欧美杂交杨Pnd-LRR3基因克隆及其抗锈菌侵染表达

为深入探究欧美杂交杨(Populus nigra×P.deltoides)感病的分子机理,以及抗病基因在发病过程中所起的作用,克隆了欧美杂交杨LRR3蛋白编码基因Pnd-LRR3。对Pnd-LRR3基因进行了生物信息学分析,并对其在抗锈菌过程中的功能进行了研究。Q-PCR结果显示,强致病性的落叶松-杨栅锈菌(Melampsora larici-populina)E4菌株接种6 h,Pnd-LRR3基因表达量上调增幅较大,168 h为显著下调。说明Pnd-LRR3基因在E4侵染过程中起到一定抗性作用,但最终无法阻止锈菌的侵染。     

巴西橡胶NBS类抗病基因同源序列的克隆与分析

[目的]探讨巴西橡胶NBS类抗病基因同源序列的克隆与序列分析。[方法]以高抗炭疽病的巴西橡胶无性系R042为材料，根据抗病基因Prf、L6、N等的P．100p和GLPL保守结构域设计简并引物，从R042扩增具有抗病基因同源序列的片段，对分离获得的抗病基因同源序列进行Blastn、氨基酸同源性分析、序列比较和系统进化分析。[结果]从巴西橡胶高抗炭疽病无性系R042基因组DNA中分离获得1个NBS类抗病基因同源序列，经Blastn比对后发现，该抗病基因同源序列与毛果杨中的一个CC—NBS—LRR抗病蛋白的mRNA的序列同源性为66％，Blastn的E值为2e-24，推测氨基酸序列与已知抗病基因的相似性在26．9％～43．2％。[结论]序列比对与系统进化分析表明，该抗病基因同源序列具有NBS类抗病基因的所有保守序列，且与Xal的亲缘关系最近。     

The Structure and Sequence Analysis of TLR4 Gene in Cattle

Toll-like receptor 4 （TLR4） is essential for initiating the innate response to lipopolysaccharide （LPS） from Gram-negative bacteria by acting as a signal transducting receptor. In order to help in investigating TLR4 as a candidate disease-resistance gene in cows, we isolated the cDNA （GenBank accession no. DQ839566） by RT-PCR and rapid amplification of cDNA ends （RACE） experiments and analyzed the sequence characters by bioinformatics. The results showed that cattle TLR4 gene about 3 739 bp contains an open reading frame of 2 526 bp encoded 841 amino acids （aa）, 470 bp 5′ untranslated region （UTR）, and 743 bp 3′ UTR. Tissue expression profile by RT-PCR indicated that TLR4 gene expresses in mammary glands, liver, muscle, duodenum, fats, uterus, kidneys, hearts, lungs, pancreas, and ovary. TLR4 protein domain predicted by bioinformatics consists of signal peptide, transmembrane helices domain, 3 sorts of leucine-rich repeat domains （LRR, LRR-TYP, and LRRCT）, and a toll-interleukinl-resistance domain （TIR）. Leucine-rich repeat domains were related with recognizing a broad of pathogen-associated molecular patterns （PAMP） from pathogen, and TIR domain for downstream signaling transduction was most conservative （98% identify） than other domains after alignment of protein from ovine, porcine, human, and mouse. In addition, a 470 bp 5′-flanking region sequence was amplified by PCR, and 15 putative DNA binding sites were predicted, but this sequence lacks TATA box, CCAAT character, and GC-rich regions.     

Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

Conserved domain such as nucleotide binding site （NBS） was found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogues （RGAs） have been isolated. A full-length cDNA, SPR1 was obtained by rapid amplification of cDNA ends （RACE） method. Sequence analysis indicated that the length of SPR1 was 3 066 bp, including a complete open reading frame of 2 667 bp encoding SPR1 protein of 888 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has a typical structure of nonT1R-NBS-LRR genes, with NB-ARC, CC, and LRR domains. The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting. Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues. The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato. The gene has been submitted to the GenBank database, and the accession number is EF428453.     

