稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果

【目的】从河南大豆根瘤的内生细菌资源中筛选对稻瘟病菌有拮抗作用的菌株,初步探讨其抑菌效果,为进一步研究其抑菌机理提供菌种资源。【方法】以稻瘟病菌为供试病原菌,采用对峙法进行拮抗性菌株筛选,显微观察法研究受抑制病原菌菌丝变化,对筛选拮抗性菌株进行细胞形态学及生理生化特性试验、16S rRNA基因测序和系统发育分析及接种防效试验。【结果】经复筛有17株内生菌拮抗效果较明显,最高抑制率为62.16%;受抑制病原菌丝呈现弯曲打结、断裂、原生质浓缩等畸形状态。拮抗性筛选过程中内生菌快速生长形成生物薄膜,包埋菌丝并使其断裂。拮抗菌株分布在7属9种,稻瘟病拮抗性大豆根瘤内生菌呈现种属多样性。防效试验表明内生菌处理组稻苗发病率和病情指数均显著降低,防治效果最高达74.19%。【结论】大豆根瘤内生拮抗性菌株具有种属多样性,拮抗性菌株处理组稻苗发病率和病情指数均显著降低,防治效果显著,为进一步研究其抑菌机理提供菌种资源。     

产PK和NRP类抗生素苦豆子内生放线菌分子筛选及抗生素类型鉴定

【目的】从19株苦豆子内生拮抗放线菌中筛选PKSⅠ、PKSⅡ和NRPS基因阳性菌株,并对其产抗生素种类进行初步鉴定,为苦豆子内生放线菌资源的合理开发和利用提供理论依据。【方法】分别以PKSⅠ、PKSⅡ和NRPS基因引物对19株拮抗菌株进行特异性扩增,筛选阳性菌株;以7种抗生素标准样品为对照,采用TLC和HPLC法对阳性菌株所产抗生素类型进行鉴定。【结果】PKSⅠ、PKSⅡ和NRPS基因阳性菌株率分别为47.4%、10.5%和21.1%;9株内生放线菌发酵液中各有1个峰的洗脱时间与麦迪霉素的洗脱时间一致,菌株NDZKDS69的发酵液中有4个峰的洗脱时间分别与麦迪霉素、乙酰螺旋霉素、替考拉宁和土霉素标准样品的洗脱时间一致。【结论】苦豆子内生放线菌中链霉菌属(Strepomyces)菌株是大环内脂类、芳香环类和非核糖体多肽类抗生素的丰富菌源;分子指纹图谱和化学指纹图谱检测结果一致,且建立的TLC和HPLC法检测抗生素的方法简便、快捷、灵敏、重复性良好。     

药用野生稻拮抗稻瘟病内生细菌的筛选、鉴定及发酵条件优化

【背景】稻瘟病是水稻的三大重要病害之一，每年对水稻生产都会造成较大的损失，生物防治稻瘟病已成防治该病害的重要手段，而目前鲜少有报道从药用野生稻中分离内生菌用以防治稻瘟病。【目的】从药用野生稻内生菌中挖掘对稻瘟病具有拮抗作用的微生物资源。【方法】采用分离法从勐遮药用野生稻中先分离出内生菌，再通过平板对峙法筛选对稻瘟病菌具有拮抗作用的菌株，通过形态学和16S rRNA基因序列分析对菌株进行鉴定，同时筛选该菌株的最适培养基、pH、培养温度和摇床转速。【结果】筛选得到拮抗菌株Z5，在LB培养基上对稻瘟病菌菌丝生长的抑制率达到96.43%，经形态学和16S rRNA基因序列分析，初步鉴定为短小芽孢杆菌(Bacillus pumilus)。在NYBD、CM、LB、NA和YSP这5种培养基中，菌株Z5的最适培养基为NYBD培养基、最适pH值为8.0-9.0、最适培养温度为35℃、最适摇床转速为250 r/min。【结论】短小芽孢杆菌Z5对稻瘟病菌的生长具有较好的抑制作用，且该菌株耐高温、较耐酸碱，作为稻瘟病生防制剂研发具有一定的价值。     

