草莓果实FaRIPK1蛋白的酵母外源表达及验证

[目的]类受体蛋白激酶在植物的生长发育及果实成熟过程中发挥重要作用,而植物中已鉴定的类受体激酶绝大多数属于LRR型.为了得到草莓中LRR型蛋白激酶FaRIPK1,从而进一步研究其功能.本实验采用真核表达的方法诱导表达FaRIPK1蛋白质,以期确定其最佳的诱导表达时间.[方法]以‘童子一号’草莓果实为试验材料,扩增出FaRIPK1基因的功能区序列,构建真核表达载体pPICZB-RIPK1后转入酵母表达菌株X-33诱导表达,通过在0,12,24,36 h处取样检测FaRIPK1蛋白质的表达情况并采用Ni-NTA琼脂糖树脂亲和层析柱纯化得到目的蛋白,纯化后的FaRIPK1蛋白质再进行Western-blot验证.[结果]重组表达菌株经过BMMY诱导培养基诱导24 h后FaRIPK1蛋白质的表达量达到最高值,此时最利于FaRIPK1蛋白质的纯化.Western-blot验证表明所得蛋白质确为FaRIPK1蛋白质.[结论]确定了诱导表达后24 h为FaRIPK1蛋白质真核表达的最佳时间,为进一步研究FaRIPK1蛋白质的功能奠定基础.     

Natural history of Arabidopsis thaliana and oomycete symbioses

Molecular ecology of plant–microbe interactions has immediate significance for filling a gap in knowledge between the laboratory discipline of molecular biology and the largely theoretical discipline of evolutionary ecology. Somewhere in between lies conservation biology, aimed at protection of habitats and the diversity of species housed within them. A seemingly insignificant wildflower called Arabidopsis thaliana has an important contribution to make in this endeavour. It has already transformed botanical research with deepening understanding of molecular processes within the species and across the Plant Kingdom; and has begun to revolutionize plant breeding by providing an invaluable catalogue of gene sequences that can be used to design the most precise molecular markers attainable for marker-assisted selection of valued traits. This review describes how A. thaliana and two of its natural biotrophic parasites could be seminal as a model for exploring the biogeography and molecular ecology of plant–microbe interactions, and specifically, for testing hypotheses proposed from the geographic mosaic theory of co-evolution.     

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.     

拟南芥LRR-Extensin蛋白家族的研究

LRR-Extensin(LRX)是一种定位于植物细胞壁中的蛋白,其结构中含有富含亮氨酸的重复序列和伸展蛋白结构域。LRX蛋白的功能众多,除了促进细胞生长外,还影响营养生长、形态发生、授粉受精等,随细胞基质不同具有不同的功能,并表现出高度的组织、器官和细胞特异性。介绍了拟南芥中LRX的分类、结构特征、表达形式及其在根毛和花粉发育中的作用。     

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

Two sets of degenerate oligonucleotide primers were designed according to amino acid conservedregions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site andleucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine proteinkinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified fromthree wild rice species in Yunnan Province, China. The DNA fragments from amplification have been clonedinto the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. ,and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Ory-za officinalis Wall. and TR19 from Oryza rufipogon Griff. belong to the same class and homology of theirsequences are 100%. The result shows that the sequences of the same class disease resistance gene analogueshave no difference among different species of wild rice. 5 classes STK disease resistance gene analogues werealso obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. Bycomparison analysis of amino acid sequences, we found that the obtained disease resistance gene analogues havevery iow identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative diseaseresistance genes that have not been isolated so far.     

玉米Rp3基因LRR结构域的多态性及进化分析

玉米扩锈病基因Rp3是来源于玉米B73自交系中的一类NBS-LRR抗病基因。研究中提取了87个玉米自交系基因组,在52个自交系中扩增到Rp3基因的LRR结构域片段。利用生物信息学对LRR结构域的核酸多态性、选择压力及不同杂种优势群中的遗传差异等参数进行分析。Rp3基因的LRR结构域在核酸水平上变异程度较高,核苷酸多态性π值为0.062 36,存在较多的多态性位点;Ka/Ks值的估算发现该基因的LRR结构域总体上受到纯化选择作用。Rp3基因的LRR结构域在5种杂种优势群中的遗传多态性存在明显差异。     

植物LRR类受体蛋白激酶的研究进展

植物中富亮氨酸重复片断类受体蛋白激酶(LRR RLK)属于跨膜类受体蛋白激酶,由胞外LRR结构域、单次跨膜区以及胞内激酶结构域三部分组成。在不同植物中,LRR RLK作为信号识别受体参与CLV、BR、Ax21等信号转导过程,在植物生长发育、激素调节以及逆境应答反应等方面中发挥重要的作用。综述了近年来LRR RLK的相关调控功能及其作用机制的研究进展,并为进一步探索LRR RLK的功能及应用提供参考。     

SON-PCR扩增抗禾谷孢囊线虫基因LRR区及序列分析

根据与抗禾谷孢囊线虫(Heterodera avenae)基因Cre3的NBS(nucleotide binding site)编码区高度同源的Rccn4 序列设计3条3'端嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法从含有源于易变山羊草(Aegilops varibilis)的抗禾谷孢囊线虫基因的小麦(Triticum aestivum)-易变山羊草染色体小片段易位系E-10中获得了长度为1264 bp的Rccn-L(GenBank登录号为DQ124933)，它将Rccn4 3'端延伸了1209 bp,编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构。其编码一个342个氨基酸残基的蛋白质，含有NBS(nucleotide binding site)-LRR(leucine-rich repeats) 类抗性基因LRR区的保守模体,呈现XXLXXLXXL重复。首次将SON-PCR方法成功地运用到植物基因组学研究中，为植物基因克隆又提供一方法。