水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用invivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定

由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害。通过筛选18000个XooTn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11。TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中。对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB。Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移。将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失。证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用。     

水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定水稻黄单胞菌 TAIL-PCR hrp基因簇 hpaB基因

由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害.通过筛选18000个Xoo Tn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11.TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中.对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB.Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移.将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失.证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用.     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

斑马鱼sat1.a突变体的鉴定

目的在我们的前期研究工作中,通过Tol2转座子介导的插入突变,筛选到了一批组织特异性表达绿色荧光蛋白GFP的斑马鱼品系。其中一个品系Tol2:20141221t的GFP在神经系统中表达,但还没有鉴定到Tol2转座子插入到基因组什么位置,造成了哪个基因的突变。本文的主要研究目标就是对这一由Tol2转座子插入诱导的斑马鱼突变品系进行鉴定。方法使用交错式热不对称PCR(thermal asymmetric interlaced PCR,TAIL-PCR)鉴定Tol2转座子插入的基因组位置;利用原位杂交检测被突变的基因的时空表达是否与该品系GFP表达具有一致性;筛选鉴定纯合体突变体,并进一步分析该基因突变造成的发育缺陷。结果在该品系中,Tol2转座子插入到了亚精胺/精胺N1-乙酰基转移酶1a(spermidine/spermine N1-acetyltransferase 1a,sat1.a)的第八个内含子区域,致使sat1.a基因转录提前终止。我们筛选到了sat1.a纯合突变体,但是没有检测到明显的发育缺陷。结论筛选鉴定的Tol2转座子介导的斑马鱼sat1.a突变体没有明显的发育缺陷,但可以作为研究神经系统发育的有力工具。     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以miniTn5gfp-km转座子中nptII片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc）非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR （TAIL-PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL-PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简便高效的新方法。     

植物功能基因组研究中的基因敲除技术

综述了T-DNA插入突变和转座子插入突变等基因敲除技术的原理及其在植物功能基因组研究中的应用,并简要介绍了向国际有关机构索要或购买拟南芥插入突变体的方法.     

利用酿酒酵母转座子文库筛选MTM1基因缺失表型相关基因

【目的】MTM1基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1基因的缺失会严重影响锰超氧化物歧化酶活性,并损伤线粒体功能,使酵母在非发酵培养基上不能生长。为加深对MTM1基因功能及其相关基因的研究,尝试利用转座子文库筛选MTM1基因缺失表型相关基因,寻找哪些位置的转座子插入能挽救MTM1基因缺失导致的生长缺陷。【方法】因MTM1基因的缺失造成的损伤不可逆,直接转入文库无法筛选得到MTM1基因缺失表型相关基因,本研究利用外源MTM1基因菌株和mTn-lacZ/LEU2酿酒酵母转座子文库进行筛选,寻找能挽救mtm1突变体生长缺陷的转座子插入位点。【结果】发现转座子插入HSL1和TPS2基因能挽救mtm1突变体的生长缺陷。【结论】我们的结果为深入了解MTM1基因的功能提供了线索。     

十字花科黑腐病菌中影响致病相关基因XC3814表达的基因鉴定

十字花科黑腐病菌8004菌株的XC3814基因与致病性和胞外多糖合成有关。文章将XC3814的启动子与报告基因sacB融合, 构建了XC3814的表达报告质粒pL3814sac。将该质粒导入野生型菌株8004, 获得了报告菌株8004/pL3814sac。利用转座子EZ::Tn5对报告菌株的基因组进行随机诱变, 分离到3株耐蔗糖的突变体。分析发现其中的1株突变体是由EZ::Tn5插入到编号为XC3882的未知功能的基因所产生的。将由XC3814启动子与报告基因gusA融合得到的报告质粒pGUS3814分别导入8004菌株和XC3882的转座子Tn5gusA5插入突变体, 测定比较pGUS3814的GUS表达水平, 结果显示在XC3882突变体背景下GUS的表达水平比在野生型背景下降低81.3%, 表明XC3814基因的表达水平受XC3882基因的影响。     