东乡野生稻内生放线菌分离及菌株S123次级代谢产物分析

【目的】从东乡野生稻(Oryza rufipogon)中分离和鉴定内生放线菌,对其进行抗菌活性筛选,并分析高抗菌活性菌株S123的次级代谢产物。【方法】采用S培养基对东乡野生稻内生放线菌进行分离、纯化,并构建16S rRNA基因序列系统发育进化树进行菌株鉴定。以琼脂扩散法和菌丝生长速率法进行抗菌活性筛选,同时设计简并引物检测菌株I型聚酮合酶(PKS-I)基因。对具广谱抗菌活性的菌株S123进行分批大量发酵,运用多种色谱方法对发酵产物进行分离、纯化,利用MS和NMR分析鉴定化合物的结构。【结果】从东乡野生稻中共分离到11株内生放线菌,分别属于链霉菌属(8株)和假诺卡氏属(3株)。其中有8株具有抗菌活性,8株呈现I型PKS阳性。从高抑菌活性菌株S123中分离到化合物Nigericin和17-O-demethylgeldanamycin,其中Nigericin对金黄色葡萄球菌、枯草芽孢杆菌及水稻纹枯病菌均有抑制活性。【结论】对东乡野生稻内生放线菌进行了分离、鉴定和抗菌活性筛选,并从中分到两种与I型PKS基因相关活性的化合物Nigericin和17-O-demethylgeldanamycin,为研究东乡野生稻内生放线菌的多样性和次级代谢产物的分离提供依据。     

百部内生放线菌的分离、分类及次级代谢潜力

【目的】以对叶百部块根为材料分离内生放线菌,并对分离菌株进行分类、抗菌活性和次级代谢产物合成基因研究。【方法】样品经过严格的表面消毒,选用4种培养基分离百部内生放线菌;分离菌株通过形态观察和16S rRNA序列分析进行分类鉴定;采用琼脂移块法测试分离菌株的抗菌活性;通过PCR检测分离菌株的PKS/NPRS和卤化酶基因;使用HPLC-UV/VIS-ESI-MS/MS分析发酵产物。【结果】从6个样品中获得18株内生放线菌,分属链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、假诺卡氏菌属(Pseudonocardia)和甲基杆菌属(Methylobacterium)。分离菌株绝大部分具有抗菌活性和次级代谢产物合成基因,其中13株对耐药金黄色葡萄球菌和/或绿脓杆菌有拮抗活性,17株具有PKS/NRPS基因,8株菌具有卤化酶基因,且卤化酶阳性代表菌株的发酵产物具有抗细菌活性和卤代化合物特征。【结论】百部作为一种传统中药,其内生放线菌以链霉菌和小单孢菌为主,在次级代谢产物合成方面具有很好的潜力,可作为一类重要微生物资源进行活性产物开发。     

烟草根黑腐病拮抗内生细菌的筛选及其抑菌作用

【目的】从烟草根、茎中分离获得对烟草根黑腐病菌(Thielaviopsis basicola)有较好控病作用的内生细菌。【方法】采用平板对峙培养,测定分离获得的306个内生细菌对烟草根黑腐病菌的抑制作用;通过菌株形态特征、生理生化特性及16S rDNA序列对菌株进行分类鉴定。【结果】筛选获得6个菌株对烟草根黑腐病菌有较强拮抗作用,室内测定其对病原菌的抑菌带宽达6.5 mm-11.0 mm,温室控病效果达11.9%-77.1%,其中来自茎部的内生细菌T295菌株对烟草根黑腐病的防效达77.1%;无菌滤液实验表明,拮抗内生细菌T295无菌滤液在实验浓度范围内对病菌菌丝生长、孢子萌发有较好地抑制作用。【结论】菌株T295对烟草根黑腐病具有较好的防治作用,经鉴定为枯草芽孢杆菌(Bacillussubtilis)。     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

