植物根结线虫基因组学研究进展

根结线虫是世界农业生产中危害最大的植物病原之一,目前仍缺乏安全有效的防治措施。深入揭示寄生线虫与植物之间互作的分子机制,利用生物技术进行抗性育种被认为是最有前景的抗线虫策略。在根结线虫基因组学研究方面,目前已经构建了北方根结线虫AFLP遗传连锁图谱,南方根结线虫和北方根结线虫基因组测序也已完成;基因组的注释和比较基因组学分析,较全面地描述了根结线虫的遗传组成;以差异表达分析和比较基因组学为主的方法鉴定了大量的重要基因;以RNA干扰、植物转化和蛋白互作为主的根结线虫基因功能研究也取得了一些进展。本文就根结线虫基因组学研究予以综述,并进一步探讨其研究方向和可持续抗线虫新策略的发展前景。     

根结线虫多样性研究进展

根结线虫属(Meloidogyne spp.)是专性内寄生物,寄主范围广,全球分布,给世界农业生产造成严重为害。生殖方式主要有孤雌生殖和两性融合。系统发育学研究指出,种间杂交可能在根结线虫多样化的进程中起着重要作用。越来越多的证据表明,基因组范围内的序列重复和水平基因转移为根结线虫的多样性提供了遗传基础。南方根结线虫(Meloidogyne incognita)和北方根结线虫(Meloidogyne hapla)基因组测序的完成和注释也为研究根结线虫的多样性提供了重要线索。根结线虫的多样性给鉴定和防治工作带来了巨大挑战,需要寻找更加有效的方法。     

江苏进出口园林植物寄生线虫调查及检疫处理技术

1997－1998年，对南京口岸进口的荷兰、加拿大、美国、韩国、台湾和香港等国家和地区41批（次）园林植物检疫中，1次截获鳞球茎类线虫、19交截获咖啡短体线虫、草莓芽叶线虫、克鲁克剑线虫、毛刺尾线虫、南方根结线虫、花生根结线虫、北方根结线虫和其它根结线虫等，进口的园林植物中危险性疫情发生率高达34％。对江苏5个主要出口园林植物生长基地进行的植物寄生线虫种类调查，在59种经常性出口的园林植物中，发现27属植物寄生线虫，其中已鉴定出13种。对红花木莲苗上的南方根结线虫、牡丹苗上的腐烂茎线虫进行的检疫处理试验表明，用几种农药混配的处理液加热后处理植物的根部，可以有效地杀死这两种植物寄生线虫，保证货物安全出口。     

四种植物寄生根结线虫的区别及防治

目前世界上植物寄生根结线虫中,南方根结线虫（Ｍｅｌｏｉｄｏｇｙｎｅ ｉｎｃｏｇｈｉｔａ）、爪哇根结线虫（Ｍ．ｊａｖａｎｉｃａ）、花生根结线虫（Ｍ． ａｒｅｎａｒｉａ）、北方根结线虫（ Ｍ． ｈａｐｌａ）这 ４个种,分布最广泛、危害寄主最多、最重,占整个根结线虫引起农作物损失的９０％以上。为了帮助基层的植保工作者鉴别这４种线虫,本文列出４种常见根结线虫的检索表如下,供参考。 一、４种最常见根结线虫检索表 Ⅰ（Ａ）会阴花纹有一个高背弓,无明显侧线;（Ｂ）雄虫头部有一个中央凹陷的唇盘,这个唇盘高出中唇;（Ｃ…     

根结线虫病的病原鉴定

根结线虫是植物寄生线虫中最重要的一类病原线虫,寄主范围广,分布普遍,根结线虫侵染植物(或品种),给农作物造成很大的经济损失。其为害特点是寄生专化性。因此,对根结线虫进行准确的鉴定是非常重要的。作者从广东省的广州市、坪石、新兴等13个县、市采集了31种作物根结线虫病的病     

禾谷孢囊线虫(Heterodera avenae)纤维素结合 蛋白基因(Ha-cbp-1)的克隆和序列分析

 禾谷孢囊线虫(Heterodera avenae)是中国小麦上的重要病原线虫。纤维素结合蛋白基因是一种重要的植物线虫寄生和致病相关基因。用同源克隆的方法从禾谷孢囊线虫寄生前二龄幼虫中克隆出一种纤维素结合蛋白新基因Ha-cbp-1（GenBank 注册号GQ178086）cDNA序列。Ha-cbp-1基因cDNA包含1个开放阅读框,编码131个氨基酸残基的蛋白质,预测蛋白由1个长度为18氨基酸残基的信号肽和1个纤维素结合区域（CBD）组成。Ha-cbp-1的基因组DNA序列含有2个内含子。预测的禾谷孢囊线虫纤维素结合蛋白（HA-CBP-1）序列与大豆孢囊线虫纤维素结合蛋白（HG-CBP-1）序列有60%的同一性和76%的相似性,与甜菜孢囊线虫纤维素结合蛋白（HS-CBP-1）序列有60%的同一性和75%的相似性。本研究首次从禾谷孢囊线虫中成功克隆出CBP蛋白基因。     

