贝莱斯芽孢杆菌生防次级代谢产物研究进展

贝莱斯芽孢杆菌(Bacillus velezensis)是生防芽孢杆菌中的重要代表,作为微生物农药的核心菌种,已被广泛应用于作物病害生物防治。贝莱斯芽孢杆菌具有植物内生性,其生防作用机制主要包括产生次级代谢产物对抗植物病原物;改善宿主植物根际微生物群落,促进宿主营养和生长;激发宿主植物产生防御反应,诱导植物获得系统抗性。其中,产生次级代谢产物是其最重要的生防作用机制。贝莱斯芽孢杆菌含有多个编码生物合成次级代谢产物的基因簇,其中包括编码聚酮化合物合酶(PKS)和非核糖体肽合成酶(NRPS)的基因簇,同时存在核糖体途径合成次级代谢产物基因簇。通过非核糖体途径可产生脂肽类化合物、聚酮类化合物、二肽和铁载体;通过核糖体途径产生小菌素、细菌素、羊毛硫抗生素。这些具有生物活性的次级代谢产物成为了天然新药和候选抗生素的储存库,对于解析生防菌作用机制具有重要意义。本文综述了贝莱斯芽孢杆菌的命名与更迭,产生次级代谢产物的类型、合成与调控基因以及靶标病原菌,以期为生防菌株的改良和生物农药的研发提供参考。

根结线虫诱导的巨型细胞及其形成机制

根结线虫(Meloidogyne spp.)是一种专性寄生线虫,具有分布广泛、寄主繁多和危害严重的特点。每年给农作物造成巨大的经济损失,是危害农作物最严重的线虫之一。侵染植物时,根结线虫会诱导根尖部位的维管束细胞重新分化形成多核的巨型细胞,将其作为线虫的取食位点和线虫生长发育过程中营养物质的唯一来源。因此,巨型细胞的形成和维持对线虫的生长和繁殖至关重要。本文从根结线虫诱导寄主植物巨型细胞的形成及调控机制,重点介绍了巨型细胞的结构特征、巨型细胞的细胞周期变化、营养物质运输、激素调节,以及寄主植物的识别和防御和表达模式,并探讨了根结线虫效应蛋白在巨型细胞形成过程中的功能。通过对巨型细胞形成和调控机制及线虫-植物相互分子机制等多层次的研究进展进行解析,揭示根结线虫的致病机理,以期为后续研究根结线虫防治创新性策略提供启发和思路。

组学技术在生防芽胞杆菌的应用进展

芽胞杆菌(Bacillus spp.)是全世界应用最广泛的生防微生物菌种资源之一,在基因组学成为普遍性基础学科的时代,转录组、蛋白质组、代谢组等多组学技术的联用成为深入揭示芽胞杆菌生物学特性和生防机制研究的重要手段。目前NCBI数据库共计发布了10813个芽胞杆菌全基因组序列,其中装配注释完整的共1842个,占总数量的17.04%。对芽胞杆菌基因组与比较基因组的分析发现,次级代谢产物基因簇是芽胞杆菌基因组中最具有保守性,又具有特异性的部分,它们既是天然产物的重要来源,也在细菌间以及细菌与环境的相互作用和生命周期中扮演着重要角色。截至目前,对于芽胞杆菌的相关研究,多采取整合组学模式和生物信息学计算方法,聚焦于芽胞杆菌的抗性形成、生物膜形成、定殖、促生、诱导植物抗逆性等机制。随着研究的不断深入,芽胞杆菌生防过程中的基因调控机制、蛋白质表达和代谢途径将进一步解析,这将有助于发现新的生防活性物质和优化生防策略。本文综述了转录组、蛋白质组、代谢组等多组学技术在芽胞杆菌上的研究进展,以期为芽胞杆菌生防作用机制的解析以及生防菌株改良的深入研究提供参考。

刺角瓜营养成分及抗氧化活性的分析与评价

利用高效液相色谱仪、紫外分光光度计及全自动氨基酸分析仪等对甜瓜属新种质刺角瓜的营养成分含量进行了测定,并对其抗氧化活性进行了评价。结果表明:刺角瓜富含VC、多酚、多糖及氨基酸等营养成分,其中VC含量为125mg·kg^(-1),多酚含量为314 mg·kg^(-1),可溶性总糖、可溶性多糖含量分别为2.7%、0.5%,游离总氨基酸、必需氨基酸含量分别为9 908、3 120 mg·kg^(-1),水分及灰分分别占鲜质量的95.0%、0.6%;刺角瓜具有一定的抗氧化活性,其对DPPH半清除率IC50为0.060 g·m L^(-1),对羟自由基半清除率IC50为0.036 g·m L^(-1)。

