早酥梨病程相关基因非表达子1基因（NPR1）克隆及原核表达

本研究以成年早酥梨(Pyrus bretschneideri cv.Zaosu)叶片为材料,通过RT-PCR克隆早酥梨病程相关基因非表达子1基因(NPR1)并获得其全长序列1771 bp,开放阅读框为1761 bp,编码586个氨基酸(GenBank登录号:FJ769372).利用NCBI/Blastp和ClustalX软件进行相似性分析表明,目的基因编码蛋白质序列与日本梨(p.pyrifolia)、秋子梨(P.ussuriensis)、苹果(Malus xdomestica)、烟草(Nicotiana tabacum)和拟南芥(Arabidopsis thaliana)的NPR1蛋白相似性分别为99%、98%、98%、67%和59%.将其连接pGEX-4T-1原核表达载体并转化大肠杆菌(EScherichia coli)BL21,经IPTG诱导表达获得大小约为91 kD的目的融合蛋白,融合蛋白主要以包涵体形式表达,表达量约占总蛋白17%.     

Plot-scale manipulations of organic matter inputs to soils correlate with shifts in microbial community composition in a lowland tropical rain forest

Little is known about the organisms responsible for decomposition in terrestrial ecosystems, or how variations in their relative abundance may influence soil carbon (C) cycling. Here, we altered organic matter in situ by manipulating both litter and throughfall inputs to tropical rain forest soils, and then used qPCR and error-corrected bar-coded pyrosequencing to investigate how the resulting changes in soil chemical properties affected microbial community structure. The plot-scale manipulations drove significant changes in microbial community composition: Acidobacteria were present in greater relative abundance in litter removal plots than in double-litter plots, while Alphaproteobacteria were found in higher relative abundance in double-litter and throughfall reduction plots than in control or litter removal plots. In addition, the bacterial:archaeal ratio was higher in double-litter than no-litter plots. The relative abundances of Actinobacteria, Alphaproteobacteria and Gammaproteobacteria were positively correlated with microbial biomass C and nitrogen (N), and soil N and C pools, while acidobacterial relative abundance was negatively correlated with these same factors. Bacterial:archaeal ratios were positively correlated with soil moisture, total soil C and N, extractable ammonium pools, and soil C:N ratios. Additionally, bacterial:archaeal ratios were positively related to the relative abundance of Actinobacteria, Gammaproteobacteria, and Actinobacteria, and negatively correlated to the relative abundance of Nitrospira and Acidobacteria. Together, our results support the copiotrophic/oligotrophic model of soil heterotrophic microbes suggested by Fierer et al. (2007).     

桃蚜组织蛋白酶基因CTSB-16D和CTSB-348的克隆、表达谱及对不同环境胁迫的响应

为了探索组织蛋白酶基因CTSB-16D和CTSB-348在桃蚜Myzus persicae(Sulzer)响应高低温和UV-B胁迫中的作用, 通过RT-PCR技术克隆了桃蚜CTSB-16D和CTSB-348基因, 利用生物信息学方法分析了它们的序列特征;采用实时荧光定量PCR(quantitative real-time PCR, RT-qPCR)技术检测了2个基因在桃蚜不同发育阶段的相对表达量及经高温、低温和UV-B胁迫不同时长的无翅成虫中的相对表达量。克隆获得了桃蚜CTSB-16D(GenBank登录号:MZ962352)和CTSB-348(GenBank登录号:MZ962353)基因, 序列长度分别为1 096 bp和1 101 bp, 开放阅读框(ORF)分别为1 017 bp和1 023 bp, 分别编码338个和340个氨基酸, 蛋白相对分子量为38.14 kD和37.52 kD, 等电点(pI)为5.46和5.99。系统发育分析表明, 桃蚜CTSB-16D和CTSB-348均与豌豆蚜Acyrthosiphon pisum (XP_003246386.1、NP_001119608.1) CTSB亲缘关系最近。RT-qPCR分析表明, CTSB-16D和CTSB-348在桃蚜不同发育阶段均有表达, 分别在1龄若虫和无翅成虫中的表达量最高。高、低温和UV-B胁迫对CTSB-16D和CTSB-348基因的表达具有明显的诱导作用, 且表达量均随时间的延长呈先上升后下降的趋势;4℃胁迫下, CTSB-16D和CTSB-348表达量均在90 min时达到峰值;36℃胁迫下, CTSB-16D和CTSB-348表达量均在30 min时达到峰值;UV-B胁迫下, CTSB-16D和CTSB-348表达量分别在60 min和120 min时达峰值。桃蚜CTSB-16D和CTSB-348基因在不同发育阶段差异表达, 且响应温度和UV-B的胁迫, 推测两个基因参与桃蚜的生长发育并在桃蚜响应环境胁迫中发挥了重要作用。     