郑州市绿地草坪重要植物寄生线虫鉴定

郑州市部分绿地草坪草存在植株矮小，长势衰弱，叶片枯黄等现象，推测其可能受植物线虫危害。为明确危害郑州市绿地草坪草的寄生线虫种类以及分布情况，2020—2021年，从郑州市12县/区辖区内的30个公园绿地随机采集136份草坪草疑似发病样品。采用浅盘法和漂浮法分离样品中的植物线虫，利用形态学和分子生物学鉴定相结合的方法，对分离到的植物线虫进行种类鉴定。结果表明，18个地区中的98份样品中检测到植物寄生线虫，检出率高达72.1%，覆盖调查地区的60.0%。同时，发现不同公园或绿地之间土壤中线虫数量差异悬殊，其中上街东虢湖公园线虫密度可达98.68条/100 m L土，而新郑龙湖城市湿地公园线虫密度仅为1.10条/100 mL土。种类鉴定结果显示，本次调查共检测到9种重要草坪草植物寄生线虫：小叶螺旋线虫（Helicotylenchus microlobus）、花生茎线虫（Ditylenchus arachis）、马铃薯腐烂茎线虫（D. destructor）、茎线虫属线虫（Ditylenchus sp.）、菲利普孢囊线虫（Heterodera filipjevi）、南方根结线虫（Meloido...     

土壤中线虫孢囊的三种分离方法及综合评价

土壤中线虫孢囊的三种分离方法及综合评价郑经武，程瑚瑞，方中达（南京农业大学植保系２１００９５）孢囊线虫是植物寄生线虫中很重要的一个类群，从种类数量及危害程度上仅次于根结线虫。土壤中线虫孢囊的分离方法很多，目前应用最普遍的是Ｆｅｎｗｉｃｋ漂浮器法，以后...     

RNAi基因沉默技术及其在植物抗病性改良中应用的策略

RNAi (RNA interference) 是一种由dsRNA参与、对靶基因表达进行干扰或沉默的现象。由此发展起来的RNAi基因沉默技术已成为当今植物基因功能研究和遗传改良的一个重要手段。该技术已经在靶向病原物(真菌、细菌、病毒和线虫)基因沉默方面得到了广泛的应用,并且产生了一批抗病性增强的转基因植物。人工设计和合成的amiRNAs和ata siRNAs的成功研发加快了RNAi技术的应用。本文对RNAi基因沉默机制、RNAi技术研发进展及其在植物抗病性遗传改良中的应用进行综述,并对其应用策略进行探讨。     

牛蒡上发生的根结线虫种类鉴定

2007～2009年,对我国牛蒡主产区的牛蒡及其根际土壤中的植物寄生线虫进行调查研究,发现根结线虫危害最为严重。根据根结线虫的形态学特征和分子生物学检测方法对其进行鉴定,结果表明,我国牛蒡主产区根结线虫种类为北方根结线虫(Meloidogyne hapla)。     

玉米大斑病菌黑色素合成酶基因4HNR的克隆及其启动子预测

 4HNR (1,3,6,8-tetra-HN reductase) gene of melanin biosynthesis in Setosphaeria turcica had been cloned successfully by RT-PCR in this study. Both sequences of DNA and cDNA of 4HNR were 807 bp and there was no intron in the sequences. This gene only had single copy in genome through Southern blotting analysis. A 2 285 bp for the flanking sequence of 5' had been obtained and it had promoter structure through the software analysis.     

Complete genome characterization of Sugarcane yellow leaf virus from India: Evidence for RNA recombination

Yellow leaf (YL) caused by Sugarcane yellow leaf virus (SCYLV) has become a serious constraint for sugarcane production in different countries. Worldwide seven genotypes have been reported, with five based on complete and two based on partial genome characterization. We have previously reported the occurrence of three different SCYLV genotypes in India based on their partial genome sequences. A further four SCYLV isolates from sugarcane from Coimbatore (in India) were characterized after complete genome sequencing (~ 5,875 nt). These isolates (SCYLV-IND) exhibited amino acid (aa) sequence differences of 29.2–31.8, 28.1–34.4 and 30.7–33.4 % with REU, HAW-PER and BRA in partial ORF0 sequences, respectively. Similarly IND isolates have 21.4–23.7, 22.5–25.0 and 21.4–23.9 % aa sequence differences with REU, HAW-PER and BRA, respectively in partial ORF1. However, the difference was found to be least in ORF5. The genotype reported from China, CHN1 shared a very close relationship with IND isolates with minimum differences of 4.3–5.3 %, 4.8–5.8 % and 2.5–3.0 % in ORF0, 1 and 5 in aa sequences, respectively and 4.4–5.3 % in complete nucleotide sequences. Phylogenetic analyses showed a separate lineage for IND isolates. Evidence of recombination was found in ORF1 to ORF5 with the maximum number of sites occurring in ORF2. The high incidence of SCYLV recombination suggests that recombination plays an important role in SCYLV evolution.     

