Study on histopathology of nuclear polyhedrosis virus ofZethenia rufescentaria Motsch

IntroductionZOtheni8ruf8scent8rtaMotsch.isoneofthemainpestsinlarchpIantationinNortheastChina.Inrecentyears,thepestcauseddisasterintheforestareaofHeilongjiangProvince.TheneedlesofIarchinsomeareawerebeeneatenupcompIetelyandthegrowthoflarchpIantationwasseriousIyaffected.Thenu-clearpoIyhedrosisvirusofZruf8scent8rinMotsch(ZrNPV)wasobtainedfromtheIarvaeofZruf6s-cent8finMotsch,in1991.ZrNPVwasmainpathogenOftheIarvaeofZrufescentBfisMotsch.TOxicitymeasuringindoorsandinvestigationinfieldprov…     

Effect ofCronartium quercuum on cell chromosome of Xingkai Lake pine

The microscopical observation and karyoltype analysis of embryo root cells of healthy Xingkai Lake pine and Xingkai Lake pine infected byCronartium quercuum were conducted. The results showed that the dividing phase of embryo root cells decreased and the viscidity of cells in dividing phase increased when Xingkai Lake pine was infected byC. quercuum. The karyoltype changed from the contract 4A to 4B, but the number of cell chromosome and karyoltype component did not change. (Responsible Editor: Zhu Hong)     

白蜡虫蜡酯合酶在昆虫细胞Sf9中的表达

[目的]本研究欲利用Bac-to-Bac表达系统,在Sf9细胞中表达WS.[方法]将WS编码序列克隆到质粒pFastBac^TMHT B后,转化大肠杆菌DH10Bac^TM,经抗性和蓝白斑筛选,获得重组杆粒rBacmid/EpelWS.将rBacmid/EpelWS转染Sf9细胞.[结果]免疫荧光标记检测表明:WS在受感染细胞的细胞质中表达.[结论]本研究成功表达了白蜡虫WS,为白蜡虫WS的生化特性研究奠定了基础.     

miR396在落叶松体细胞胚胎中的功能研究

[目的]探讨miR396在落叶松体细胞胚胎发生中的功能.以期该研究能为解决体胚发育中子叶畸形胚与生根率低的问题提供新的视角,同时也为进一步揭示体胚发育的分子调控网络做基础铺垫.[方法]构建pre-miR396超表达载体,通过农杆菌介导侵染,将pre-miR396转入落叶松胚性细胞中,筛选稳定抗性细胞系.在DNA与RNA水平上,对抗性细胞系进行的鉴定、检测,同时统计比对其与野生细胞系间发芽率、叶片数目以及生根率的差异.[结果]在抗性细胞系基因组中,扩增出HTP与miR396的特异性片段,且无农杆菌Virg基因片段.RNA表达水平上,抗性系pre-miR396与miR396表达皆增加,而靶基因LaGRFs的表达量下调.统计表明抗性系胚的发芽率、生根率、叶片数目畸形率分别为70.60%、0.60%、37%,而野生型分别为92.50%、10.55%、4%,抗性系胚的发芽率、生根率显著降低(Sig.=0.000),叶片数目畸形率显著增加(Sig.=0.000).[结论]miR396在落叶松体细胞胚胎发生过程中,可能通过负调节靶基因LaGRFs的表达或者通过LaGRFs影响与它相互作用的基因,参与体胚子叶原基细胞代谢,从而影响了子叶与叶片的发育;通过负调节LaGRFs或者其它与根部发育相关的靶基因,影响了根端分生区细胞的活动,参与调控根的发育.     

Study on Cytoplasmic polyhedrosis virus onDendrolimus superans

A strain of Cytoplasmic polyhedrosis virus (CPV) was separated from the infected larva during the research of integrated pest management ofDendrolimus superans. The morphology, bioassay, histopathology and field-test for this CPV were studied. The size of CPV is 0.16 μm×1.57 μm and the virion is 16.0 nm×58.1 nm. The L_C50 to the 3rd and 5th instar larva ofDendrolimus superans were 2.81×10⁴ PIB/mL and 7.17×10⁴ PIB/mL respectively. The polyhedrosis were formed after midgut of larva were infected for 72 h. A large amount of polyhedrosis was formed after 144 h. The mortality was more than 82% and average mortality was 84.62% when using 1.17×10⁸ PIB/mL virus suspension to control the pest in field test. The project was supported by the Natural Sciences Foundation of Heilongjiang Province. (Responsible Editor: Chai Ruihai)     