永兴岛白穗软珊瑚共附生放线菌筛选及部分活性次级代谢产物的鉴定

【目的】从白穗软珊瑚中分离和鉴定共附生放线菌,运用PCR技术对所分离的放线菌进行I型聚酮合酶(PKS)筛选,研究其次级代谢产物。【方法】使用11种培养基对白穗软珊瑚共附生放线菌进行分离、鉴定,构建16S rRNA基因系统发育进化树,以基于I型PKS的KS基因设计的简并引物对所分离放线菌进行基因筛选,对阳性菌株用3种培养基发酵检测,对目标菌株进行放大规模发酵分离鉴定次级代谢产物。【结果】从白穗软珊瑚中分离到20株共附生放线菌,包括链霉菌属10株、迪茨氏菌属2株和盐水孢菌属8株,筛选获得18株I型PKS阳性菌株,并从菌株Salinospora arenicola SH04中分离到化合物rifamycin S和rifamycin W。【结论】首次从珊瑚共附生环境中分离得到海洋专属性稀有放线菌盐水孢菌属,并以I型PKS基因筛选为指导,分离鉴定了聚酮类化合物rifamycins,为研究软珊瑚共附生可培养放线菌的多样性和基于基因筛选指导分离次级代谢产物提供了可靠依据。     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

基于化学遗传学筛选产萘醌类化合物内生放线菌

【目的】为探究含具有抗肿瘤活性的美登素的滑桃(Trewia nudiflora)种子中内生放线菌的多样性,以及从内生放线菌中寻找萘醌类化合物产生菌。【方法】利用放线菌富集筛选培养基对经消毒处理的滑桃种子进行内生菌分离,根据菌落形态及16S rRNA基因序列分析进行菌种的分类鉴定。通过对所分离到的内生放线菌拮抗模式病原细菌(金黄色葡萄球菌、铜绿假单胞菌)、作物病原真菌(小麦赤霉菌、水稻纹枯病致病菌等)活性检测,以及萘醌类化合物合成关键基因为探针定向筛选萘醌类化合物产生菌。【结果】从分离到的100余株滑桃种子内生菌中鉴定出66株以链霉菌为主的放线菌,发现Streptomyces sp.HTZ27菌株含有目标基因,经固体发酵、化合物分离纯化、鉴定后,发现该菌发酵产物中有呋喃萘醌I,得率接近5 mg/L。【意义】本研究采用的化学遗传学方法可有效提高筛选目标化合物产生菌的效率,所筛选到FNQ I产生菌为深入研究呋喃萘醌类化合物生物合成与调控、抗肿瘤分子机理以及产业化应用等创建了有利条件。     

东乡野生稻内生细菌Fse32拮抗作物病害真菌及对水稻的促生活性

【背景】植物种子是植物内生菌筛选的重要原料,从中能够分离得到具有巨大应用价值的内生菌株。【目的】为发掘优良的种子内生细菌资源,对分离自东乡野生稻种子的内生细菌Fse32进行鉴定并研究其抗病原真菌和促生活性。【方法】通过形态学观察、生理生化特征和16SrRNA基因序列分析进行菌种鉴定,采用拮抗试验检测抑制病原真菌的活性,通过促生能力测定试验、水稻种子萌发及盆栽试验评价该菌株的促生效果。【结果】内生细菌Fse32鉴定为唐菖蒲伯克霍尔德氏菌,命名为Burkholderia gladioli Fse32。拮抗试验结果显示,菌株Fse32对禾谷镰孢菌(Fusarium graminearum)、水稻纹枯病菌(Rhizoctoniasolani)、核盘菌(Sclerotiniasclerotiorum)、大豆核盘菌(Sclerotinialibertiana)、尖孢镰刀菌(Fusariumoxysporum)和辣椒疫霉病菌(Phytophthoracapsici)均有较好的抑制作用,吲哚乙酸(indole-3-aceticacid,IAA)产率为17.95mg/L,能产铁载体,其A/Ar比值为0....     

Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence

Several chemical constituents are important to the fragrance of cooked rice. However, the chemical compound 2-acetyl-1-pyrroline (AP) is regarded as the most important component of fragrance in the basmati- and jasmine-style fragrant rices. AP is found in all parts of the plant except the roots. It is believed that a single recessive gene is responsible for the production of fragrance in most rice plants. The detection of fragrance can be carried out via sensory or chemical methods, although each has their disadvantages. To overcome these difficulties, we have identified an (AT)₄₀ repeat microsatellite or simple sequence repeat (SSR) marker for fragrant and non-fragrant alleles of the fgr gene. Identification of this marker was facilitated through use of both the publicly available and restricted access sequence information of the Monsanto rice sequence databases. Fifty F₂ individuals from a mapping population were genotyped for the polymorphic marker. This marker has a high polymorphism information content (PIC = 0.9). Other SSR markers linked to fragrance could be identified in the same way of use in other populations. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.     

