表达Harpin蛋白质Hpa1功能片段的转基因小麦对赤霉病的抗性

来自水稻黄单胞菌的harpin蛋白质Hpa1有促进植物生长、诱导植物抗病性的功能,Hpa1序列10~42氨基酸片段（Hpa1_10-42）的活性比全长分子高1.3~7.5倍。为研究Hpa1_10-42在转基因小麦体内表达以后对赤霉病的影响和评价转基因小麦抗病水平与应用潜力,对6个小麦转基因系进行了测定。结果显示,Hpa1_10-42在转基因系T₃~T₅代呈现稳定表达,用禾谷镰刀菌一个分离物进行接种以后,转基因系发生赤霉病的程度较非转基因小麦显著降低,且转基因系T₃~T₅代赤霉病降低的趋势一致。另外,转基因系小麦病害减轻的程度,与在非转基因小麦上使用杀菌剂的效果相当,表明Hpa1_10-42转基因表达对小麦赤霉病有防治作用。     

大豆RSC4抗病候选基因Glyma.14G204700的克隆及其生物信息学分析

为明确大豆抗大豆花叶病毒（soybean mosaic virus，SMV）SC4株系的基因位点R_SC4（resistance to SMV strain SC4，R_SC4）的候选基因Glyma.14G204700在大豆不同抗性品种中的序列结构特征和保守结构域，以齐黄1号、科丰1号、大白麻和南农1138-2共4个大豆品种为材料，通过基因克隆获得大豆Glyma.14G204700基因的cDNA全长序列，并采用生物信息学方法分析其序列特征和编码蛋白的理化特性及结构特征。结果表明，大豆Glyma.14G204700基因在4个大豆品种中的cDNA全长为4 719~4 776 bp，编码1 295~1 307个氨基酸，预测蛋白的分子量为148.38~149.33 kD，等电点为5.53~5.61，均为具较强亲水性的非分泌蛋白。Glyma.14G204700蛋白含有植物抗病基因家族蛋白的保守功能域——核苷酸结合域和富含亮氨酸重复结构域。该蛋白的二级结构主要由α-螺旋、无规则卷曲、延伸链和β-折叠组成，所占比例分别为59.45%~61.08%、28.65%~30.15%、8.11%~8.88%和1.61%~2.16%。启动子序列分析发现该基因含有脱落酸、低温及干旱等多种逆境胁迫响应元件。表明大豆R_SC4抗病候选基因Glyma.14G204700在大豆不同抗性品种中具有不同的等位变异，优异等位变异的鉴定和开发可为抗SMV大豆育种提供基础材料。     

四个李属坏死环斑病毒分离物的检测鉴定及CP基因序列分析

采用RT-PCR方法,从北京昌平、云南昆明、陕西西安和浙江海宁的樱桃和月季罹病叶片中检测到李属坏死环斑病毒,为分析我国李属坏死环斑病毒分子生态学特性,克隆了病毒分离物的CP基因并进行序列分析。结果显示,北京分离物的CP基因长675nt、编码224aa,而西安、昆明和海宁分离物的CP基因均为681nt、编码226aa。序列相似性分析表明,4个分离物的CP基因之间具 有很高的同源性, 各个分离物间的核苷酸同源性在94.8%~99.3%之间, 氨基酸同源性在94.2%~98.7%之间。序列比对与系统发生树的分析结果显示,北京分离物属于GroupⅡ组,昆明、西安和海宁分离物属于Group Ⅰ组。     

水稻黑条矮缩病毒外壳蛋白基因的克隆、原核表达及抗体制备

为探讨水稻黑条矮缩病毒（Rice black-streaked dwarf virus,RBSDV）的种群变异,采用RT-PCR方法从感染RBSDV山东分离物RBSDV-JN1的水稻中克隆该病毒的S10片段,并进行序列分析,进而构建包含RBSDV-JN1的CP基因的原核表达载体,导入大肠杆菌Escherichia coli BL21（DE3）诱导表达;并以表达的融合蛋白为抗原,制备病毒的多克隆抗体。结果显示：S10片段全长为1 801 bp,包含1个1 677 bp编码框,编码558个氨基酸的外壳蛋白（CP）;与GenBank已注册的19个RBSDV的CP基因序列相比较发现,病毒各分离物间核苷酸的序列相似性为90.5%~99.8%,氨基酸的序列相似性为95.9%~100.0%;地理位置较近的分离物间序列相似性较高,RBSDV种群分布呈现区域性差异。原核表达载体pET-RBSDV-CP经IPTG诱导,获得了分子量约为63 kD带有His标签的目的蛋白。用该蛋白制备的多克隆抗体经ELISA、Western blot和Dot-blot ELISA检测显示,效价为1：81 000,且具有良好的特异性。     