与丈夫同型的地中海贫血孕妇的产前诊断结果、妊娠和新生儿结局

目的探讨与丈夫同型的地中海贫血(地贫)孕妇的产前诊断结果、妊娠和新生儿结局。方法选择与丈夫同型的1069例地贫孕妇作为研究对象。收集所有病例的胎儿产前诊断结果、妊娠结局及新生儿指标。结果837例同型α地贫孕妇中,胎儿产前诊断为静止型α地贫140例(16.73%),轻型α地贫283例(33.81%),中间型α地贫117例(13.98%),重型α地贫93例(11.11%);225例同型β地贫孕妇中,胎儿产前诊断为轻型β地贫110例(48.89%),中间型或重型β地贫68例(30.22%);7例同型的αβ复合型地贫孕妇中,胎儿产前诊断为轻型α地贫、轻型β地贫复合静止型α地贫各2例(28.57%),轻型β地贫复合轻型α地贫、轻型β地贫复合中间型α地贫、中间型β地贫复合静止型α地贫各1例(14.29%)。所有胎儿产前诊断为重型α地贫的孕妇,以及92.65%产前诊断为中间型或重型β地贫的孕妇,均终止妊娠。中重型地贫组人工终止妊娠率高于对照组和轻型地贫组,剖宫产率高于轻型地贫组,低体重儿发生率高于对照组(均P<0.05)。结论通过对与丈夫同型的地贫孕妇进行产前诊断,有效阻止了重型地贫患儿的出生。胎儿中重型地贫或可影响妊娠及新生儿结局,应加强地贫孕妇围生期保健,做好产前诊断工作。

淡紫拟青霉次级代谢产物的研究进展

淡紫拟青霉是子囊菌门、肉座菌目、蛇形虫草科、Purpureocillium属的一种丝状真菌。它是一种广谱性很强的生防菌,不仅对植物寄生线虫、蚜虫、红蜘蛛、温室粉虱和切叶蚁等有很好的防治作用,而且对多种植物病原真菌、细菌乃至病毒也有抑制作用。淡紫拟青霉分泌的几丁质酶与丝氨酸蛋白酶能够降解线虫表皮的蛋白质和几丁质组成成分,利于侵入并破坏细胞成分;也能内生于植物,产生效应物对其它真菌产生拮抗作用;也能定殖于植物根际,产生次级代谢产物,对真菌、线虫等产生抑杀作用。目前对淡紫拟青霉次级代谢产物的研究主要有非核糖体肽类、生物碱及聚酮化合物等的合成调控、活性作用和分子结构确定。淡紫拟青霉所产的非核糖体肽类化合物有白灰制菌素A和B,生物碱有拟青霉酰胺,聚酮化合物有Acremoxanthone C和Acremonidin A。综述了淡紫拟青霉次级代谢产物的研究进展,讨论了淡紫拟青霉次级代谢产物的研究意义,最后展望了淡紫拟青霉的研究前景,以期为相关研究者了解淡紫拟青霉提供参考。

神经炎症在锰中毒中的作用及对氨基水杨酸钠对其治疗作用研究进展

实验研究发现,过量锰暴露能刺激神经系统产生大量的炎症因子,发生炎症反应,以至神经中毒表现加重。流行病学调查显示,长期慢性锰暴露能引起神经功能紊乱,可能与帕金森病、阿尔茨海默病的发生关系密切。神经炎症是锰中毒及其他神经退行性疾病重要的致病机制,小胶质细胞和星形胶质细胞功能失衡与神经炎症的发生发展密切相关。对氨基水杨酸钠不仅有螯合金属的特性,而且有非特异性抗炎作用,可能对锰中毒性神经炎症有治疗作用,对锰中毒治疗有积极意义。

