Broad-spectrum Blast Resistance Gene Pi-k h Cloned from Rice Line Tetep Designated as Pi54

Blast disease caused by Magnaporthe oryzae is one of the important biotic stresses of rice. So far more than 85 blast resistance genes have been identified of these more than 14 have already been cloned. A broad spectrum rice blast resistance gene Pi-k ^h was cloned from the rice line Tetep. The gene was named Pi-k ^h based on the earlier reports on its genetic analysis in various rice lines. However, with the advances in molecular genetics and genomics of rice, the Pik locus has now been mapped more precisely. Since there are two reports on the mapping of Pi-k ^h gene from different rice lines, there is some confusion in the naming of this gene. In this report the name of Pi-k ^h gene cloned from the rice line Tetep has been designated as per the standard guidelines of Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) and its physical location on rice chromosome 11, which is ～2.5 Mbp away from the Pik locus mapped recently. Hence Pi-k ^h gene cloned from Tetep is now designated as Pi54.     

药用野生稻转育后代一个抗白叶枯病新基因的定位

从药用野生稻渗入后代选育的水稻株系B5表现为高抗褐飞虱、白背飞虱和白叶枯病。对B5与籼稻品种明恢63杂交组合的187个重组自交系(RILs)进行了抗白叶枯病接种鉴定,采用分离集团分析法(Bulked Segregant Analysis,BSA),在第1染色体上筛选到与水稻抗白叶枯病基因相连锁RFLP分子标记。利用RILs抗病性表现型鉴定资料和构建的分子标记连锁图谱,将抗白叶枯病基因定位在第1染色体短臂的C904和R596之间,这两个分子标记间遗传距离为1．3cM。该基因对RILs群体抗病性变异的贡献率为52．96％,是一效应值较大的主效基因。这一抗白叶枯病基因不同于已报道的抗白叶枯病基因的位点,因此将其命名为Xa29(t)。     

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.     

稻属NBS-LRR类基因的克隆及抗稻瘟病基因的表达

利用已克隆植物的R基因NBS序列中保守基序设计简并引物进行PCR扩增是克隆NBS有效的方法。从广东普通野生稻HLW028中克隆出3条序列,经同源性分析均为NBS,序列号为DQ272573～DQ272575。从NCBI中下载普通野生水稻和功能已知的水稻NBS—LRR类基因,与本试验所提交的NBS进行聚类分析。这些NBS可分为5大类,其中DQ272574是一个新的NBS基因类型。从野生水稻中克隆NBS基因存在P—loop、RNBS。A、kin-2、RNBS．B、RNBS—C和PLAL的基序。RT—PCR结果表明,培矮64中的Pikh的表达量比2种野生稻中高。从培矮64中扩增出1个完整读码框的cDNA。     

海南普通野生稻稻瘟病的抗性鉴定与评价

在苗期应用自然诱发鉴定法对海南普通野生稻(Oryza rufipogonGriff.)41个居群的410份材料进行了2年的稻瘟病(rice blast)抗性鉴定,结果表明:经过初鉴和复鉴,410份海南普通野生稻中有21份表现高抗,占5.1%,117份表现抗,占28.5%,说明海南普通野生稻具有较好的稻瘟病抗性。     

Evolutionary Dynamics of the Genomic Region Around the Blast Resistance Gene Pi-ta in AA Genome Oryza Species