防御假单胞菌双突变体H78ΔrsmA/E中hmgA基因对藤黄绿菌素生物合成的抑制

【背景】防御假单胞菌(Pseudomonas protegens) H78是分离于油菜根际的一株生防菌,其能合成藤黄绿菌素(pyoluteorin,Plt)等多种广谱抗生素,H78的rsmA/E双突变体中Plt合成被完全抑制。【目的】通过转座子诱变技术,筛选H78ΔrsmA/E双突变体中重新激活Plt合成的下游调控因子。【方法】通过同源重组的方法在pltL基因下游插入红色荧光蛋白(redfluorescentprotein,RFP)基因来指示Plt操纵子表达的激活情况;利用转座子随机插入突变、半随机PCR技术筛选并定位目标基因;通过基因回补等方法进一步验证基因功能。【结果】从约2万株H78ΔrsmA/E的转座子突变体中筛选到一株高产Plt和某种黑色素的菌株,并确定其插入位点为hmgA基因,hmgA基因回补能重新抑制H78ΔrsmA/E的Plt合成。【结论】假单胞菌双突变体H78ΔrsmA/E中hmgA基因对Plt的合成存在强烈抑制作用,是潜在的RsmA/E下游调控基因。本研究为进一步阐明Plt合成的调控机制与网络及通过基因工程提高Plt产量奠定了基础。     

RaxH/RaxR: a two-component regulatory system in Xanthomonas oryzae pv. oryzae required for AvrXa21 activity

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, one of the most serious diseases in rice. X. oryzae pv. oryzae Philippine race 6 (PR6) strains are unable to establish infection in rice lines expressing the resistance gene Xa21. Although the pathogen-associated molecule that triggers the Xa21-mediated defense response (AvrXa21) is unknown, six rax (required for AvrXa21 activity) genes encoding proteins involved in sulfur metabolism and Type I secretion were recently identified. Here, we report on the identification of two additional rax genes, raxR and raxH, which encode a response regulator and a histidine protein kinase of two-component regulatory systems, respectively. Null mutants of PR6 strain PXO99 that are impaired in either raxR, raxH, or both cause lesions significantly longer and grow to significantly higher levels than does the wild-type strain in Xa21-rice leaves. Both raxR and raxH mutants are complemented to wild-type levels of AvrXa21 activity by introduction of expression vectors carrying raxR and raxH, respectively. These null mutants do not affect AvrXa7 and AvrXa10 activities, as observed in inoculation experiments with Xa7- and Xa10-rice lines. Western blot and raxR/gfp promoter-reporter analyses confirmed RaxR expression in X. oryzae pv. oryzae. The results of promoter-reporter studies also suggest that the previously identified raxSTAB operon is a target for RaxH/RaxR regulation. Characterization of the RaxH/RaxR system provides new opportunities for understanding the specificity of the X. oryzae pv. oryzae-Xa21 interaction and may contribute to the identification of AvrXa21.     

The molecular basis of disease resistance in rice

The rice gene Xa21 conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo), was isolated using a map-based cloning strategy. Compared with previously cloned genes, the structure of Xa21 represents a novel class of plant disease R genes encoding a putative receptor kinase (RK). This article proposes a model for the mode of action of Xa21 and summarizes our current knowledge of the modular basis of resistance in rice to bacterial leaf blight and blast.     

水稻黄单胞菌黄色素合成相关基因的克隆与鉴定

用硫酸二乙酯(DES)诱变水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)和条斑病细菌(Xanthomonas oryzae pv. oryzicola, 简称Xooc),分别得到5株和13株黄色素缺失突变体,其中来自Xooc的M6和M12 还丧失了对水稻的致病性和在烟草上激发过敏反应的能力。以Xooc黄色素缺失突变体M51为受体菌交叉互补从Xoo JXOIII基因文库中筛选出一个黄色素合成相关的基因克隆pA341,以Xoo黄色素缺失突变体M1071为受体菌,从Xooc RS105基因文库中获得了一个黄色素合成相关的基因克隆pA270。功能互补显示,18株黄色素缺失突变体中的10株能分别被pA341和 pA270互补后正常产生黄色素,但这两个克隆不能同时互补同一株黄色素缺失突变体。能被pA341互补的黄色素缺失突变体M6没有恢复对水稻的致病性和在烟草上激发过敏反应,表明黄色素合成相关基因与hrp基因间不存在相关性。斑点杂交结果表明,pA270与pA341之间没有同源性。pA270亚克隆结果显示,与黄色素合成相关的基因约11.6kb大小,以基因簇的形式存在,不仅决定了黄色素的产生,还影响黄色素合成的数量和质量（吸收峰）。在紫外光条件下,黄色素能够提高菌体的存活率,提示黄色素对病原细菌有保护作用。     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response