Classical and molecular genetics of <Emphasis Type="Italic">Bremia lactucae</Emphasis>, cause of lettuce downy mildew

Lettuce downy mildew caused by Bremia lactucae has long been a model for understanding biotrophic oomycete–plant interactions. Initial research involved physiological and cytological studies that have been reviewed earlier. This review provides an overview of the genetic and molecular analyses that have occurred in the past 25 years as well as perspectives on future directions. The interaction between B. lactucae and lettuce (Lactuca sativa) is determined by an extensively characterized gene-for-gene relationship. Resistance genes have been cloned from L. sativa that encode proteins similar to resistance proteins isolated from other plant species. Avirulence genes have yet to be cloned from B. lactucae, although candidate sequences have been identified on the basis of motifs present in secreted avirulence proteins characterized from other oomycetes. Bremia lactucae has a minimum of 7 or 8 chromosome pairs ranging in size from 3 to at least 8 Mb and a set of linear polymorphic molecules that range in size between 0.3 and 1.6 Mb and are inherited in a non-Mendelian manner. Several methods indicated the genome size of B. lactucae to be ca. 50 Mb, although this is probably an underestimate, comprising approximately equal fractions of highly repeated sequences, intermediate repeats, and low-copy sequences. The genome of B. lactucae still awaits sequencing. To date, several EST libraries have been sequenced to provide an incomplete view of the gene space. Bremia lactucae has yet to be transformed, but regulatory sequences from it form components of transformation vectors used for other oomycetes. Molecular technology has now advanced to the point where rapid progress is likely in determining the molecular basis of specificity, mating type, and fungicide insensitivity.     

昆虫基因组组装大小偏差的原因解析

为明确昆虫基因组组装大小产生偏差的原因,利用流式细胞术估测来自6目10科的21种常见农业昆虫的基因组大小,同时从动物基因组大小数据库收集和整理1 345个经流式细胞术估测的昆虫基因组大小信息,并从NCBI、GigaDB、DDBJ、i5k workspace@NAL、InsectBase和VectorBase等14个物种遗传信息数据网站获取536种昆虫的基因组组装信息进行比较分析。结果表明,收集的昆虫中有202种同时具有流式细胞术估测的基因组大小和基因组组装大小的信息,以更接近真实值的流式细胞术估测基因组大小为参照,比较发现其中42种昆虫的基因组组装大小偏大,98种昆虫的基因组组装大小偏小,而62种昆虫的基因组组装大小和经流式细胞术估测大小相似。基因组组装大小比经流式细胞术估测大小更大的物种,通过Wilcoxon秩和检验发现显著具有更多的重复序列,但与GC含量、contig N50及基因组测序和组装策略并无显著相关性。综合分析认为,在大多数情况下昆虫基因组组装大小更小,表明组装并不完整,但在重复序列占比较高的情况下,昆虫基因组的组装出现了冗余,导致组装大小更大。     

哈茨木霉突变体T-DNA插入位点侧翼序列的克隆及其插入序列分析

利用分子生物学技术扩增T-DNA插入突变体中T-DNA侧翼未知序列是克隆突变相关基因、研究T-DNA整合方式的基础,TAIL-PCR是扩增已知序列侧翼未知序列的有效方法,本研究根据哈茨木霉的基因组特点,引用和设计了12条随机AD引物,用这些引物分别与3条右边界嵌套特异引物进行组合对哈茨木霉突变子的T-DNA侧翼未知序列进行扩增,发现不同的随机引物的扩增效率差异很大,以AD5的扩增效率最高,能扩增出80%的T-DNA插入位点的侧翼序列。随机挑取哈茨木霉T-DNA插入突变子52个,选用引物AD5对T-DNA插入位点的侧翼序列进行TAIL-PCR扩增并分析扩增序列发现,在获得的42条侧翼序列中7条只含有质粒序列、33条对应着单一的侧翼序列T-DNA、另外2条序列相同。34条T-DNA侧翼边界序列中13条保存着完整的右边界序列,21条T-DNA边界序列存在一定程度的缺失现象,说明农杆菌转化哈茨木霉的过程中T-DNA右边界存在一定程度的剪切。该研究的进行对明确农杆菌介导的木霉遗传转化过程中T-DNA的整合方式具有一定意义,为研究农杆菌转化木霉的机理奠定了实验基础。研究也精细确定了33个不同突变株的T-DNA插入位置,为后续的基因功能研究奠定基础。     