The race-specific resistance gene Pi-ta has been effectively used to control blast disease, one of the most destructive plant diseases worldwide. A single amino acid change at the 918 position of the Pi-ta protein was known to determine resistance specificity. To understand the evolutionary dynamics present, we examined sequences of the Pi-ta locus and its flanking regions in 159 accessions composed of seven AA genome Oryza species: O. sativa, O. rufipogon, O. nivara, O. meridionalis, O. glaberrima, O. barthii, and O. glumaepatula. A 3364-bp fragment encoding a predicted transposon was found in the proximity of the Pi-ta promoter region associated with the resistance phenotype. Haplotype network analysis with 33 newly identified Pi-ta haplotypes and 18 newly identified Pi-ta protein variants demonstrated the evolutionary relationships of Pi-ta haplotypes between O. sativa and O. rufipogon. In O. rufipogon, the recent directional selection was found in the Pi-ta region, while significant deviation from neutral evolution was not found in all O. sativa groups. Results of sequence variation in flanking regions around Pi-ta in O. sativa suggest that the size of the resistant Pi-ta introgressed block was at least 5.4 Mb in all elite resistant cultivars but not in the cultivars without Pi-ta. These findings demonstrate that the Pi-ta region with transposon and additional plant modifiers has evolved under an extensive selection pressure during crop breeding.PLANT resistance (R) genes have evolved to fight against a wide range of pathogens in a race-specific manner where a particular R gene in a plant recognizes the corresponding avirulence (AVR) gene in a pathogen race (Flor 1971). Thus far, a number of R genes have been identified and characterized from diverse plant species. Most characterized R genes to date encode putative proteins with nucleotide binding sites (NBS) and leucine-rich repeats (LRR) (Hulbert et al. 2001). Most R genes are highly polymorphic and diversified, which is consistent with the ability to interact with diverse random molecules encoded by diverse pathogen AVR genes (Meyers et al. 2003; Bakker et al. 2006; Shen et al. 2006).Blast disease, caused by the filamentous ascomycete Magnaporthe oryzae B.C. Couch [formerly M. grisea (T. T. Hebert) M. E. Barr] (Rossman et al. 1990; Couch and Kohn 2002), has been one of the major constraints to stable crop production. Currently, Oryza sativa and M. oryzae have been an excellent model pathosystem for uncovering the molecular coevolution mechanisms of host–pathogen (Valent et al. 1991; Talbot 2003). At least 80 race-specific R genes that confer resistance to specific pathogen races have been described in rice germplasm (Ballini et al. 2008). Eleven blast R genes (Pi-ta, Pib, Pi2/Piz-t, Pi5, Pi9, Pi21, Pi36, Pi37, Pi-d2, Pikm, and Pit) have been cloned, and most of them, except Pi21 and Pi-d2, were also predicted to encode receptor proteins with NBS (Chen et al. 2006; Fukuoka et al. 2009; Jia et al. 2009b). In most cases, blast R genes are members of small gene families with a single family member required for resistance. Pikm and Pi5 are exceptions that require two members of the same gene family for Pikm- and Pi5-mediated resistance, respectively (Ashikawa et al. 2008; Lee et al. 2009). Recently, a retrotransposon was predicted to be involved in the Pit resistance (Hayashi and Yoshida 2009).The evolutionary dynamics and mechanisms of resistance mediated by Pi-ta is one of the best-studied R-genes. Pi-ta has been effectively deployed in the United States and around the globe for controlling blast disease (Bryan et al. 2000; Jia et al. 2000; Jia 2003; Jia et al. 2004a,b; Huang et al. 2008; Jia and Martin 2008; Wang et al. 2008; Jia et al. 2009a). Pi-ta encodes a predicted cytoplasmic protein with a centrally located NBS and a highly interrupted LRR domain (referred to as the LRD) at the carboxyl terminus that recognizes the corresponding avirulence gene AVR-Pita, triggering race-specific resistance. A single amino acid substitution, serine (Ser) to alanine (Ala) at the position of 918, in the LRD of the Pi-ta protein was demonstrated to determine the direct interaction with AVR-Pita and the resistance specificity to blast pathogen M. oryazae (Bryan et al. 2000; Jia et al. 2000). The resistant Pi-ta allele (Ala-918) was found in O. sativa and its ancestor O. rufipogon (Jia et al. 2004b; Huang et al. 2008). Surveys of Pi-ta nucleotide sequences with limited accessions of Oryza species have revealed that the degree of nucleotide diversity is higher at the intron of the Pi-ta gene (Jia et al. 2003; Huang et al. 2008; Wang et al. 2008; Yoshida and Miyashita 2009). Huang et al. (2008) further suggested that a selective sweep occurred recently at the Pi-ta gene in O. rufipogon, but the extent of selection around the Pi-ta genomic region has not been demonstrated in either O. rufipogon or O. sativa.Knowledge of the historical introduction of the Pi-ta gene can help to understand the extent of selection at the Pi-ta locus. The landraces Tadukan and Tetep, containing Pi-ta and other blast R genes in chromosome 12, have been used as breeding parents for preventing blast disease worldwide. Tadukan was confirmed to be the Pi-ta donor for various Asian japonica cultivars (Rybka et al. 1997) whereas Tetep was the Pi-ta donor for the U. S. cultivars (Gravois et al. 1995; Moldenhauer et al. 1998; McClung et al. 1999; Gibbons et al. 2006; Moldenhauer et al. 2007). Recently, the large introgressed chromosomal segments surrounding the Pi-ta locus were identified in backcross BC₅ progenies and elite rice cultivars (Jia 2009). This suggests that the broad spectrum of the Pi-ta resistance in the United States may include the effects of other loci in the Pi-ta region, inherited as a “superlocus.” Toward this end, Ptr(t), a nuclear gene that is required for the Pi-ta-mediated resistance, was also mapped at the Pi-ta region (Jia and Martin 2008). Further determination of DNA sequences around the Pi-ta gene should help to determine the minimal genomic region that is essential for Pi-ta-mediated resistance.The two cultivated rice species, O. sativa and O. glaberrima, belong to the AA genome of Oryza species. O. rufipogon and O. nivara are wild progenitors of the Asian rice O. sativa, whereas O. barthii is a wild progenitor of the African cultivated rice O. glaberrima (Linares 2002; Yamanaka et al. 2003; Londo et al. 2006). The comparison of R-gene diversity between cultivated rice and its wild ancestors is important to understand the selection effects of crop domestication and breeding.The objectives of this study were (1) to characterize distributions of the Pi-ta allele in O. sativa and to detect the potential presence/absence of polymorphism(s) associated with the resistance phenotype; (2) to examine the molecular evolution and patterns of selection in the Pi-ta gene in O. sativa and O. rufipogon; (3) to analyze molecular diversity around the Pi-ta locus in AA genome Oryza species; and (4) to understand the pattern and extent of selection for Pi-ta-mediated resistance in Oryza species during crop domestication.     