大豆干种子中大豆花叶病毒的RT-PCR检测

 应用改进的SDS-酚氯仿法,在沉淀RNA前先加入1/4体积无水乙醇、1/10体积5mol/L乙酸钾沉淀多糖,然后再用异丙醇沉淀RNA,成功地从大豆干种子中提取了大豆花叶病毒(SMV)总RNA;应用RT-PCR技术对大豆种子中携带的SMV进行了检测,同时以DAS-ELISA方法作比较,建立了能直接以大豆干种子为检测对象的SMV快速、灵敏、特异的RT-PCR检测技术。     

栽培密度对高产大豆根系生长及花荚形成的影响

为探明栽培密度对高产大豆根系生长和花荚形成的影响规律,田间试验设置21.0（D₁）、30.0（D₂）、48.0（D₃）×104株·hm^-2 3种栽培密度,研究了新大豆27号、新大豆8号在0~60 cm土层中根系生长、开花、结荚过程及产量的变化。结果表明,相对于D1,D2、D3密度下新大豆27号总根干重增加了23.68%、18.48%,总根长增加了9.33%、26.72%,新大豆8号总根干重增加了27.25%、19.71%,总根长增加了6.94%、30.80%,且增量以40~60 cm为主;增加至密度D₃时,新大豆27号开花和结荚高峰出现时间较D₁分别推迟了2、8 d,新大豆8号推迟了2、6 d,但二者单位面积的总花数、总荚数、总腔数和总粒数分别较D₁提高了51.92%、10.29%、7.66%、23.05%和49.47%、31.91%、35.40%、16.11%;总花数与始荚期根干重、根长度呈正相关关系,新大豆27号R²值分别为0.8477^*、0.9106^*,新大豆8号R²值分别为0.7531、0.9993^**;总腔数与始粒期根干重、根长度呈正相关关系,新大豆27号R²值分别为0.6954、0.9837^**,新大豆8号R²值分别为0.7902^*、0.9277^*;且根长度与总花数、总腔数的关系比根干重更密切。合理密植显著提高大豆籽粒产量,是密植促进了大豆根系生长,增加了单位面积总根长和深层根量,进而增加了单位面积花数、荚数、腔数和粒数的结果。     

臭氧胁迫对大豆根系激素含量和活性氧代谢的影响

以大豆（Glycine max.）为试验材料,探讨臭氧(O₃)浓度升高〔(80±10) nL·L^-1)对大豆根系内源激素含量及活性氧代谢系统的影响。研究表明：在大豆整个生育期内,O₃胁迫处理下的大豆根系脱落酸（ABA）含量、玉米素核苷（ZR）含量与对照相比显著（P<0.05）升高,生长素（IAA）含量显著（P<0.05）降低,内源激素间平衡改变,IAA/ABA、AR/ABA及(IAA+ZR)/ABA比值显著（P<0.05）降低,超氧阴离子产生速率、MDA含量、POD活性和脯氨酸含量与对照相比显著（P<0.05 ）升高,SOD和CAT活性显著（P<0.05）降低。O₃胁迫在一定程度上可以改变内源激素的含量及激素间的平衡比例,提高大豆的抗氧化能力,但是,长时间的胁迫作用将导致抗氧化系统氧化损伤,从而使膜脂过氧化程度加重,对大豆表现为伤害效应。     

淡色生赤壳菌Bo-1菌株拮抗物质的分离纯化、解析及活性分析

从浙江省金华市郊水稻内生菌中筛选到1株拮抗水稻白叶枯病菌（Xanthomonas oryzae pv.oryzae,Xoo） P6生理小种的淡色生赤壳菌Bionectria ochroleuca Bo-1菌株。该菌株的菌体和发酵液对Xoo P6生理小种均表现出较强的拮抗能力。以P6生理小种作为指示菌进行活性跟踪,通过分步萃取、硅胶柱层析、高效液相色谱等,分离纯化获得拮抗活性物质结晶;对所获得的结晶物进行核磁共振氢谱分析和质谱分析,通过解析与比对,该物质分子量为344.0835,分子式为C₁₈H₁₆O₇,鉴定为松萝酸（usnic acid）。将纯化的松萝酸以20%叶枯唑（bismerthiazol）和3%中生菌素（zhongshengmycin）为对照,采用96孔板法测定对Xoo P6生理小种的抑菌活性,最低抑制浓度为200 μg/mL,表明松萝酸具有较强的抑菌活性,但较中生菌素弱,与20%叶枯唑效果相近。     

