升高后降低的趋势,且在羽化后第7天达到最高值,约为羽化第1天的23倍。研究结果为揭示Bt Ec R在烟粉虱整个生长发育过程中的作用提供了重要依据。     

Kerala wilt disease phytoplasma: Phylogenetic analysis and identification of a vector, Proutista moesta

Kerala wilt disease of coconut palm is a major threat of coconut production in Kerala caused by phytoplasma. The genomic DNA purified from the insect tissues of Proutista moesta (PM) and Stephanitis typica (ST) was subjected to PCR assay using the primer combination P1/P6, P1/P7 and P4/P7. The amplified products resolved a prominent band of 650 bp for the universal primer P4/P7 and no bands were noticed for the primer pairs P1/P6 and P1/P7 combination. Since P4/P7 amplifies the 16S–23S intergenic spacer region of 16SrRNA gene, the PCR product 650 bp of the insect PM indicate the phytoplasma DNA. The presence of 650 bp for the primer P4/P7 in the genomic DNA isolated from P. moesta indicates the vectoral ability of the insect. No sign of amplification was noticed in the case of ST for the three sets of primers suggesting the inability of this insect as vector. The amplified product 650 bp from the genomic DNA of KWD palms as well as the insect tissues of P. moesta was gel purified and sequenced. The sequential similarity of 650 bp of both KWD phytoplasma and the insect phytoplasma supports the transmission of phytoplasma through the vector PM. Moreover, the sequence of 650 bp was compared with other sequences of 26 coconut phytoplasmas so far reported internationally and a cladogram was prepared for determining the phylogenetic status. It is obvious from the cladogram that the KWD disease phytoplasma is evolutionarily closest to coconut phytoplasma of coconut lethal yellowing of Mexican palms within the group 16SrIV. Phylogenetically, KWD phytoplasma is grouped in the new subgroup 16SrIV-C subsequent to the groups 16SrIV-A and 16SrIV-B for Mexican coconut lethal yellowing and Tanzanian coconut lethal decline, respectively. The restriction enzyme analysis of the PCR product 650 bp using the enzymes AluI, BclI, HindIII and RsaI further supports the phytoplasmic nature of DNA. This data records the first finding of the vector of Kerala wilt disease by detecting KWD phytoplasma in insect tissue of PM by PCR based methods. Moreover, the study reveals the phylogenetic status of KWD phytoplasma compared to other coconut phytoplasmas internationally.     

进境玉米中玉米内州萎蔫病菌的PCR检测

 为了快速准确检测进境玉米样品中的玉米内州萎蔫病菌Clavibacter michiganensis subsp. nebraskensis(Cmn), 根据GenBank中Cmn的16S-23S序列设计引物CM1/CM4和引物PSM1/CM3。引物PSM1/CM3仅能从供试的4株Cmn菌株中扩增获得208 bp的预期产物, 而其他36株对照菌株均不能扩增出预期条带。灵敏度测试结果表明引物CM1/CM4和PSM1/CM3组合的巢式PCR方法的检测灵敏度高于常规PCR, 检测灵敏度可达40 fg DNA或6.8 CFU目标细菌。常规PCR和巢式PCR方法对进境美国玉米样品的阳性检出率分别为8%和24%, 试验结果表明所建立的PCR方法可用于玉米样品中Cmn的快速检测。     

淡紫拟青霉γ-actin基因cDNA全长的克隆与序列分析

利用同源克隆的策略对淡紫拟青霉(Paecilomyces lilacinus)γ-actin基因进行克隆,获得了淡紫拟青霉γ-ac-tin基因cDNA全长。该基因cDNA的ORF长1128bp,编码375个氨基酸,具有actin基因的保守特征序列。由该基因推导的氨基酸序列与多个子囊菌的γ-actin蛋白序列相似性达98%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。     

番茄叶霉病高抗基因Cf-9的CAPS标记创建

根据番茄叶霉病高抗基因Cf-9的基因序列设计3对特异扩增引物,以近等基因系和本组的多重PCR检测材料为试材,扩增Cf-9基因1～2867 bp之间的单拷贝片段,3对引物分别扩增出560 bp,1 000 bp,1 080 bp的特异片段。分别用限制性内切酶TaqⅠ,HindⅢ,HinfⅠ,EcorⅠ酶切,限制性内切酶TaqⅠ酶切特异引物SCAR1扩增的560特异片段具有酶切多态性,抗病品种酶切出450 bp,330 bp,290 bp的特异片段,感病品种酶切出450 bp,290 bp的特异片段。初步建成番茄叶霉病高抗基因Cf-9的CAPS标记,经近等基因系及其杂交种检验,结果稳定可靠,可用于番茄Cf-9抗病基因辅助选育。     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

棉铃虫V-ATP酶亚基A基因克隆及表达谱分析

为探索棉铃虫Helicoverpa armigera液泡型ATP酶(vacuolar-type proton ATPase,V-ATP酶)亚基A对棉铃虫生长发育的影响及其在Bt杀虫机制中的作用,采用PCR结合RACE技术克隆了棉铃虫V-ATP酶亚基A基因序列,通过实时荧光定量PCR技术测定了其在棉铃虫不同发育历期和幼虫肠道不同组织中的表达量;并比较了4龄幼虫取食含Cry1Ac蛋白饲料后中肠中该基因表达量的变化。结果显示,棉铃虫V-ATP酶亚基A基因全长2 578 bp(Gen Bank登录号KP090287),开放阅读框1 863 bp,编码621个氨基酸。V-ATP酶亚基A高度保守,不同物种间氨基酸序列同源性大于90%。V-ATP酶亚基A在棉铃虫整个生育期都有表达,在4龄幼虫体内表达量最高,是卵期的3.00倍;在幼虫肠道不同组织中,中肠中表达量最高,是后肠中的2.65倍。4龄棉铃虫幼虫取食含Cry1Ac蛋白的人工饲料后,中肠V-ATP酶亚基A的表达受到抑制,表达量为对照的0.39~0.81倍。表明V-ATP酶亚基A基因可能参与棉铃虫的生长发育和代谢过程,并可能与抵御Cry1Ac的毒杀作用有一定关系。     

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.