谷子抗病基因同源序列的克隆与分析

谷子锈病是谷子上的一种流行性强、毁灭性大的病害,严重影响谷子生产.种植抗病品种是防治锈病最经济有效的方法,但谷子抗锈病种质资源非 常贫乏,而且高抗锈病的材料其农艺性状又很差.很难通过传统育种方法培育抗锈病品种,因此克隆谷子抗锈病基因尤为重要.目前克隆到的许多植 物抗病基因编码的氨基酸序列都有一定的保守结构域.根据抗病基因保守结构域,已克隆的抗病基因主要分为5类,其中最主要的是NBS-LRR(nu- leotide-binding site leucine-rich epeat)和STK(Se-rine／Threonine protein kinase)类.因而根据抗病基因保守结构域设计引物,从植物的DNA中扩增植物的抗病基因同源序列RGA(resistance geneanalogs)更加快捷有效,目前通过RGA方法克隆植物抗病基因已有报道[1,2].     

甘蔗NBS-LRR类抗梢腐病基因的筛选及定量表达分析

本文旨在探讨核苷酸结合位点和富含亮氨酸重复(NBS-LRR)类基因参与甘蔗抗梢腐病菌侵染应答机制,为后续克隆关键抗病基因以及研究抗病机理提供理论依据。试验利用Illumina高通量转录组测序技术检测高抗梢腐病品种‘粤糖94-128’和高感梢腐病品种‘桂糖37号’接种前后NBS-LRR类抗梢腐病基因的表达情况,然后设计引物对显著差异表达基因进行不同接种时期下的荧光定量PCR验证。结果表明,16个NBS-LRR类基因在甘蔗叶片受到梢腐病菌侵染后持续上调表达,但表达趋势存在两种情况,13个基因在诱导后7 d显著或极显著上调表达,3个基因在诱导7 d后上调表达不显著,在诱导14 d后才显著上调表达。据此可将其分为瞬时基础防御基因(0~7 d)和滞后特异性防御基因(14~21 d),确定NBS-LRR类基因参与甘蔗防御梢腐病菌的侵染。     

小菜蛾GluCl受体α亚基cDNA基因克隆和序列分析

利用RT-PCR和RACE技术对小菜蛾[Plutella xylostella(L.)]4龄幼虫谷氨酸门控的氯离子通道(GluCl)受体cDNA全长进行了克隆和序列分析。通过设计简并性上、下游引物扩增出小菜蛾GluCl受体α亚基基因192bp的cDNA片段,根据得到的片段序列设计3′和5′特异性引物采用RACE技术进行3′和5′末端的克隆,获得了小菜蛾GluCl受体的cDNA的完整序列,GenBank登录号为GQ221939。该基因全长2144bp,其开放阅读框(ORF)1344bp,编码447个氨基酸。相似性分析表明:它与果蝇和赤拟谷盗的相似性均为73%,与埃及伊蚊的相似性为75%,与东亚飞蝗的相似性为79%;氨基酸分析后显示它具有GluClα亚基的典型特征,表明克隆的cDNA序列是小菜蛾GluClα亚基基因的序列。     

小麦LRR类抗病相关基因及其cDNA 3′末端的克隆与分析

抗病基因克隆对于培育抗病作物品种和研究病原物与寄主之间相互作用机制具有重要意义。目前,已经从多种植物中成功克隆了48个抗病基因[1]。从这些克隆的基因及其所揭示的产物结构和功能看,已克隆的植物抗病基因在氨基酸水平上表现了一定程度的相似性,在一些区段高度保守[2,3],这一特性使得同源克隆成为可能,目前已广泛用于植物抗病基因及其同源物的分离。其中Lr10是利用该技术获得成功克隆的首次报道[4]。cDNA末端快速扩增技术(rapid amplification ofcDNA ends,RACE),是一种从低丰度转录本中快速扩增cDNA5′和3′末端简单而有效的方…     

1个小麦NBS类抗病基因同源cDNA序列的克隆与鉴定

 利用cDNA末端快速扩增技术对小麦抗叶锈病近等基因系TcLr35中所获得的抗病基因同源片段S2A2 5'端和3'末端序列进行扩增,并根据拼接序列设计特异性引物,进行全长基因的扩增,获得了1个通读的NBS类抗病同源基因S2A2 cDNA序列,该序列全长为3476 bp,编码866个氨基酸序列。经BLASTp比较,该片段含有NB-ARC保守结构域和多个LRR结构域,与已知植物抗病基因I2C-1、L6、RPS2等相应区域相一致。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr35小麦中成功获得了抗病同源基因,这为最终克隆小麦抗叶锈病基因Lr35奠定了基础。     