长枝木霉TL16菌株对南方根结线虫的防治效果

为探明TL16菌株对南方根结线虫的防治效果,先利用长枝木霉TL16(Trichoderma longibrachiatum)孢子悬浮液测定对南方根结线虫(Meloidogyne incognita)卵和二龄幼虫的离体作用。进一步采用盆栽试验探讨TL16对黄瓜根结线虫病的防治效果。结果表明:长枝木霉菌株TL16孢子悬浮液抑制线虫卵的孵化(53.58%~69.05%),其中,处理后9 d,7.5×10^6 cfu/mL的TL16孢子悬浮液对线虫卵孵化的抑制率最高(69.05%)。然而,孢子悬浮液对二龄幼虫无明显的致死作用(0.96%~1.65%)。同时,盆栽试验发现,浓度为7.5×10^6 cfu/mL的TL16孢子悬浮液对黄瓜根结线虫的防效最佳(55.00%)。

The Potential Geographic Distribution of Radopholus similis in China

The burrowing nematode （Radopholus similis） is one of the main quarantine pests in China, and the risk of invasion posed by this nematode is becoming more and more serious with regard to the international trade being intensified day by day. It is urgent to analyse the potential geographic distribution of R. similis in China. Genetic algorithm for rule-set prediction modeling system （GARP） and maximum entropy （MAXENT）, the two niche models which have been widely used to predict the potential geographic distribution of alien species, were used to predict the distribution of R. similis in China. We also presented a model comparison of the results by both threshold-dependent and threshold-independent evaluations. It has been shown that the two niche models could be used to predict the potential distribution of R. similis reliably. The potential distribution of R. similis should be constricted within the south of China, such as Hainan, Guangdong, Guangxi, Fujian, Yunnan provinces, and Taiwan of China. The MAXENT gives a better prediction than that of GARP. R. similis can be introduced to China by flowers and nursery stock＇s international shipping. The predicted results indicate that R. similis can occur in south coastal area of China and Yunnan Province, which are the main flower and nursery stock＇s import-export areas in China. Consequently, a strong quarantine program is needed at the ports of such areas to prevent the pest from being introduced to China.

Chinese leek(Allium tuberosum Rottler ex Sprengel) reduced disease symptom caused by root-knot nematode

Root-knot nematodes（RKNs,Meloidogyne spp.） are obligate biotrophic parasites that settle close to the vascular tissues in plant roots.The diseases resulting from RKN infections cause serious damage to agricultural production worldwide.In the present paper,the resistance of Chinese leek（Allium tuberosum Rottler ex Sprengel） against RKNs,its suppressive effect on nematode disease,its nematicidal activity and its component profile were studied to identify an efficient disease control method.In soil heavily infected by nematodes,Chinese leek showed strong resistance to RKNs.Additionally,the gall indexes of cucumber plants rotated with Chinese leek and of tomato plants intercropped with Chinese leek were reduced by 70.2 and 41.1%,respectively.In a pot experiment,the gall indexes of Chinese leek extract-treated tomato and cucumber plants were reduced by 88.9 and 75.9%,respectively.In an in vitro experiment,the mortality rate of a RKN（Meloidogyne incognita J2） treated with Chinese leek extract was significantly higher than that of the control.The gas chromatography-mass spectrometry（GC-MS） analysis revealed that glycosides,carboxylic acids,ketones and organic sulfides are the main components in the Chinese leek extract.This study revealed that Chinese leek possesses a high resistance to RKNs,has strong nematicidal activity against M.incognita and can significantly reduce the incidence of disease caused by nematodes.

Comparative genomics provide a rapid detection of Fusarium oxysporum f. sp. conglutinans

Fusarium oxysporum f. sp. conglutinans （Foc） is the causal agent of Fusarium wilt disease of Brassica oleracea. A rapid, accurate, and reliable method to detect and identify plant pathogens is vitally important to integrated disease management. In this study, using a comparative genome analysis among Fusarium oxysporum （Fo）, we developed a Foc-specific primer set （Focs-l/Focs-2） and established a multiplex-PCR assay. In the assay, the Focs-1/Focs-2 and universal primers for Fusarium species （W106PJF106S） could be used to detect Foc isolates in a single PCR reaction. With the optimized PCR parameters, the multiplex-PCR assay showed a high specificity for detecting Foc and was very sensitive to detect as little as 100 pg of pure Foc genomic DNAor 1 000 spores in 1 g of twice-autoclaved soil. We also demonstrated that Foc isolates were easily detected from infected plant tissues, as well as from natural field soils, using the multiplex-PCR assay. To our knowledge, this is a first report on detection Fo by comparative genomic method.