新疆巩留县杏果实斑点病病原菌鉴定

[目的]鉴定引起新疆巩留县杏树果实斑点病病原菌,研究造成杏树果实病害的原因,为当地杏树防治工作提供依据。[方法]采用常规组织分离法分离得到罹病杏真菌菌株,利用传统形态学观察和分子系统学分析相结合的方式对所分离出菌株进行分类鉴定及其致病性检测。[结果]杏果病斑处的病原菌在显微镜下观察到分生孢子形态与经PDA培养基培养后观察到分生孢子形态均与Thyrostroma carpophilum（Lév.） B.Sutton所产生分生孢子一致;将分离获得的3株真菌的rDNA-ITS片段测序后与NCBI参考序列进行多重序列比对的结果显示,其序列与T.carpophilum一致性为100%;在基于ITS基因序列构建的系统发育树中,3株菌与T.carpophilum聚在同一分支。在接种了T.carpophilum后,杏果实和叶片均产生明显病斑并且从其所产生的病斑上再次分离到所接菌,满足柯赫氏法则。[结论]从新疆巩留县杏果实病斑处分离获得的3株真菌,经鉴定为引起杏穿孔病的病原菌T.carpophilum。这是该菌首次在该地区发现并报道。     

木麻黄青枯病菌的分离及强致病菌株的筛选

[目的]筛选出强致病菌株用于木麻黄抗病育种研究工作。[方法]在广东沿海木麻黄青枯病发病区采集病根,开展病原菌两种不同分离方法的比较研究,对分离出的31个病株进行16s rRNA测序鉴定及致病性测定。[结果]采用稀释分离法及根系溢出法在TTC培养基上共分离出了31个病原菌株,根系溢出法操作简便,杂菌含量低,分离率在60%左右,可作为常规稀释分离法的补充。31个菌株进行分子鉴定,只有22个菌株扩增出了特异性条带,经测序比对确定这22个菌株为青枯菌。青枯菌株致病性测定结果显示菌株致病性在无性系间、菌株间及菌株与无性系间的交互作用均具有极显著差异(P0.01),不同接种方法间菌株致病性相关系数值较小,介于0.496 6~0.731 0之间,即室内水培接种与小苗盆栽接种不存在密切的直线相关关系。[结论]综合选择在不同无性系及不同接种方法中均具有较强致病性的GL-2、H、M、TC-1、F、Q菌株作为下一步木麻黄种质资源抗性鉴定及抗病育种研究试验菌株。     

Neuroprotective cadinane sesquiterpenes from the resinous exudates of Commiphora myrrha

Three new cadinane sesquiterpenes, commiterpenes A−C (1−3), were isolated from the resinous exudates of Commiphora myrrha. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HRESIMS, 1D and 2D NMR). All the isolated sesquiterpenes showed neuroprotective effects against MPP⁺-induced neuronal cell death in SH-SY5Y cells.     

大花蕙兰转齿兰环斑病毒外壳蛋白基因及检测

[目的]通过植物转基因技术获得抗病毒大花蕙兰种质资源,优化转化体系和鉴定方法.[方法]本研究克隆了齿兰环斑病毒外壳蛋白基因,并构建了该基因的pBI121表达载体,用根癌农杆菌介导法转化大花蕙兰,尝试以巢式PCR方法检测转基因再生植株.[结果]优化了大花蕙兰遗传转化体系,建立了利用巢式PCR技术检测转基因大花蕙兰植株的方法,获得了32株转基因株(系).[结论]优化了以类原球茎为外植体的农杆菌介导转化大花蕙兰的方法,确定以5%~10%类原球茎存活时的抗生素(卡那霉素)浓度为筛选浓度;获得了转ORSV CP基因大花蕙兰植株;对大花蕙兰转基因植株检测时,巢式PCR较普通PCR更灵敏、准确.     

