一个二粒小麦LRR类受体蛋白激酶基因的克隆和鉴定

 Near the HMW-glutenin gene of wheat (Triticum aestivum), there is a locus (temporarily named TaXa) encoding LRR-receptor-like protein kinase, which is homologous to disease resistance protein Xa21 of rice (Oryza sativa). Through RT-PCR approach, a cDNA clone of ZS2002 was isolated from the orthologous locus of TaXa in Triticum turgidum. ZS2002 was 3 081 bp long and encoding a peptide composed of 1 026 amino acid. The protein included N-terminal conserved sequence, LRR domains, a transmembrane region and a serine/threonine protein kinase domain. ZS2002 was expressed in root, stem, leaf and spike. The transcribing in seedling leaves was significantly enhanced by Blumeria graminis f.sp. tritici. TaXa gene might play a role in powdery mildew resistance reaction in Triticum.     

引起玉米粗缩病的水稻黑条矮缩病毒山东分离物的分子特性

 Four isolates of Rice black-streaked dwarf virus (RBSDV) were collected from the maize plants showing rough dwarf symptom in Linyi and Tai'an,Shandong province.The S10 genomic sequences of these isolates were determined and compared with those of 14 other RBSDV isolates.All of the four sequences were 1 801 base pairs (bp) long including the 5'-UTR of 21 bp and the 3'-UTR of 103 bp.They all contained an open reading frame of 1 677 bp (22-1698),encoding the coat protein (CP) of 558 amino acids.The sequences of these four RBSDV isolates and those of the major cp gene of 14 other isolates available in the GenBank were divided into two groups in the phylogenetic tree.Recombination analysis indicated that the isolate Lym2 was likely a recombinant of isolates Lym1 and Zhjs.     

水稻条纹病毒NS2基因原核表达产物多克隆抗体制备及受侵染水稻和灰飞虱体内NS2检测

 The NS2 gene of Rice stripe virus (RSV) was amplified by RT-PCR, cloned into pGEM-T vector and sequenced. The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3) pLysS. SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG. The recombinant NS2 protein was purified with Ni²⁺-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit. NS2 protein was successfully detected in small brown planthopper (Laodelphax striatellus) at 1:1 600 dilution of the total protein of single planthopper and in infected rice (Oryza sativa) at 1:800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

苏云金芽孢杆菌晶体蛋白对北方根结线虫生物测定方法的建立和杀虫效果评价

 A bioassay procedure was developed to assess the toxicity of Bacillus thuringiensis crystal protein against Meloidogyne hapla,a root-knot nematode,under laboratory conditions.Reproducibility and precision of the bioassay results were optimal when forty 2nd stage juveniles were incubated in the dissolved crystal protein solution at 25℃,pH9.0 for 7 days.The juveniles were stained with 1% KMnO₄ for 2 hours or methylene blue solution for 1 hour to distinguish living and dead ones.By the bioassay procedure,the LC₅₀ value of strain YBT-1532 crystal protein against M.hapla was determined as 0.304±0.086 mg/mL(LC₅₀±1.96SE).Moreover,the strain YBT-1532 showed toxicity to Caenorhabditis elegans,a free-living nematode.All results indicated that YBT-1532 is a toxic strain to plant-parasitic nematode,and has the potential to control plant-parasitic nematode.     

土壤中烟草根黑腐病菌的实时定量PCR检测技术研究

 Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.     

香蕉线条病毒ORFⅡ基因的原核表达及抗体制备

 ORFⅡ gene of Banana streak virus GuangDong isolate (BSV-GD) was amplified from a BSV-GD recombinant plasmid by PCR, and the gene was expressed by being cloned into prokaryote expression vector pET-28b (+). The fusion protein was about 16.5 kDa in size and was soluble with SDS-PAGE analysis. The purified protein was obtained by using the histidine labeling kit of N-terminus of protein. The antiserum was obtained by immunizing healthy rabbits with the purified protein. Western blot and ELISA analysis showed that the special antiserum of BSV possessed high titer, which was tested as 1:51 200. The study was a base for further research on BSV including ORFⅡ gene function and virus detection.     

菰黑粉菌MAPK同源基因UeKss1的克隆及表达

菰黑粉菌作为茭白植株体内的内生真菌,其二型态转换与茭白孕茭密切相关,而MAPK途径在真菌二型态转换中具有重要调控作用。本研究在菰黑粉菌中克隆得到了一个MAPK基因UeKss1,通过与酵母菌中的MAPK途径蛋白进行聚类分析表明其属于Kss1的同源蛋白。进一步的系统进化分析表明,UeKss1与黑粉菌属真菌的Kss1同源性最高,达80%以上,且它的丝/苏氨酸双特异性蛋白激酶催化结构域高度保守。不同碳源诱导下菰黑粉菌的生长特征及UeKss1表达分析发现,只有蔗糖培养基能诱导菰黑粉菌菌丝形成,而在PDA、葡萄糖和麦芽糖培养基中,该菌都保持酵母型生长;UeKss1基因的表达量在PDA和葡萄糖培养基中变化不显著,但在麦芽糖培养基中,该基因的表达随着培养时间的延长持续上调表达,而在蔗糖培养基中,UeKss1的表达量虽然也呈现上升趋势,但是远小于麦芽糖的诱导表达量。对UeKss1进行的原核表达纯化,经Western blot验证得到纯度较高的UeKss1蛋白。研究结果可为UeKss1基因的功能研究,阐明其在菰黑粉菌二型态转换中的作用机制提供帮助。     