辣椒LRR类抗病基因同源序列的克隆与分析

【目的】克隆和分析抗、感疫病辣椒材料的抗病基因同源序列,为辣椒抗疫病相关基因的克隆奠定基础。【方法】根据番茄抗叶霉病基因Cf2和Cf9 C端的LRR类保守结构域设计简并引物,对6个不同抗、感疫病的辣椒种质材料基因组DNA进行抗病基因同源序列的PCR扩增。【结果】得到27个抗病基因同源序列(RGAs),其在GenBank中的登录号为EF064260~EF064286。其中,21个RGAs与番茄抗叶霉病基因Cf2和Cf9有较高的相似性。感病材料的辣椒基因组中也存在抗疫病相关RGAs。【结论】上述21个RGAs可能与辣椒抗疫病作用有关,可为辣椒抗疫病相关基因的克隆提供依据。     

小麦属Xa 21-类似蛋白激酶基因的克隆及与同源位点序列比较

对小麦属Xa21-类似蛋白激酶基因进行了克隆及与同源序列比较研究.在小麦(Triticum aestivum)高分子量麦谷蛋白基因位点附近有一个编码LRR-类受体蛋白激酶的基因位点(暂标记为TaXa),编码蛋白与水稻(O ryza sativa)抗病蛋白Xa 21类似.通过反转录PCR途径,从普通小麦和二粒小麦(Triticum turgidum)的TaXa同源位点分离了3个cDNA克隆,ZS 860(GenBank查询号:EF 394367),ZS 2000(GenBank查询号:EF 394368)和ZS 2001(GenBank查询号:EF 394369).TaXa位点的祖先基因可能编码1 028个氨基酸组成的多肽.一个完整的TaXa蛋白包括N-端保守区、LRR结构域、一个跨膜区和位于C—端的丝氨酸—苏氨酸蛋白激酶功能域.在基因进化过程中,由于碱基代换、缺失和插入导致了开放读码框的改变,使该位点基因的编码多肽缩短.本研究对小麦属TaXa同源位点编码氨基酸序列和1个大麦(Hordeum vulgare)中的同源蛋白进行了详细比较.     

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…     

木薯一个假定的NBS类抗病基因的获得及其功能分析

根据已克隆植物NBS类抗病基因的保守结构域设计一对简并引物,以抗细菌性枯萎病木薯种质E1340基因组DNA为模板,通过PCR扩增获得大小约0.5 kb的产物。该产物克隆、测序后比对木薯基因组数据库,分别获取其上下游各1.7 kb和2.0 kb的序列,进行基因预测。预测结果表明,扩增到的序列位于一个预测基因内,该基因命名为SNB1。序列分析表明,该基因编码986 aa,具有NBS类抗病基因的结构特征,是一个假定的抗病基因,可能在木薯抗细菌性枯萎病过程中发挥重要作用。     

Comparative proteomic analysis of cucumber roots infected by Fusarium oxysporum f. sp. cucumerium Owen

Cucumber Fusarium wilt (CFW), caused by the soil-borne fungus Fusarium oxysporum f. sp. cucumerium, is a serious disease in cucumber (Cucumis sativus) production worldwide. For the efficient control of the pathogenic fungi, a better understanding of its interaction and associated resistance mechanisms at the molecular level is required. Here, we report a comparative proteomics analysis of total root protein isolated from infected cucumber root of susceptible bulk (SB) and resistant bulk (RB) of cucumber generation F2. Two-dimensional gel electrophoresis (2-DE) coupled with MS/MS approaches identified 15 over-accumulated proteins from the RB plants. Identified proteins are mainly involved in defense and stress responses, oxidation reduction, metabolism and transport and other process. These proteins are likely to be a part of resistance-related protein network, playing different roles in cucumber disease resistance. Three vital clues regarding wilt resistance of C. sativus are gained from this study. First, jasmonic acid and redox signaling components were found in response to F. oxysporum infection in resistant plants. Second, the LRR family protein may play an important role in the defense reaction against CFW. Third, biotic and abiotic stress-related proteins were induced by the CFW fungus F. oxysporum, indicating the activation of common stress pathway.