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

Expression of mitogen-activated protein kinase double-stranded RNA in cucumber has no apparent effect on the diversity of rhizosphere archaea

The expression of mitogen-activated protein kinase （mapk） double-stranded RNA in cucumber is effective in controlling infestations of the root-knot nematode Meloidogyne incognita. However, little is known about the ecological effects of transgenic plants. Here, we analyzed the diversity of 16S rDNA genes derived from the rhizosphere archaea of transgenic cucumber plants as an indicator of ecological change. A total of 17 and 18 operational taxonomic units were detected in the rhizospheres of non-transgenic cucumber and mapk dsRNA-expressing plants, respectively. No significant difference was observed between the two groups according to Shannon and Simpson indices. In soil samples of the two rhizospheres, the dominant group was Crenarchaeota at the phylum level, with Staphylothermus, Methanococcus, Pyrodictium and Sulfolobus the abundant taxa at the genus level. These results suggest that expressing mapk double-stranded （ds） RNA in cucumber has no apparent effect on the diversity of rhizosphere archaea, and provide powerful evidence for the ecological safety of transgenic cucumber expressing mapk dsRNA.

A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Rediscovery and analysis of Phytophthora carbohydrate esterase(CE) genes revealing their evolutionary diversity

A continuous co-evolutionary arms-race between pathogens and their host plants promotes the development of pathogenic factors by microbes, including carbohydrate esterase(CE) genes to overcome the barriers in plant cell walls. Identification of CEs is essential to facilitate their functional and evolutionary investigations; however, current methods may have a limit in detecting some conserved domains, and ignore evolutionary relationships of CEs, as well as do not distinguish CEs from proteases. Here, candidate CEs were annotated using conserved functional domains, and orthologous gene detection and phylogenetic relationships were used to identify new CEs in 16 oomycete genomes, excluding genes with protease domains. In our method, 41 new putative CEs were discovered comparing to current methods, including three CE4, 14 CE5, eight CE12, five CE13, and 11 CE14. We found that significantly more CEs were identified in Phytophthora than in Hyaloperonospora and Pythium, especially CE8, CE12, and CE13 that are putatively involved in pectin degradation. The abundance of these CEs in Phytophthora may be due to a high frequency of multiple-copy genes, supporting by the phylogenetic distribution of CE13 genes, which showed five units of Phytophthora CE13 gene clusters each displaying a species tree like topology, but without any gene from Hyaloperonospora or Pythium species. Additionally, diverse proteins associated with products of CE13 genes were identified in Phytophthora strains. Our analyses provide a highly effective method for CE discovery, complementing current methods, and have the potential to advance our understanding of function and evolution of CEs.

A novel strategy to enhance resistance to Cucumber mosaic virus in tomato by grafting to transgenic rootstocks

Cucumber mosaic virus（CMV） can infect a wide range of host species. For the lacking of CMV resistant varieties of tomato, RNA interference（RNAi） can be used as a fast and effective method for the generation of transgenic resistant varieties. In this current study, five intron-spliced hairpin RNA（ihp RNA） plant expression vectors aimed at five genes of CMV have been constructed. Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with expression vectors. Highly resistant generations of transgenic plants were employed as rootstocks and grafted onto non-transgenic tomatoes that resulted in the successful transfer of resistance to the scions. Using a novel method of plant cuttings for rootstock propagation, we obtained large quantities of disease-resistant material. Further, this method produces scions that can remain undetectable for transgenic resistance marker genes that may provide novel approaches to evade collective concerns about genetically-modified organism（GMO） biosafety.