小麦抗叶锈病基因Lr38的RGA分析初报

小麦抗叶锈病基因Lr38位于中间偃麦草 (Agropyron intermedium)第7组的一条染色体上,是抗性很强的基因,国内外至今尚未发现对 Lr38有毒性的菌株,是一个应用潜力很大的抗病基因。通过对目前已克隆的抗病基因结构分析,大部分抗病基因存在一些保守区域,利用保守域序列设计简并引物,PCR扩增抗病基因相似序列(Re- sistance gene analogs,RGA),来进行抗病基因克隆是一条很好的途径。本研究根据已克隆基因的蛋白激酶类保守序列设计简并引物RAF3和RAR4,对TcLr38及感病亲本Thatcher进行PCR扩增,旨在明确与小麦抗叶锈病基因Lr38相关的蛋白激酶,为克隆Lr38奠定基础。     

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。     

心叶烟NPR1基因全长cDNA序列的克隆及表达分析

 NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。     

我国栽培番茄部分品种LescPtoF基因序列多态性的初步研究

根据已报道的番茄抗细菌性斑点病基因Pto家族成员之一LescPtoF的序列特征设计一对引物,通过PCR扩增,从我国10个抗病栽培番茄品种和3个感病品种获得13个同源序列。Blast结果表明,2个来自抗病品种和2个来自感病品种的核苷酸序列均和报道的LescPtoF完全一致。其余9个核苷酸序列和LescPtoF序列相似性最低为91%,它们之间相似性最低为87%。具有完整读码框的11个核苷酸序列所编码氨基酸与LescPtoF蛋白相似性最低为84%,11个氨基酸序列之间相似性最低也为84%。研究结果表明我国栽培番茄品种LescPtoF基因同源序列的分布具有多态性。     

本氏烟NbWRKY40亚家族转录因子抗病相关功能研究

 植物WRKY类转录因子的IIa亚类广泛参与调控植物的生物胁迫和非生物胁迫过程。本研究根据拟南芥和普通烟中具有抗病和抗胁迫功能的IIa亚类WRKY40转录因子的序列,利用基因同源克隆法,获得本氏烟中NbWRKY40基因。序列分析表明在本氏烟中有5个属于IIa亚类的基因,其序列之间具有高度相似性。qRT-PCR检测发现NbWRKY40基因是受寄生疫霉侵染诱导上调表达的基因,通过VIGS技术研究该类基因在本氏烟中的抗病相关功能,用半活体营养型寄生疫霉进行抗病性试验,结果表明NbWRKY40基因的沉默降低了本氏烟对寄生疫霉的抗性,且在侵染点的细胞坏死程度增强,过氧化氢积累和胼胝质沉积都明显减少。NbWRKY40基因沉默同样降低了本氏烟对死体营养型灰霉菌的抗性。检测NbWRKY40基因沉默后4个不同抗病信号通路下游基因的表达,发现基因沉默导致参与抗病和SA途径的PR1b、PR2b基因受疫霉侵染诱导表达的水平明显下降。综上,推测NbWRKY40基因可能依靠SA介导的信号通路参与调控本氏烟抗病性。     

Identifying resistance gene analogs associated with resistances to different pathogens in common bean

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.     

Cloning and characterization of an NBS-LRR resistance gene from peanuts (Arachis hypogaea L.)

The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes and is one of the largest gene families in plant genomes. In this study, resistance gene analogs (RGAs) were isolated from peanuts based on the NBS domain. A full-length cDNA, PnAG₃, was obtained by rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the length of PnAG₃ was 1882 bp, which included a complete open reading frame of 1335 bp that encoded for the PnAG₃ protein composed of 444 amino acids. Multiple analyses showed that this protein had homology with known resistance proteins, the highest being 48.01% with a resistance protein from Arachis cardenasii. The polypeptide has a typical non-TIR-NBS-LRR gene structure. Real-time fluorescence quantitative PCR analysis showed that after Aspergillus flavus infection, expression of the PnAG₃ gene in J11 (A. flavus-resistant species) increased by 16.68, 11.16 and 25.96 in the seed coat, kernel and pericarp, respectively. However, it only increased 2–3 times in JH1012 (A. flavus-sensitive species). Cloning of the putative resistance gene from peanut provides a basis for studying the structure and function of peanut disease resistance-related genes and disease resistance genetic breeding in peanuts.     