大豆对灰斑病菌15号小种的抗病基因定位及标记检测

为明确大豆对灰斑病菌15号小种的抗性位点,以大豆抗病品种垦丰16、感病品种绥农10及其杂交F₂、F₃代群体为试验材料,在接种鉴定的基础上,运用SSR标记技术及分离群体组群分析法(BSA法)对垦丰16抗病基因进行了定位,并应用108份大豆新品系对标记进行了符合性检测。结果表明,垦丰16对15号小种的抗性受1对显性基因控制,抗病基因位于大豆染色体组的J连锁群上,将该基因定名为Rcs15。用Mapmaker/Exp 3.0 b进行连锁分析,获得了5个与抗病基因紧密连锁的SSR标记:Satt 529、Satt 431、Sat_151、Satt 547和Sat_224,标记与抗病基因间的排列顺序和遗传距离为Sat_151-10.7 cM-Satt 529-18.5 cM- Rcs15-6.7 cM-Satt 547-7.8 cM-Sat_224-10.7 cM-Satt 431。标记符合性检测结果显示,Satt 547和Sat_224的检测准确率达到85%以上,可用于分子标记辅助选择育种和抗源筛选。     

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more sensitive. This effect is probably due to the ability of AFAL to interact with the bacterial cell wall where we observed that AFAL induced macroscopic change. The maximum bacterial growth inhibition was approximately 78% when incubated with Gram-negative strains, and as high as 92% percent for the Gram-positive one. The antibacterial effect of flavonoids (rutin, quercetin and morin) was also observed using low concentrations against both bacterial strains. Prior incubation of both with AFAL at high concentrations increases the inhibitory effect of flavonoids on bacterial growth. The potential use of AFAL as a control agent against the root-knot nematode Meloidogyne incognita was investigated as well, showing anti-nematode properties involving both egg hatching and motility. In the juvenile second-stage, AFAL showed reduction in larval mobility when measured against a control group. The results suggest that AFAL is effective against M. incognita and could be used as a component of integrated pest management programs. These data also suggest that lectins probably play a role in plant defense not only against invertebrate phytopathogens, herbivores and fungi but also against bacteria.     

菜豆普通细菌性疫病菌在土壤和植株残体中的越冬能力

为评估菜豆普通细菌性疫病菌地毯草黄单胞杆菌菜豆致病变种或褐色黄单胞菌褐色亚种在土壤及植物残体中的越冬能力,对采自黑龙江、内蒙古、山西、河北及新疆的18块菜豆生产田的20份土壤及14份植物残体样品进行病原菌分离和鉴定。在MT选择性培养基上有12个土壤样品和13个植株残体样品提取液产生典型的类似黄单胞菌菌落。选取29个分离物进行致病性测定,有27个分离物对菜豆品种"英国红"致病。利用地毯草黄单胞杆菌菜豆致病变种和褐色黄单胞菌褐色亚种的特异性引物X4c/X4e及褐色黄单胞菌褐色亚种特异性引物Xf1/Xf2对29个分离物进行多重PCR检测,其中17个分离物为地毯草黄单胞杆菌菜豆致病变种,10个分离物为褐色黄单胞菌褐色亚种。结果表明,菜豆普通细菌性疫病菌可以在黑龙江、内蒙古、山西、河北的一些菜豆种植区的土壤及植株残体中越冬存活。     