Innate immunity to microorganisms relies on the specific sensing of pathogen-associated molecules by host recognition receptors. Whereas studies in animals have largely focused on the recognition of extracellular pathogen-associated molecules by the TLR (toll-like receptor) superfamily, few studies have been carried out in plants, and it is not understood how these molecules are secreted or modified. The rice Xa21 gene encodes a receptor-like kinase that provides immunity against strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae carrying AvrXa21 activity. We identified four X. oryzae pv. oryzae genes that are required for AvrXa21 activity. raxA, raxB, and raxC encode proteins with similarity to a membrane fusion protein, an ATP-binding cassette transporter, and an outer membrane protein, respectively, of bacterial type I secretion systems. The fourth gene, raxST, encodes a sulfotransferase-like protein. Sequence analysis of three naturally occurring X. oryzae pv. oryzae strains no longer recognized by Xa21 revealed alterations in the raxST and raxA genes. The raxC gene complemented an Escherichia coli tolC mutant for secretion of a double glycine-leader peptide confirming the function of raxC in type I secretion. These results indicate that bacterial type I secretion is necessary for Xa21-mediated recognition and immunity and further suggest that type I secretion and modification of pathogen-associated molecules play an important role in triggering the innate immune response in rice.     

Epigenetic inheritance in rice plants

BACKGROUND AND AIMS: Epigenetics is defined as mechanisms that regulate gene expression without base sequence alteration. One molecular basis is considered to be DNA cytosine methylation, which reversibly modifies DNA or chromatin structures. Although its correlation with epigenetic inheritance over generations has been circumstantially shown, evidence at the gene level has been limited. The present study aims to find genes whose methylation status directly correlates with inheritance of phenotypic changes. METHODS: DNA methylation in vivo was artificially reduced by treating rice (Oryza sativa ssp. japonica) seeds with 5-azadeoxycytidine, and the progeny were cultivated in the field for > 10 years. Genomic regions with changed methylation status were screened by the methylation-sensitive amplified polymorphysm (MSAP) method, and cytosine methylation was directly scanned by the bisulfite mapping method. Pathogen infection with Xanthomonas oryzae pv. oryzae, race PR2 was performed by the scissors-dip method on mature leaf blades. KEY RESULTS: The majority of seedlings were lethal, but some survived to maturity. One line designated as Line-2 showed a clear marker phenotype of dwarfism, which was stably inherited by the progeny over nine generations. MSAP screening identified six fragments, among which two were further characterized by DNA blot hybridization and direct methylation mapping. One clone encoding a retrotransposon gag-pol polyprotein showed a complete erasure of 5-methylcytosines in Line-2, but neither translocation nor expression of this region was detectable. The other clone encoded an Xa21-like protein, Xa21G. In wild-type plants, all cytosines were methylated within the promoter region, whereas in Line-2, corresponding methylation was completely erased throughout generations. Expression of Xa21G was not detectable in wild type but was constitutive in Line-2. When infected with X. oryzae pv. oryzae, against which Xa21 confers resistance in a gene-for-gene manner, the progeny of Line-2 were apparently resistant while the wild type was highly susceptible without Xa21G expression. CONCLUSIONS: These results indicated that demethylation was selective in Line-2, and that promoter demethylation abolished the constitutive silencing of Xa21G due to hypermethylation, resulting in acquisition of disease resistance. Both hypomethylation and resistant trait were stably inherited. This is a clear example of epigenetic inheritance, and supports the idea of Lamarckian inheritance which suggested acquired traits to be heritable.     

Isolation and characterization of rice mutants compromised in<Emphasis Type="Italic"> Xa21</Emphasis>-mediated resistance to<Emphasis Type="Italic"> X. oryzae</Emphasis> pv.<Emphasis Type="Italic"> oryzae</Emphasis>

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 (Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.Communicated by D.J. Mackill