苏云金芽胞杆菌杀虫晶体蛋白Cry1D新基因的克隆与特性

为扩充鳞翅目害虫杀虫基因资源，本研究从苏云金芽胞杆菌BN23-5中克隆得到一个新的cry基因，并对其进行鉴定和分析。该基因为一个完整的cry1D基因，全长3501 bp，编码1166个氨基酸残基。该氨基酸序列是一个新的Cry氨基酸序列，与Cry1Db1的同源性最高，为86%，命名为Cry1Dd1（登录号为KJ728844）。将该基因插入穿梭表达载体pSTK中，转入BT无晶体突变株HD73^-中进行表达。结果表明，cry1Dd1基因能在BT无晶体突变株中表达，并形成菱形伴孢晶体。SDS-PAGE验证其分子量为132.2 kD，与预测的大小相符。生物活性测定表明，Cry1Dd1晶体蛋白对小菜蛾的幼虫具有杀虫活性，LC₅₀为13.1 μg/mL；能明显抑制甜菜夜蛾幼虫的生长；但对棉铃虫幼虫没有杀虫活性。对cry1Dd1基因序列进行分析，cry1Dd1包含8个block保守区域，这和目前其他的cry基因相似；Cry1Dd1蛋白的活性区域为N端的37~593位氨基酸残基。     

苹果炭疽叶枯病菌致病相关基因CgNVF1的功能初步分析

苹果炭疽叶枯病是主要由胶孢炭疽菌(Colletotrichum gloeosporioides)引起的一种苹果重要叶部病害,严重威胁着苹果树的生长。本研究从构建的苹果炭疽叶枯病菌T-DNA突变体库中筛选获得一株致病力缺失的突变菌株A3083,采用hiTAIL-PCR方法克隆了该突变体T-DNA插入位点的右翼序列;通过与胶孢炭疽菌基因组序列比对分析,发现T-DNA插入位点位于1个预测的CGGC5_9603基因内,并将该基因命名为CgNVF1。CgNVF1基因全长2252 bp,含有2个内含子,编码709个氨基酸。CgNVF1定位于细胞质,在苹果炭疽叶枯病菌菌丝、分生孢子和附着胞中均有表达。通过构建CgNVF1敲除菌株和CgNVF1互补菌株,并结合表型分析,证实CgNVF1基因在苹果炭疽叶枯病菌附着胞形成及致病中具有重要的作用。     

胶孢炭疽菌CgSho1基因的克隆与功能分析

HOG-MAPK(high osmolarity glycerol mitogen-activated protein kinase)信号途径是真菌MAPK途径中参与渗透压响应的一条重要通路,在植物病原菌生长发育及致病过程中发挥着重要的作用。Sho1(synthetic high osmolarity-sensitive protein1)是HOG-M APK信号途径上游的一个重要感受器,在不同真菌中常具有不同的功能。本研究从胶孢炭疽菌中克隆了Sho1的同源基因,命名为Cg Sho1,该基因编码一个291个氨基酸的蛋白,含有4个跨膜结构域和一个SH3功能域。利用同源重组的方法获得了该基因的敲除突变体,与野生型相比,敲除突变体表现为营养生长缓慢,菌丝稀疏且疏水性增强,产孢量下降,对氧化压力和渗透压更加敏感,致病力明显减弱。上述结果表明,Cg Sho1参与调控胶胞炭疽菌的营养生长、分生孢子产量、氧化应激反应、渗透压响应及致病性。     

寄生于南方根结线虫卵的长梗木霉几丁质酶基因TlChi46的克隆

为进一步筛选高效寄生线虫真菌和阐明其寄生线虫卵的机理,本研究从湖北省烟草南方根结线虫雌虫分离到1株具高效生防潜力的菌株HBF1。形态学、rDNA-ITS和翻译延长因子tef1-α序列分析鉴定该菌为长梗木霉菌Trichoderma longibrachiatum,其对南方根结线虫卵第10 d寄生率为80.45%。通过简并引物设计和RACE技术克隆其几丁质酶基因,分析该基因序列及与其他几丁质酶的同源性。该菌是1株可以产生几丁质酶的南方根结线虫卵寄生真菌,第10 d几丁质酶的活性达到高峰,为24.88μmol/h/mL。本研究首次从长梗木霉HBF1菌株中克隆到1个几丁质酶基因TlChi46,该基因DNA全长1 793 bp,含3个内含子和1个1 272 bp的开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.23。同源性比对表明和昆虫寄生菌几丁质酶的亲缘关系较远。从长梗木霉HBF1中克隆得到的几丁质酶基因编码的几丁质酶的功能域可供进一步研究,高效产几丁质酶并有效寄生南方根结线虫卵的长梗木霉对南方根结线虫具有良好的生防潜力。     