截形叶螨抗哒螨灵种群抗性遗传方式的研究

通过对室内选育出的截形叶螨敏感品系和抗哒螨灵品系的杂交和回交试验表明:所测得的截形叶螨正交F1SR(SS♀×RR♂)和反交F1RS(RR♀×SS♂)代的显性度DSR和DRS分别为0.40和0.60,表明抗性由不完全显性基因控制;两个D值(DRS和DSR)95%置信限有重叠,并且经t检验,两个D值不存在显著差异(P0.05),证明截形叶螨对哒螨灵的抗性遗传为常染色体控制;回交F2代(BC1RS和BC1SR)的实际死亡率和期望值经χ2检验,无显著性差异(χ2=9.72,df=9,P0.05),表明抗性遗传由单基因控制。     

竹花叶病毒浙江麻竹分离物全基因组序列及siRNA分析

正已知有3种病毒可在自然条件下侵染竹类植物,即竹花叶病毒(bamboo mosaic virus,BaMV)~[1]、樱桃坏死锈斑驳病毒(cherry necrotic rusty mottle virus,CNRMV)和苹果茎沟病毒(apple stem grooving virus,ASGV)~[2,3]。其中,BaMV是最早在巴西的金竹(Bambusa vulgaris Cv.)和孝顺竹     

First report on the whole genome sequence of Pseudomonas cichorii strain JBC1 and comparison with other Pseudomonas species

Pseudomonas cichorii, a plant pathogen that infects a wide range of host plants worldwide, causes several diseases in economically important vegetable crops. Availability of the genome sequences of pathogens can greatly enhance research necessary for the advancement of disease management programmes. Despite the significance of P. cichorii, its whole genome sequence has not been reported previously. The genome sequence of P. cichorii JBC1, described for the first time in this study, is 5 986 012 bp with an average GC content of 58·1% and has 5174 coding sequences (CDS). The genes related to virulence, transport mechanisms, phytotoxic compounds, and secondary metabolite products were analysed and the genome was compared to eight other Pseudomonas species to understand the diversity at species level. Despite the high similarity (up to 80·85%), significant diversity was found among the different Pseudomonas species at the genome level. A comparison of JBC1 pathogenicity island (PAI) regions indicated that the P. viridiflava UASWS0038 PAI has more similarity than the P. syringae PAI region, and the analysis revealed significant divergence at PAI regions among the Pseudomonas species, providing an insight into the differences in host specificity and degree of virulence. In addition, JBC1 encodes antibiotic resistance and tolerance to heavy metals, and two different prophage segments were inserted at three different regions. The genome sequence of JBC1, which was deposited into the NCBI GenBank (accession no. CP007039 ), will be a reference sequence for other P. cichorii strains and a useful resource for further research.     

葡萄蚕豆萎蔫病毒中国分离物基因组全长序列分析

 葡萄蚕豆萎蔫病毒(grapevine fabavirus,GFabV)是近年报道的葡萄新病毒,与葡萄褪绿斑驳、皱缩等症状发生相关,严重影响葡萄长势。本研究采用小RNA测序结合Sanger测序方法分别对表现褪绿斑驳的‘贝达’和无症状的‘赤霞珠’葡萄中的GFabV分离物进行基因组全长序列测定,获得了GFabV分离物LN_BETA2和LN_CXZ的基因组全长序列,其RNA1基因组全长分别为5 824 bp和5 823 bp,RNA2基因组全长分别为3 132 bp和3 135 bp。LN_BETA2与LN_CXZ的RNA1编码的多聚蛋白核苷酸序列之间的同源性为81.7%,与其他GFabV分离物多聚蛋白核苷酸同源性分别为73.4%~99.5%和73.5%~82.5%;LN_BETA2和LN_CXZ的RNA2编码的多聚蛋白核苷酸同源性为83.8%,与其他GFabV分离物多聚蛋白核苷酸同源性为75.0%~83.3%和68.2%~98.6%。系统进化树分析结果显示,在RNA1基因组中,LN_BETA2划分在组3,LN_CXZ形成单独的分支。RNA2基因组中,LN_BETA2和LN_CXZ分别划分在组3和组5中。本研究首次报道了GFabV中国分离物基因组全长序列,可为今后GFabV生物学特性及致病性研究提供工作基础。