药用野生稻体细胞杂交后代对条纹叶枯病的抗性鉴定

目的：从药用野生稻体细胞杂交后代中筛选对水稻条纹叶枯病具有抗性的水稻种质。方法：利用苗期接种法对药用野生稻体细胞杂交后代进行表型抗性评价,利用ELISA技术对供试材料进行感病率检测。结果：34份F1个体中,Y37、Y39、Y42和Y45的抗病级别都为高抗,它们的感病率分别为5.7%、5.7%、5.7%和4.3%,表现高抗水稻条纹叶枯病。结论：筛选到4个高抗条纹叶枯病水稻材料。     

水稻品种‘沈农606’抗稻瘟病基因定位和连锁的SRAP片段分析

选用抗稻瘟病水稻品种‘沈农606’为抗病亲本与感病品种‘丽江新团黑谷’配制杂交组合.鉴定亲本、F_1正反交及其F_2群体的抗病性的结果表明,‘沈农606’的抗性受一对显性基因控制.采用相关序列扩增多态性(SRAP)和简单序列重复(SSR)标记,以及分离体分组混合分析法(BSA)将该基因定位于8号染色体上,其与SRAP标记m5e1-500的遗传距离为2.8 cM,与SSR标记RM25的遗传距离为9.8 cM,暂命名为Pi-SN606.m5e1-500序列位于8号染色体上,它能编码大于40个氨基酸的阅读框有2个,在NCBI网站上没有比对到同源性序列。     

水稻Pib基因及其连锁RFLP标记在栽培稻、药用野生稻和玉米中的比较物理定位

比较遗传学研究表明 ,禾本科不同基因组之间存在着广泛的同线性和共线性。对水稻 (OryzasativaL .)这一模式植物与其他禾本科植物的原位杂交定位可以揭示禾本科植物基因组的共同特点和进化规律 ,为建立禾本科遗传大体系积累资料。实验以图位克隆法分离的水稻Pib基因 (10 .3kb)和与之连锁的RFLP标记为探针 ,研究了Pib及与其连锁的RFLP标记在供试种中的同源性和物理位置。Southern杂交结果表明 ,Pib在玉米 (ZeamaysL .)基因组中有同源序列。进一步利用单色和双色荧光原位杂交技术确定了Pib在栽培稻 (O .sativassp .indicacv .Guangluai4)、玉米和药用野生稻 (O .officinalisWallexWatt)染色体上的物理位置。定位结果表明 ,Pib基因和与之连锁的RFLP标记在这 3个供试种基因组中具有同线性。     