平榛ChWRKY28基因克隆及表达模式分析

[目的]研究平榛ChWRKY28基因序列特征及其在不同非生物胁迫下的表达规律.[方法]以平榛为试材,采用RACE-PCR方法进行基因克隆;利用实时荧光定量PCR方法检测基因在不同组织及不同非生物胁迫下的表达模式.[结果]表明:克隆得到的WRKY基因,全长1 342 bp,基因内部包含1个长963 bp的完整开放阅读框,编码320个氨基酸残基,命名为ChWRKY28.构建的系统发育树表明:该序列与拟南芥AtWRKY28及杨树PtrWRKY93的关系最近,相似性分别为49%和60%.基因表达分析表明:ChWRKY28在雄花序、雌花芽及茎中均有表达,但在茎部(皮)中的表达量高于雄花序和雌花芽中的表达量,具有组织表达特异性;低温、干旱及盐胁迫均能诱导ChWRKY28基因的表达,但受诱导程度存在差异.亚细胞定位分析结果表明:ChWRKY28蛋白分布在细胞核内,是一个核蛋白.[结论]推测ChWRKY28基因可能参与植物响应非生物胁迫的信号转导过程.     

Irbic acid, a dicaffeoylquinic acid derivative from Centella asiatica cell cultures

3,5-O-dicaffeoyl-4-O-malonilquinic acid (1) (irbic acid) has been isolated for the first time from cell cultures of Centella asiatica and till now it has never been reported to be present in the intact plant. Evidence of its structure was obtained by spectroscopic analyses (MS/NMR). Besides 1, cell cultures produce also the known 3,5-O-dicaffeoylquinic acid, chlorogenic acid, and the triferulic acid 2 (4-O-8′/4′-O-8″-didehydrotriferulic acid). Biological activities were evaluated for compound 1, which showed to have a strong radical scavenging capacity, together with a high inhibitory activity on collagenase. This suggests a possible utilization of this substance as a topical agent to reduce the skin ageing process.     

Differences in aphid transmissibility and translocation between PVYN and PVYO isolates

The aphid transmissibility and within-plant movement of two PVY isolates belonging to PVY^O and PVY^N strains were studied. Potato plants were infected by Myzus persicae either 1 week after emergence or 30 or 45 days after emergence. Infected leaves, petioles of infected leaves, and different segments of the stems of plants infected 1 week after emergence were ELISA-tested 7, 11 and 14 days after infection. Segments of the plants infected 30 or 45 days after emergence were ELISA tested 30 days after infection. Normalised ELISA values were used for statistical analyses. Virus infection of progeny tubers was assessed by post-harvest inspection. The ability of 15 aphid species to transmit the virus isolates was also examined. There was no difference in the aphid transmissibility of the two isolates 7 or 11 days after exposure to M. persicae. However, normalised ELISA values were significantly affected by both isolate and sampling date 11 and 14 days after M. persicae transmission. The level of infection of different stem segments was significantly affected by both isolate and growth stage of the plant at infection. The proportions of infected leaves, stem segments and progeny tubers were all significantly higher for PVY^N than for PVY^O. PVY^N was more effectively transmitted by a number of different aphid species than was PVY^O. Newly described vectors of PVY^N include Schizaphis graminum, Aphis fabae cirsiacanthoides, Aphis spiraecola, Myzus ligustri, Aphis fabae, Aphis spiraephaga, Myzus cerasi, Macrosiphum rosae, Diuraphis noxia, Aphis pomi and Rhopalosiphum padi, while those of PVY^O include Aphis fabae cirsiacanthoides, Myzus cerasi and Myzus ligustri.     

Cytotoxicity of chemical constituents from the stems of Dalbergia parviflora

Three new compounds, dalberpene (1), dalparvinene B (2) and dalparvone B (3), as well as 12 known compounds were isolated from the heartwood of Dalbergia parviflora. Isoflavanone 13 showed strong cytotoxicity against KB, MCF-7 and NCI-H187 cell lines with IC₅₀ values ranging from 3.5 to 5.4 μg/mL and it was inactive against normal cells.     