枯草芽胞杆菌对离体荔枝果实霜疫霉病、炭疽病的防治效果

为了进一步明确从土壤中筛选得到的拮抗细菌OR-1、OR-2、OR-3、ON-6的分类地位和生防效果,采用形态学观察、理化特性结合分子生物学方法,鉴定这4株细菌皆为枯草芽胞杆菌Bacillussubitilis.拮抗菌对荔枝霜疫霉菌、炭疽菌菌丝生长均具有明显抑制作用,ON-6菌株的发酵液对菌丝生长的抑制率最高,分别为92.34％和70.36％,其次是OR-1菌株.拮抗菌处理组褐变指数均小于对照组以及杀菌剂处理组,且差异达显著水平(P＜0.05),OR-1菌株防褐变效果最好.常温下,拮抗菌发酵液对离体鲜果病害防治效果优于对照组和杀菌剂处理组,其中ON-6菌株的防治效果最佳;4℃低温储藏处理40天后,拮抗菌发酵液处理组的防治效果高于对照和杀菌剂处理组,且差异显著(P＜0.05),以ON-6和ON-3菌株的防治效果最佳.表明拮抗菌发酵液对荔枝鲜果上的霜疫霉菌和炭疽病菌有一定的抑制作用.     

芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框（GenBank登录号为JQ309841）,编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。     

进境油菜籽中黑胫病菌和茎基溃疡病菌的检测

为准确鉴定从进境澳大利亚油菜籽样品中分离的真菌分离物,利用形态学特征、PCR检测、序列分析以及致病性测试等方法对分离物6382-43和6382-51进行了鉴定试验。结果表明,分离物6382-43的形态特征和油菜茎基溃疡病菌Leptosphaeria maculans相似,菌丝生长较慢,菌落边缘不规则,不产生色素。油菜茎基溃疡病菌特异性引物LmacF/LmacR检测为PCR阳性;ITS区序列和油菜茎基溃疡病菌的序列相似性为99.8%;接种幼嫩油菜子叶产生油菜茎基溃疡病的典型症状。分离物6382-51的形态特征和油菜黑胫病菌L.biglobosa相似,菌丝生长较快,菌落边缘规则,产生色素;油菜黑胫病菌特异性引物LbigF/LmacR检测为PCR阳性;ITS区序列和油菜黑胫病菌的序列相似性为100%;接种幼嫩油菜子叶产生油菜黑胫病的典型症状。根据分离物的形态特征、PCR检测结果、序列分析以及致病性测试结果,将进境澳大利亚油菜籽样品中的真菌分离物6382-43和6382-51分别鉴定为油菜茎基溃疡病菌Leptosphaeria maculans和油菜黑胫病菌L.biglobosa。     

Agrobacterium-mediated transformation efficiency is altered in a novel rice bacterial blight resistance cultivar and is influenced by environmental temperature

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium. The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana. Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium-mediated transformation (OsMPKs, OsVIP1s and OsPR1s) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium-mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.     

枣镰翅小卷蛾成虫的寄主趋向和产卵选择

采用固相微萃取(SPME)和GC-MS分析研究木枣和酸枣挥发物成分,并测试枣镰翅小卷蛾触角电位(EAG)反应、寄主趋向和产卵选择,以探明枣镰翅小卷蛾的寄主选择机制。结果显示,在萌芽期,木枣和酸枣嫩叶的挥发成分均为罗勒烯、2-甲基-2-菠烯、α-法呢烯和邻苯二甲酸二丁酯4种,但相对含量稍有不同。枣镰翅小卷蛾成虫对木枣和酸枣2种寄主都有强烈的EAG反应,且同一寄主上雌蛾的EAG反应值极显著地高于雄蛾,其EAG值是雄蛾的3.3倍。枣镰翅小卷蛾对木枣表现强烈的趋向反应,而对酸枣的趋向不明显,且雌虫的趋向反应显著高于雄虫。木枣上的产卵量显著高于酸枣,且木枣枣吊上的单雌产卵量为307.9粒,极显著地高于酸枣枣吊上的产卵量(182.9粒)。研究表明,枣镰翅小卷蛾雌蛾在寄主选择中起主导作用,木枣是其嗜好寄主。     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

河北和山东鸭梨果实上链格孢菌鉴定

为了查清造成鸭梨果实储藏期黑斑病的链格孢Alternaria的种类,从其产孢表型、分生孢子特征和分子生物学等方面对分离的链格孢菌进行了研究.经对分离到的188支Alternaria菌株的形态学鉴定,共确定了3个种,即链格孢Alternaria alternata、细极链格孢A.tenuissima和侵染链格孢A.infectoria,比例分别为41.0%、54.8%和1.6%.分子生物学研究结果表明,Altemaria大孢子种彼此间及大孢子种与小孢子种之间可以根据ITS和gpd序列差异明确区分;而供试的多数小孢子种在ITS和gpd序列上差异很小,无法区分.