紧穗野生稻的褐飞虱抗性导入栽培稻的研究

栽培品种的远缘野生种O.eichingeri (2n=24,CC染色体组)是褐飞虱的重要抗源。为了将原产乌干达的O.eichingeri两个编号材料的褐飞虱抗性导入栽培稻02428中,利用胚培养技术获得了两个组合的F.杂种,可交配力分别为0.36％和1.62％。所得F.杂种生长旺盛,分蘖力强,但高度不育,其花粉母细胞中期Ⅰ二价体数为0～4个,平均1.33～1.37。F_1植株用02428回交及套袋自交产生的BC_1F_1和F_2植株形态相似,染色体组均为AAC,花粉母细胞中期Ⅰ染色体构型为(12.02～12.18)Ⅰ (11.67～11.89)Ⅱ (0.04～0.19)Ⅲ,均表现完全不育。进一步检查了42个BC_1F_2植株和9个BC_2F_1植株的染色体数目,其变幅为24～38,从中筛选到2n=25及2n=24的植株各21个,其中5个整倍体植株对褐飞虱表现抗,说明两份紧穗野生稻载有抗性基因的染色体片段已成功转入02428中。本研究还发现一些株高及每穗粒数等明显超亲的材料,这可能与染色体组A与C上某些基因的互作有关。     

人类新基因WDR70的克隆及初步研究

运用基于基因组数据库的特定基因同源新基因的克隆策略得到一个人类新基因WDR70 ,该基因编码一个包含 12个WD4 0结构域的蛋白。WDR70的cDNA序列长 2 2 6 6bp ,预测编码蛋白含 6 30个氨基酸 ,理论分子量为 70× 10 3 u ,染色体定位为 17p13.1。以小鼠胚胎为模型进行整体原位杂交 ,结果显示WDR70基因在 8.5d小鼠胚胎中没有表达 ,而在 9.5d和 10 .5d的小鼠胚胎的脑部有特异表达。由此推断该基因对胚胎期脑部的发育有重要的影响。     

拟南芥水杨酸受体表达与在体转录水平的研究

目的:从拟南芥叶中克隆水杨酸结合蛋白(SA binding protein 2,SABP2,也称水杨酸受体)基因sabp2进行异源表达并测定其活性.方法:从拟南芥叶RNA中通过反转录PCR扩增sabp2,将PCR产物克隆至载体pMD - 19T simple中,经测序验证后,再基于pET28a构建重组表达载体,转化至大肠杆菌BL21( DE3)并表达,检测重组蛋白的活性.另一方面,对sabp2在拟南芥中转录水平进行了研究.结果:PCR获得792bp的sabp2基因,并成功构建异源表达载体pET28a - sabp2.优化结果表明,在0.4mmol/L IPTG诱导下20℃培养8h,表达产物活性较强,具天然SABP2的特征性酯酶活性.该基因在拟南芥叶中转录模式呈SA应激性和组织特异性.结论:sabp2成功表达,不仅为筛选SA受体拮抗剂提供新的原核体系,而且为探讨SA与SABP2相互作用在植物防御过程中时空变化奠定基础.     

The Drosophila bag of marbles Gene Interacts Genetically with Wolbachia and Shows Female-Specific Effects of Divergence

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.     

Characterization of an Endoglucanase from Pseudomonas fluorescens subsp. cellulosa Produced in Escherichia coli and Regulation of the Expression of Its Cloned Gene

Several enzymatic properties of an endoglucanase produced in Escherichia coli by a gene from Pseudomonas fluorescens subsp. cellulosa were investigated. Gel filtration revealed a single peak of M_r 36,000 with endoglucanase activity. The pH optimum of the enzyme was 7.0. Carboxymethyl cellulose and barley β-glucan (mixed β-1,3 and 1,4 linkages) were good substrates, but not laminarin (β-1,3 linkages), amylose, filter paper, microcrystalline cellulose (Avicel), or cellotriose. The mode of action was typical of an “endo”-acting enzyme. Taken together, these properties do not correspond to those of any of the endoglucanases described in P. fluorescens subsp. cellulosa. Consequently, the gene was designated egIX. The enzyme was sensitive to end-product inhibition by cellobiose but was only moderately inhibited by glucose. The enzyme was formed constitutively in E. coli throughout the growth phase. Urea had no effect on endoglucanase synthesis, but glucose acted as a catabolite repressor. The formation of the enzyme in E. coli was partially dependent on cyclic AMP.     

Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance

Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.