The CC-NBS-LRR Subfamily in Pinus monticola: Targeted Identification, Gene Expression, and Genetic Linkage with Resistance to Cronartium ribicola

ABSTRACT To investigate disease resistance gene analogs (RGAs) encoding coiled-coil-nucelotide-binding site-leucine-rich repeats (CC-NBS-LRR) proteins in western white pine, degenerate primers targeting the conserved motifs in the NBS domain were designed to amplify RGAs from genomic DNA and cDNA. Sixty-one distinct RGAs were identified with identities to well-known R proteins of the CC-NBS-LRR subfamily. These RGAs exhibited variation of putative amino acid sequences from 13% to 98%, representing a complex CC-NBS-LRR subfamily. A phylogenetic tree constructed from the amino acid sequence alignment revealed that these 61 RGAs were grouped with other CC-NBS-LRR members from angiosperms, and could be further divided into six classes with an identity threshold of 68%. To map RGAs, RGA polymorphisms and a modified amplified fragment length polymorphism (AFLP) method with incorporated sequences from the NBS domain were used to reveal NBS or NBS-AFLP markers. RGA polymorphism study revealed that three off the identified RGAs were not linked to the Cr2 gene imparting resistance to white pine blister rust. However, the AFLP strategy, using bulk segregant analysis (BSA) and haploid segregation analysis, identified 11 NBS-AFLP markers localized in the Cr2 linkage, the closest two to the gene being 0.41 cM and 1.22 cM away at either side. Eight of these markers showed significant amino acid sequence homologies with RGAs.     

甘肃省主要小麦生产品种及高代品系的抗白粉基因推导

［目的］ 采用基因推导法对目前甘肃省小麦主要生产品种及高代品系进行抗白粉基因分析,为甘肃省白粉病的抗病育种和品种使用及布局提供依据。［方法］ 利用21个小麦白粉菌株,结合品种系谱对甘肃14个小麦生产品种及28个高代品系进行抗白粉基因推导。［结果］ 14个生产品种中,1个含Pm8,1个含Pm4b,4个含未知抗病基因,其余8个对所有供试菌株全部表现感病;28个高代品系中,5个含Pm8〖STBZ〗,2个含Pm〖STBX〗3〖STBZ〗a,1个含Pm〖STBX〗4〖STBZ〗b,1个含Pm〖STBX〗30〖STBZ〗,5个含未知抗病基因,其余14个对所有供试菌株全部表现感病。［结论］ 目前甘肃小麦生产品种及高代品系中缺乏抗白粉病基因,一旦条件适宜,小麦白粉病在甘肃地区极易发生和流行,应该引起生产管理部门和育种专家的注意和重视。     

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.)

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. RESULTS: Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. CONCLUSION: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.     

几个常用内参基因在淀粉酶产色链霉菌1628抗药性突变株中稳定性的评估

淀粉酶产色链霉菌Streptomyces diastatochromogenes 1628是一株重要的生防菌株,其代谢产物对多种植物病原真菌具有较强的抑制效果。为了筛选在其不同抗药性高产突变株中均能稳定表达的内参基因,选择16S rRNA、sigB、hrdB、thyA、gyrB和rpoA共6个常见内参基因,分别探究它们在野生型菌株、链霉素抗性突变株、利福平抗性突变株和巴龙霉素抗性突变株中的表达情况,并用geNorm软件和NormFinder软件对结果进行分析。结果表明,使用不同软件分析得出的最佳内参基因略有差异。geNorm软件认为在上述4株菌株中,表达最稳定的内参基因分别是gyrB、gyrB、thyA、rpoA;而NormFinder软件认为rpoA基因在所有菌株的表达都是最稳定的。利用平均等级的算法平衡两个软件的分析差异,最终确定rpoA基因为表达最稳定的内参基因。通过检测toyG基因的表达水平,发现以rpoA作为内参基因能得到更合理的结果。     

116个小麦品种（系）抗叶锈基因Lr9-Lr26、Lr19-Lr20的复合PCR检测

[目的]建立简单、快速、有效的小麦抗叶锈基因复合PCR体系,从而提高分子标记辅助选择效率。[方法]以28个‘Thatcher’为背景的近等基因系和16个已知基因载体品系作为试材,测试了小麦抗叶锈病基因Lr9、Lr26、Lr19和Lr20的STS标记特异性,通过优化PCR反应体系和循环条件,构建了抗叶锈基因Lr9-Lr26和Lr19-Lr20的复合PCR检测体系。对116个小麦品种(系)所含有的抗叶锈病基因进行了分子检测。[结果]供试品种均不含有Lr9和Lr20,47个品种含有Lr26(基因频率为40.5%),‘中梁22’含有Lr19。经反复验证,Lr9-Lr26和Lr19-Lr20复合PCR技术检测结果可靠,且与上述单个分子标记检测结果一致。[结论]建立的Lr9-Lr26和Lr19-Lr20的复合PCR检测体系可以准确、稳定、高效地检测小麦抗叶锈基因Lr9、Lr26、Lr19和Lr20。