麻疯树BRI1基因的鉴定及其在不同发育时期花蕾中的表达分析

[目的]研究油菜素内酯受体BRI1的同源基因(JcBRI1)在麻疯树花发育过程中的作用。[方法]利用RT-PCR技术克隆JcBRI1基因的CDS,以pET-30a(+)质粒为框架构建原核表达载体转化至大肠杆菌进行诱导表达,随后利用LC-MS/MS对表达产物进行质谱鉴定,并以氨基酸序列为基础分析JcBRI1蛋白质结构。利用荧光定量PCR分析麻疯树JcBRI1基因在雌雄花发育的关键时期的表达水平,初步确定其在麻疯树花发育过程中的表达模式。[结果]克隆获得了JcBRI1基因的CDS,长度为3 591 bp。SDS-PAGE检测结果表明:JcBRI1基因在大肠杆菌BL21(DE3)和Rosetta(DE3)中均能表达,但在低温环境的表达量更高。原核表达产物的LC-MS/MS鉴定结果表明:JcBRI1氨基酸序列与预期的一致,JcBRI1基因的表达产物为麻疯树BRI1蛋白。对JcBRI1在麻疯树花器官不同发育时期的表达水平进行检测分析,结果表明:JcBRI1基因的表达量在雌花发育的第一个时期即大孢子母细胞时期就达到了最高值,在随后几个时期持续下调;然而,其在雄花各个时期的表达量并无明显差异,表明其很有可能参与了麻疯树雌花大孢子母细胞发育过程。[结论]麻疯树JcBRI1基因为LRR-RLKs家族成员BRI1的同源基因,很有可能参与麻疯树雌花大孢子母细胞的发育过程,至于JcBRI1在麻疯树大孢子母细胞发育过程中的具体作用机制还需进一步研究。     

neo-Clerodane diterpenes from Ajuga ciliata Bunge and their neuroprotective activities

Three new (1–3) and three known (4–6) neo-clerodane diterpenes have been isolated from the whole plants of Ajuga ciliata Bunge. The structures of the new compounds were elucidated as (12S)-1β,6α,19-triacetoxy-18-chloro-4α,12-dihydroxy-neo-clerod-13-en-15,16-olide (1), (12S,2′S)-12,19-diacetoxy-18-chloro-4α,6α-dihydroxy-1β-(2-methylbutanoyloxy)-neo-clerod-13-en-15,16-olide (2), and (12S)-6α,18,19-triacetoxy-4α,12-dihydroxy-1β-tigloyloxy-neo-clerod-13-en-15,16-olide (3), on the basis of spectroscopic data analysis. All the diterpenes were evaluated for the neuroprotective effects against MPP⁺-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells and compounds 2–5 exhibited moderate neuroprotective effects.     

早竹丛枝病的调查及病原菌的分子鉴定

[目的]对早竹丛枝病进行调查及病原菌分子鉴定,为早竹丛枝病的病害诊断和防治提供科学依据。[方法]采用单株水平和单枝盘水平的2种病害分级标准对早竹丛枝病进行调查。使用植原体16S rDNA和真菌rDNA-ITS序列的特异性PCR引物,对早竹丛枝病的病原菌进行分子鉴定。[结果]调查的6块样地早竹丛枝病的平均发病率为18.59%,平均病情指数为6.67。感病的早竹DNA样品能够扩增出真菌的rDNA-ITS序列,而不能够扩增出植原体的16S rDNA序列;扩增出的序列与报道的竹针孢座囊菌的序列同源性达到99.00%,与其它真菌的序列同源性最高仅为94.00%。[结论]浙江省德清县早竹丛枝病的病原菌为竹针孢座囊菌。     

卵巢线虫(Contortylenchus genitalicola)在中国松褐天牛上的分布与作用研究

[目的]日本学者Kosaka与Ogura发现松褐天牛成虫除携带松材线虫外,雌成虫卵巢内还携带有另一种线虫,他们将其命名为卵巢线虫,并认为这种线虫是松褐天牛成虫的寄生性线虫。我国对该种线虫的研究迄今未见报道。为了证明这种线虫在我国是否存在和分布,开展了本项研究。[方法]分期分批捕获刚羽化的松褐天牛雌雄成虫,采用解剖松褐天牛成虫松树木质部及感病的松褐天牛幼虫等方法,调查卵巢线虫的存在与分布。[结果]通过调查研究,发现我国的松褐天牛成虫体内有该卵巢线虫存在,分布于松褐天牛成虫、幼虫体内和松树木质部3个部位;在松褐天牛成虫体内的卵巢线虫通过松褐天牛雌成虫产卵而接种、进入寄主树木木质部中。同时,木质部的卵巢线虫也有一部分进入松褐天牛幼虫体内寄生,另一部分仍在木质部生活,当松褐天牛幼虫再次发育为成虫时,在木质部中的卵巢线虫和已被寄生的松褐天牛幼虫体内的卵巢线虫再次进入松褐天牛成虫体内,完成循环。卵巢线虫在松褐天牛雌、雄虫体内均有分布,携带率为44.4%,其中松褐天牛雌成虫携带率为43.8%,松褐天牛雄成虫携带率为45.0%,两者间无显著差异;每头松褐天牛成虫平均携带卵巢线虫574条,其中雌成虫平均携带816条,雄成虫平均携带308条,具显著差异。初步研究表明,卵巢线虫在松褐天牛成虫体内只能完成产卵到1~4龄的幼虫阶段,不能完成一个完整的世代;在松褐天牛幼虫体内寄生和在木质部生活的卵巢线虫能完成一个完整世代,但具体过程尚不清楚;调查中未观察到卵巢线虫对松褐天牛成虫有寄生致病或致死的现象,但对松褐天牛幼虫有寄生致死的能力;卵巢线虫常与松材线虫同时存在,而且侵入松树及离开松树的方式与松材线虫相同。[结论]我国松褐天牛体内也发现有卵巢线虫存在;目前尚不能证明该线虫对松褐天牛成虫具有寄生致死性,但对松褐天牛幼虫具有一定的寄生致死性;卵巢线虫的生活史与松材线虫相似,是否与松材线虫一样对松树具有危害性以及其病理作用还有待于进一步研究。     