油菜叶片衰老相关基因的分离：ATP硫化酶cDNA的克隆和序列分析

通过筛选缩减ｃＤＮＡ文库中在油菜叶片衰老阶段表达而在绿叶中不表达的ｃＤＮＡ克隆，分离了与油菜叶片衰老有关的基因ＬＳＣ６６。ＤＮＡ序列测定和分析结果表明，ＬＳＣ６６的全长可读框架由１３８０个碱基组成，编码４６０个氨基酸，它的核昔酸序列与拟南芥菜ＡＴＰ硫化酶基因有８６．６７％的同源性，其推导的氨基酸序列与拟南芥菜ＡＴＰ硫化酶的氨基酸序列有９１．７４％的同源性。对ＬＳＣ６６基因产物在植物叶片衰老过程中的作用进行了讨论。     

杂交稻对白叶枯病菌主基因抗性的配合力分析

以８种不同胞质类型的不育系及其保持系与９个恢复系配制的７２个不育系杂种及保持系杂种为材料，研究了杂交稻对白叶枯病菌主基因抗性的配合力。结果表明，杂交稻的抗性以主基因抗性为主，但同时也受双亲中与抗性有关的其他加性、显性及上位性基因效应的影响。说明杂交稻的抗性强弱取决于双亲抗性基因互补和累加程度高低。     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

Pseudomonas putida BP25 alters root phenotype and triggers salicylic acid signaling as a feedback loop in regulating endophytic colonization in Arabidopsis thaliana

Endophytic Pseudomonas putida BP25 (PpBP25) triggered density dependent alterations on Arabidopsis thaliana Col-0 growth. Endogenous colonization of PpBP25 was found regulated within Arabidopsis that caused induction and repression of 131 and 74 plant genes, respectively. Induced genes like WRKY33, AtRLP19, ATL2, ATEXO70B2, pEARLI, RPS2, CBP60G, PLA2, CRK18, ATFBS1, DREB2A, TIR, RAP2.4, and MOS1 were components of defense and salicylic acid (SA) signaling. Development associated genes were found significantly repressed. Biased activation of phytohormone signaling with their associated fitness costs on plant growth was observed. The data suggests that PpBP25 colonization triggered expression of defense genes that restricted its own population in a feedback loop besides causing altered root phenotype.     

柑橘黄化脉明病毒的TGB基因生物信息学分析及亚细胞定位

柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)是一种正义单链RNA病毒。其基因组含有6个开放阅读框(ORF),其中ORF2、ORF3和ORF4组成了三基因连锁结构(triple gene block,TGB)。以感染CYVCV的尤力克柠檬[Citrus limon(L.)Burm.f.]植株的总RNA为模板,扩增和克隆了CYVCV的TGB基因,并开展了其编码蛋白的理化性质和分子特性等生物信息学分析;进而通过In-Fusion~?技术构建了TGB各基因的亚细胞定位载体,经农杆菌介导转化洋葱表皮细胞并在荧光显微镜下进行亚细胞定位观察。生物信息学分析结果表明,CYVCV-TGB具有与Potexvirus属病毒TGB相似的理化性质和分子特性,可能参与病毒在寄主中的运动且需要外壳蛋白的参与。亚细胞定位结果显示,TGBp1定位于细胞壁(膜)上,TGBp2和TGBp3主要定位于细胞壁(膜),少量呈星点状定位于细胞内。研究结果为揭示CYVCV在寄主中的运动机理奠定了基础。     

乳腺特异表达载体缺失片段的鉴定与修复

通过对pBC1中的乳腺特异表达启动子成分进行分析,鉴定了位于β酪蛋白启动子上游两个EcoRⅠ位点(-89~-94和-120~-125)之间的缺失片段,31bp的缺失使得该载体中的一个乳控盒元件(-112~-141)和一个乳腺因子识别序列(-92~-102)遭到破坏,并使得位于TATA框上游的所有启动子成分发生了位置改变。通过与山羊β酪蛋白启动子序列进行比较,设计并合成了缺失片段,经一系列的酶切和连接处理,使得该缺失得到了修复,修复后的载体具有所有乳蛋白表达需要的启动子调控原件。本试验结果为利用修复后的乳腺特异高效表达载体进行乳腺生物反应器的研究奠定了分子生物学基础。