编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交（suppression subtractive hybridization,SSH）的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

一个编码锌指蛋白的大豆疫霉基因Ps—zfA1的克隆与转录分析

对大豆疫霉野生菌株与紫外线诱导的卵孢子缺失突变株进行差异表达基冈分析,筛选到一个在大豆疫霉卵孢子形成过程中特异表达、编码CCHC型锌指蛋白的cDNA片段.克隆了该基因的全长序列,命名为PszfA1.Southern杂交结果显示,Ps-zfA1在大豆疫霉基凶组中只有2个拷贝.系统发育分析表明,Ps-zfA1与三角褐指藻Phaeodactylum tricornutum的锌指蛋白的序列同源性最高,且该基因编码的氨基酸序列具有一个CCHC型锌指蛋白典型的保守结构域.时实定量RT-PCR分析表明,该基因在大豆疫霉卵孢子形成过程中特异表达,且表达量随着产孢培养的时间延长而升高.     

大豆疫霉根腐病抗源筛选

由大豆疫霉菌引起的大豆疫霉根腐病是大豆生产的重要病害,该病已在我国大豆主要产区发生,并在局部地区造成较大产量损失。利用抗病品种是防治大豆疫霉根腐病最有效的方法。本研究目的是筛选大豆疫霉根腐病抗源,为病害防治和抗病品种的选育提供参考。用下胚轴创伤接种方法对120个栽培大豆品种（系）进行接种,鉴定其对10个具有不同毒力大豆疫霉菌菌株的抗性。有110个品种（系）分别抗1～10个大豆疫霉菌菌株,其中以河南大豆品种（系）对疫霉菌的抗性最丰富,安徽、湖北和山西大豆品种（系）也具有抗性多样性。120个大豆品种（系）对10个大豆疫霉菌菌株共产生57个反应型,有4个抗性反应型分别与单个抗病基因的反应型一致,有7个抗性反应型与2个已知基因组合的反应型相同,其他抗性反应型为新的类型。一些大豆品种（系）中可能存在有效的抗大豆疫霉根腐病新基因。     

Arthrobacter nitroguajacolicus腈水解酶基因的克隆和表达

腈水解酶是一类能将腈类化合物催化生成酸的氰基水解酶。目前已有多个菌种的腈水解酶基因序列被报道,如敏捷食酸菌Acidovoraxfacilis,粪产碱菌Alcaligenesfaecalis,睾丸丛毛单胞菌Comamonastestoteroni,肺炎克雷伯菌Klebsiellapneumoniae,假单胞菌Pseudomonas属菌株,红球菌Rhodococcus属菌株,但节杆菌属菌株Arthrobacternitroguajacolicus的腈水解酶基因序列尚未见报道。经由野生型酶的分离纯化,基因文库筛选及侧翼序列扩增等步骤,克隆得到该菌株的腈水解酶基因,从而为进一步研究该酶的特性及构建用于工业生产的重组菌打下基础。     

腈水解酶基因bxn的结构与功能研究

前期研究发现腈水解酶基因bxnRD127表达产物无功能,测序证明该基因结构中存在4处突变。本文对4处突变逐点进行结构与功能回复突变研究,发现其中2处突变能使基因表达产物丧失功能,1处使基因表达产物活性降低,1处对基因表达产物活性基本无影响。进一步将完全回复突变的bxn基因转化烟草,获得的转基因烟草具有抗溴苯腈除草剂特性,结果证明结构中的3处突变与活性中心功能有关。     

大豆种质对疫霉根腐病抗性特点研究

对1027份中国和国外引进的大豆种质进行了大豆疫霉(Phytophthora sojae)根腐病的抗病性鉴定评价。结果表明,中国大豆种质的抗病性高于国外引进种质;中国南方的大豆种质抗病性较北方种质强,长江流域大豆中抗病种质比率最高,其次为黄淮海流域种质,而东北地区抗病种质较少;不同省份大豆种质的总体抗病性差异明显;育成品系的抗性好于改良品种和农家种,但不同省份来源的农家种、品系和品种抗性存在差异,黑龙江材料抗病性最低,这也是该省大豆疫霉根腐病严重发生的重要原因之一;在大豆籽粒脐色为黄色和褐色的材料中,抗病种质较多。     