Identification of an active transposon in intact rice plants

A transposable element that is active in intact plants has been identified in rice (Oryza sativa L.). The 607-bp element itself, termed nonautonomous DNA-based active rice transposon (nDart), has no coding capacity. It was found inserted in the gene encoding Mg-protoporphyrin IX methyltransferase in a chlorophyll-deficient albino mutant isolated from backcross progeny derived from a cross between wild-type japonica varieties. The nDart has 19-bp terminal inverted repeats (TIRs) and, when mobilized, generates an 8-bp target-site duplication (TSD). At least 13 nDart elements were identified in the genome sequence of the japonica cultivar Nipponbare. Database searches identified larger elements, termed DNA-based active rice transposon (Dart) that contained one ORF for a protein that contains a region with high similarity to the hAT dimerization motif. Dart shares several features with nDart, including identical TIRs, similar subterminal sequences and the generation of an 8-bp TSD. These shared features indicate that the nonautonomous element nDart is an internal deletion derivative of the autonomous element Dart. We conclude that these active transposon systems belong to the hAT superfamily of class II transposons. Because the transposons are active in intact rice plants, they should be useful tools for tagging genes in studies of functional genomics.Electronic Supplementary Material Supplementary material is available for this article at     

辽宁地区水稻资源抗稻瘟病基因的检测分析

为了明确辽宁地区水稻资源中抗稻瘟病基因的分布情况及抗病效应,选取辽宁地区水稻资源176份,鉴定了抗稻瘟病基因pi21、Pi36、Pi37、Pita、Pid2、Pid3、Pi5及Pib在这些材料中的分布情况,并接种鉴定了这些材料对稻瘟病的抗性。结果表明:176份供试材料中,83份对稻瘟病表现抗病,栽培稻、杂草稻及农家种中抗病品种所占的比率分别为41.48%、1.14%及4.54%。抗稻瘟病基因pi21、Pi36和Pi37在所有参试材料中均未检测到,且分别有74份、49份、47份、52份及89份材料携带Pita、Pid2、Pid3、Pi5及Pib的抗病等位基因。抗病基因绝大部分分布在栽培种中,农家种和杂草稻中分布较少。不含有抗稻瘟病基因和只携带单个抗病基因的材料对稻瘟病的抗性均较差,而抗病基因聚合可不同程度提高材料的抗性。经检测,不含有本试验鉴定的pi21等8个已克隆抗病基因的材料共32份,其中表现抗病的占21.87%;只携带1个抗稻瘟病基因的材料为52份,表现抗病的占17.31%;携带2个抗稻瘟病基因的材料为39份,表现抗病的占69.23%,其中以携带Pita+Pi5的材料最多(14份),且均表现抗病;携带3个抗稻瘟病基因的材料为31份,表现抗病的占77.42%,以携带Pita+Pid3+Pi5的材料抗性最强;携带4个抗稻瘟病基因的水稻材料22份,表现抗病的占72.73%,携带5个抗病基因的水稻材料未检测到。     

RFLP tagging of a gene for aroma in rice

Summary We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F₃ segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Diploid somatic-hybrid plants regenerated from rice cultivars

Summary Somatic hybrid plants were obtained between rice cultivars Yamahoushi and Murasakidaikoku. Since Murasakidaikoku is a double mutant having both dominant (purple coloration) and recessive (dwarf) markers, the somatic hybrids can be easily distinguished from their parents. Protoplasts were isolated from anther-derived calli, and electrofused protoplasts were cultured without selection of hybrid cells. Out of 27 regenerated plants, 9 proved to be hybrids based on their purple coloration and normal plant type, traits which were identical to those of the sexual F₁ hybrid between the same parental cultivars. The somatic hybrids included three diploid and six triploid plants. Segregation of parental markers was observed in the selfed progenies. These results demonstrated that diploid hybrids of rice could be obtained through somatic hybridization between haploid anther-derived cells instead of by sexual hybridization.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.