大豆疫霉MAPK基因的鉴定与转录分析

 疫霉菌包括大豆疫霉等重要植物病原物,属于茸鞭生物界卵菌门,在进化上与真菌相差甚远;由于分离培养与遗传转化等相对困难,目前对其生长发育和致病机理的研究相对滞后。本研究综合运用基因组学和转录组学方法,首先对植物病原卵菌与真菌的蛋白激酶特别是MAPK进行了鉴定和比较,然后对大豆疫霉MAPK的基因结构、蛋白功能域以及转录模式等进行深入分析。结果表明,植物病原卵菌比真菌含有更多的蛋白激酶(包括MAPK),且卵菌及其近缘物种硅藻的MAPK与真菌在进化上相对独立;大豆疫霉的14个MAPK中,4个具有非典型的磷酸化唇序列,7个含有PH、C2、WW、PAS等与细胞信号转导相关的其它功能域;转录分析表明,大部分MAPK可能在大豆疫霉生长发育与致病的整个过程或特定过程中发挥重要作用。本文通过对蛋白激酶特别是MAPK的分析揭示了植物病原卵菌(与真菌相比)在细胞信号转导网络与机制上的独特性与复杂性,可为进一步研究疫霉菌MAPK的生物学功能及其信号调控机制提供参考。     

水稻VQ37基因的克隆及其在病原侵染下的表达

 VQ蛋白作为转录辅助蛋白,在植物的生长、发育和抗逆等生理过程中发挥重要的调节功能。本研究采用RT-PCR从水稻叶片中克隆了VQ37基因的完整cDNA序列。VQ37 cDNA长622 bp,具有长为546 bp的完整开放读码框,编码蛋白质长181个氨基酸,具有FxxxVHxVTG的VQ基序变体。系统进化分析表明,水稻VQ37与短花药野生稻(Oryza brachyantha)、大麦(Hordeum vulgare)和Dichanthelium oligosanthes等禾本科植物亲缘关系近;除水稻旁系同源物VQ39外,VQ37与短花药野生稻中的XP 015699121亲缘关系最近。原生质体瞬间表达实验证实VQ37定位在细胞核中。荧光定量PCR分析显示,VQ37基因的组织特异性表达和诱导表达结果与启动子顺式元件预测基本一致。VQ37在叶片中的表达丰度最高,其次是叶鞘、茎、穗、根和花,在胚和胚乳中无表达。VQ37受纹枯病菌(Rhizoctonia solani)和稻瘟病菌(Magnaporthe oryza)显著诱导,而不受白叶枯细菌(Xanthomonas oryzae pv. oryzae)诱导;与此一致的是,VQ37受真菌病原相关模式(PAMP)分子几丁质寡糖快速诱导,而不受细菌鞭毛蛋白flg22影响。茉莉酸甲酯和乙烯利能显著诱导VQ37的表达,而水杨酸对其表达无明显影响。上述结果提示,VQ37可能调控水稻对稻瘟病菌和纹枯病菌防御反应,这种调节作用可能依赖于茉莉酸/乙烯介导的信号途径,而与水杨酸信号途径无关。该研究为阐释水稻VQ37基因在水稻抗病反应中的调节功能提供了基础。     

滞育七星瓢虫酰基辅酶AΔ11去饱和酶基因的克隆及表达分析

去饱和酶（Fatty acid desaturase,FAD）是生物体不饱和脂肪酸合成中的关键酶,酰基辅酶AΔ11去饱和酶基因是其中重要的一种。本试验基于七星瓢虫Coccinella septempunctata L.转录组数据,利用RT-PCR和RACE技术从滞育七星瓢虫中克隆得到酰基辅酶AΔ11去饱和酶基因全长,并命名为CsFadΔ11（GenBank登录号：MF458996）,该基因全长1447 bp,开放阅读框（ORF）1095 bp,编码364个氨基酸,预测蛋白质分子量为42.99 kD,等电点（pI）为8.87,无信号肽。氨基酸序列分析结果表明,CsFadΔ11有3个组氨酸富集区和6个跨膜结构域,与膜翅目的内华达古白蚁Zootermopsis nevadensis同源性较高。利用实时荧光定量PCR技术研究其时间表达模式,发现CsFadΔ11基因在七星瓢虫初羽化、滞育诱导10 d、滞育诱导20 d、滞育初期、滞育后期、滞育解除期以及正常发育状态下均有所表达,且在滞育早期表达量最高,其次是在滞育解除个体中。其整体表达趋势与相应时期脂积累变化情况基本相符,推测CsFadΔ11基因参与七星瓢虫脂积累调控,并进一步影响七星瓢虫滞育。