白蜡虫ws基因RNAi载体构建及原核表达dsRNA

[目的]构建白蜡虫(Ericerus pela)蜡酯合酶(wax synthase,WS)基因干扰载体并建立其体外dsRNA(doublestranded RNA,dsRNA)原核表达体系,低成本大量制备白蜡虫ws基因的dsRNA。[方法]克隆白蜡虫蜡酯合酶基因ws片段,连入L4440载体,将重组质粒转入大肠杆菌HT115感受态细胞,经IPTG诱导获得与目的片段相对应的dsRNA。[结果]白蜡虫ws基因RNA干扰(RNA interference,RNAi)载体成功构建,重组质粒转入HT115感受态细胞经IPTG诱导后菌体成功表达dsRNA,dsRNA的平均获得量1 705 ng·m L~(-1)。[结论]该研究通过原核表达白蜡虫ws基因的dsRNA,为后续利用RNAi实验研究白蜡虫ws基因功能及作用机理奠定基础。     

避光对麻竹笋苦涩味及单宁含量、形态与分布的影响

[目的]探讨不同避光处理对麻竹笋苦涩味强度及单宁含量、形态与分布影响的机理。[方法]运用感官评定、光学显微镜、电子显微镜透射技术和磷钨酸-钨酸钠比色法,采用覆土(EP)、双层不透光套袋(CLPB)和自然生长(CK)3种不同处理,对麻竹鲜笋的口感品质进行感官评定,分析测定麻竹笋笋体单宁物质含量、形态和分布。[结果](1)不同处理的麻竹鲜笋的口感不仅均呈苦涩味,且苦涩味强度均表现为由麻竹笋的基部到笋尖逐渐增强的趋势,其苦涩味强度整体大小顺序为:CKCLPBEP。(2)麻竹竹笋壁中含有单宁物质的细胞(CWT)可被2%氯化亚铁溶液染成黑色,与不含单宁物质的细胞区别明显。(3)单宁大量分布在薄壁细胞内,少量在纤维细胞内,维管束中的筛管和导管细胞中无单宁分布。(4)单宁在薄壁细胞中主要分布于细胞质内,少量分布在液泡中,电镜下CWT中积累的单宁可分为絮状、颗粒状和块状3种类型。(5)不同处理的麻竹笋单宁含量为CK:1.15 2.67 mg·g~(-1),CLPB:1.03 1.43 mg·g~(-1),EP:0.36 1.13 mg·g~(-1),其相应的CWT密度大小顺序为:CKCLPBEP,3种处理不同部位的单宁含量与CWT密度大小均为笋尖中部基部。[结论]避光显著降低麻竹竹笋壁的苦涩味强度及单宁含量,自然光照下生长的麻竹笋苦涩味强度及单宁含量显著高于避光处理;光与竹笋中单宁物质分布和形态相关不显著,单宁形态仅与竹笋部位密切相关。覆土、套袋等避光措施可显著降低笋体单宁物质的含量,降低苦涩味,改善竹笋的口感品质,为培育低苦涩味麻竹笋提供科学的理论依据。     

基于cpDNA rps16序列分析兰考泡桐与白花泡桐和毛泡桐的遗传关系

【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。