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）

昆虫相关放线菌资源挖掘利用研究进展

昆虫是自然界物种多样性最丰富的生物类群之一,它们在漫长的进化过程中与各种功能独特的共生菌建立了紧密的共生关系。这些共生关系早期被认为是互惠互利的关系,近年来的研究还发现了偏利共生和寄生关系等。在这些共生菌中,放线菌是一类特殊的微生物,它们可存在于昆虫肠道、粪便、触角、外骨骼以及培菌昆虫的菌圃中,帮助宿主昆虫抵御病原菌、寄生虫和捕食者,在维持宿主昆虫生存和繁衍方面起到了重要作用。近年来,人们从昆虫共生放线菌产生的代谢产物中分离到多种新型化合物,这些化合物可以抑制多种动植物病原菌、肿瘤细胞以及癌细胞的增殖。因此,研究昆虫相关放线菌不仅可以解析宿主与微生物的共生机制,还可以为生物农药与生物医药的开发提供新的选择。本文对昆虫相关放线菌资源挖掘与利用进行综述,总结了不同种类昆虫相关放线菌资源和功能多样性,同时对有重要活性的昆虫相关放线菌的次级代谢产物按照结构差异进行分类研究,主要包括多肽、醌酮、内酯、生物碱及其他化合物等,探讨了这些次级代谢产物的功能多样性,为深入理解生态系统的运作,发现新的生物活性物质,以及开发新型生物农药和医药奠定基础。

对氨基水杨酸钠对锰染毒大鼠丘脑原代小胶质细胞氧化损伤及炎症反应的影响

[目的]探讨对氨基水杨酸钠(PAS-Na)对体外染锰大鼠丘脑原代小胶质细胞氧化损伤、炎症反应的影响。[方法]将提纯、鉴定后的SD大鼠丘脑小胶质细胞分别给予含不同浓度氯化锰(MnCl_2)(0、200、300、400、500μmol/L)、PAS-Na(50、150、450μmol/L)的完全培养基(DMEM+FBS)培养24 h,采用MTT法检测细胞存活率,根据结果选择染锰的毒性剂量并选定PAS-Na的无毒剂量。随后将细胞随机分为对照组、400μmol/L MnCl_2组、400μmol/L MnCl_2+(50、150、450μmol/L)PAS-Na组、450μmol/L PAS-Na组,共6组。采用MTT法检测各组小胶质细胞存活率;运用DCFH-DA探针标记,荧光倒置显微镜下观察小胶质细胞内活性氧(ROS)的产生情况;ELISA法测定炎症因子白介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α的蛋白分泌量和荧光定量PCR法测IL-1β,TNF-αmRNA表达水平。[结果]400、500μmol/L MnCl_2染毒组的细胞存活率为(85.83±12.53)%、(83.30±6.33)%,低于对照组(均P<0.05),故选择400μmol/L作为锰的染毒剂量;与对照组比较,50、150、450μmol/L PAS-Na组的细胞存活率无明显变化(均P>0.05)。400μmol/L MnCl_2+(50、150、450μmol/L)PAS-Na组的细胞存活率分别为(96.00±18.11)%、(106.13±18.32)%、(110.21±15.13)%,均比400μmol/L MnCl_2组高(均P<0.05)。与对照组比较,400μmol/L MnCl_2组细胞ROS水平、IL-1β和TNF-α的蛋白及mR NA表达水平均升高(均P<0.05)。与400μmol/L MnCl_2组比较,400μmol/L MnCl_2+(50、150、450μmol/L)PAS-Na组的ROS水平、TNF-α分泌量和IL-1βmRNA表达量均降低(均P<0.05),400μmol/L MnCl_2+(150、450μmol/L)PAS-Na组的TNF-αmRNA表达量降低(均P<0.05)。[结论]PAS-Na可减轻锰致大鼠丘脑原代小胶质细胞氧化损伤、炎症反应的程度。

“人本位”目标管理模式在甲状腺围手术期护理中的应用效果研究

目的探讨"人本位"目标管理模式在甲状腺围手术期护理中的应用效果。方法将选择2016年7月~2017年6月122例接受甲状腺切除手术的患者,按入院时间分为对照组和干预组,每组61例,对照组进行常规护理,干预组在常规护理的基础上开展"人本位"整体护理,并实施目标管理,比较两组患者疼痛强度、焦虑评分、并发症的发生率、患者满意度等指标,评价护理效果。结果干预组与对照组相比,患者术后并发症发生率、焦虑自评得分均降低,干预组术后24 h疼痛评分<3分患者多于对照组,疼痛评分>3分患者少于对照组(P<0.05),对护理服务的满意度上升,差异有统计学意义(P<0.05)。结论 "人本位"目标管理模式能够有效降低甲状腺切除术后患者并发症的发生率,减轻患者术后疼痛程度,改善患者的焦虑状态,有利于患者的术后恢复,值得临床推广。