抗大豆疫霉根腐病野生大豆资源的初步筛选

由大豆疫霉菌引起的大豆疫霉根腐病是严重影响大豆生产的毁灭性病害之一.防治该病唯一经济、有效和环境安全的方法是利用抗病品种.本研究对野生大豆资源进行抗大豆疫霉根腐病初步筛选,以期探讨野生大豆的抗性水平、分布和获得抗性野生大豆资源.通过苗期接种大豆疫霉菌对412份野生大豆资源进行抗病性鉴定,有13.4%的资源抗大豆疫霉根腐病,15.3%的资源表现为中间反应类型.对野生大豆资源的来源分析表明,抗大豆疫霉根腐病野生大豆资源在我国分布广泛,其中安徽省野生大豆资源抗性最丰富.     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

Virulence of Phytophthora sojae in the Pampeana Subregion of Argentina from 1998 to 2004

Phytophthora root rot is one of the most serious diseases of soybeans in Argentina. Surveys of commercial fields and trial plots of soybean were conducted throughout the northern Pampeana subregion (Argentina) between 1998 and 2004. A total of 193 isolates of Phytophthora sojae were collected and classified into races or virulence formulae. Among the 173 isolates tested on 8 differentials, 42 different pathotypes were detected, including 18 described races. Races 1, 4, 5, 7, 9, 13, 23 and 24 were found in both plants and soils, whereas races 2, 3, 6, 8, 11, 14, 15, 17, 43 and 44 were only isolated from plants. An additional 19 pathotypes were described from 20 isolates tested in Canada on the expanded set of 14 differential cultivars. Currently, all Rps genes/alleles associated with resistance have been defeated, indicating an increased complexity of virulence within the P. sojae populations in the region. The great increase in virulence complexity found in this study is most likely a result of a long period of continuous production of soybean cultivars with Rps genes and the extensive adoption of the no‐tillage system.     

大豆疫霉菌的分离与鉴定

采用叶碟诱捕法从2007年进口的美国大豆携带的土壤和2006年从黑龙江感病大豆田采集的土壤中分离出2株疫霉菌菌株,并对病原菌进行了形态特征、致病性、分子检测。结果表明:形态观察为疫霉属真菌;接种大豆后出现典型的大豆疫病症状;采用大豆疫霉的特异性引物PCR检测,2个菌株均能扩增出分子量为330 bp的特异性条带。结合形态、致病性测定和分子检测,2株病菌鉴定为大豆疫霉菌(Phytophthora sojaeKauf-mann et Gerdemann)。     

植物提取物对大豆疫霉菌抗菌活性的初步研究

采用Paper Disk法测定了云南省200种植物的提取物对大豆疫霉(Phytophthora sojae)的抑菌活性。结果表明,光叶合欢(Albizia lucidior)、白钩藤(Uncaria sessilifructus)及黄豆树(Albizia procera)的提取物在浓度为10μg/μL时有较好的抗菌活性,而在2.5μg/μL时只有黄豆树提取物显示出抗菌活性。并从黄豆树中分离出其抗菌的主要活性成分3-O-[α-L-arabinopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-2-acetamidO-2-deoxy-β-D-glu-copyranosyl]echinocystic acid。     

Pathotype and SSR variation in Phytophthora sojae from the Argentinean Pampas

Genetic variation in Phytophthora sojae was compared between northern (NPS) and southern (SPS) Pampeana sub-regions, in the core soya bean region of Argentina. Ninety-three isolates from plants and soil were evaluated in the present study. The pathotype for each isolate was determined using the hypocotyl technique on a set of eight differentials. For neutral genetic diversity evaluation, eight SSRs markers were utilized. Thirty pathotypes were determined, including 19 in SPS and 23 in NPS. The number of isolates for each locality and region was unevenly distributed. Pathotype variation was high for P. sojae samples compared among fields and within isolates from single fields. SSR variation studies resulted in an elevated haplotype composition, partitioned at all, but the regional levels. P. sojae populations from Argentina have evolved towards a complex epidemiological scenario, with no evidence of geographic correlation at the pathotypic nor the neutral genetic level. It is likely that future works aiming at elucidating the evolution of pathogenesis in this species will need to include non-neutral markers, specifically associated to avr loci. The results of the present work are considered a useful contribution to the adequate and sustainable management of this pathogen in the main soya bean area of Argentina.