转录组分析不同砧木对嫁接烤烟抗青枯病的影响

为研究不同砧木对烟草嫁接苗抗病性的影响,明确抗病材料云烟87(Y87)作为砧木提高接穗红大金元(HD)对青枯病抗性的机理,采用劈接加生料带缠绕法以易感青枯病品种HD作为接穗,以HD和Y87作为砧木,分别构建HD/HD和HD/Y87嫁接烤烟苗,然后测量HD/HD和HD/Y87保护性酶苯丙氨酸解氨酶(PAL)活性的变化,最后利用高通量测序技术和Illumina HiSeqTM 4000测序平台,提取5株HD/HD或HD/Y87 mRNA,反转录构建总cDNA文库,测序,过滤低质量Reads后,用TopHat2比对参考基因组,用Cufflinks和DESeq软件获得基因表达量和差异表达基因。结果发现,不同取样时间HD/Y87 PAL活性均显著高于HD/HD,4个样品分别得到48 414 474～48 697 874条Clean Reads和38 272～40 938条基因,但HD/Y87发生选择性剪切事件数高于HD/HD;比较HD/Y87和HD/HD两组间基因表达量,得到3 904条差异基因,其中3 096条基因上调,808条基因下调;在上调基因中有参与木质素合成的3条编码苯丙氨酸解氨酶基因, 5条Myb家族转录因子基因, 5条编码多酚氧化酶基因;还有2条病程相关蛋白基因和3条相应的ERF转录因子基因等。这些结果表明,Y87作为砧木能够提高接穗HD PAL活性和多种抗性基因(包含木质素、多酚及病程相关蛋白)表达量,从而提高接穗红大金元对青枯病等病害抗性。     

大豆转录因子GmMYB52的克隆、表达及结合功能分析

MYB类转录因子在植物的生长发育和逆境响应中有重要的调控作用。依据课题组前期获得的盐胁迫相关的数字表达谱(DGEP)数据,获得盐胁迫响应显著上调基因GmMYB52,利用RT-PCR方法从栽培大豆(Williams 82)中克隆该基因片段,并与已公布的Williams 82基因组数据库序列比对,该基因与GmMYB52序列一致。生物信息学分析表明,GmMYB52编码区(CDS)全长1083 bp,编码360个氨基酸。其编码的氨基酸序列具有MYB类转录因子的共同特征,其距N端110~160氨基酸残基处有MYB结构域;系统进化树分析表明,该基因编码的蛋白与GmMYB62、拟南芥AtMYBSt1、苜蓿Mt MYB52、水稻Os MYBS3及木豆Cc MYB-like protein J的亲缘关系最近;实时荧光定量PCR结果表明,大豆根部GmMYB52的转录水平受外界非生物逆境的调控,用脱落酸(ABA)和低温(4℃)处理后12 h明显上调,用氯化钠和PEG处理0.5~24.0 h后检测到GmMYB52的转录水平在50%~600%区间内呈现出先上调后下调再上调的趋势。GmMYB52为组成型表达,在大豆的幼苗期和开花期表达较多,在成熟期表达相对较低。GmMYB52在茎叶与开花期的花中表达较强,在根的表达较弱,而在成熟期的豆荚中几乎不表达。亚细胞定位的结果表明,GmMYB52定位于细胞核,符合典型转录因子的定位特征。酵母杂交系统检测表明,GmMYB52具有转录激活特征,并且能够与MYB相关顺式作用元件基序相结合。本研究结果表明,GmMYB52编码典型的MYB转录因子,具有转录激活活性及DNA结合活性,在大豆中的表达可能与大豆的非生物胁迫和ABA信号转导途径有关,推测其可能参与了大豆对非生物胁迫的响应。     

2种中草药提取物对黑木耳活性成分形成的影响

为研究2种中草药提取物对黑木耳发酵过程中活性成分产生的影响,分别向黑木耳发酵基础培养基中添加不同比例的刺五加及五味子提取液,培养6天后,采用比色法及HPLC法分析黑木耳多糖成分及腺苷含量;同时,采用HPLC法分析黑木耳发酵过程中2种中草药活性成分的含量变化。结果表明：刺五加、五味子的添加量分别为4、3 g/L对黑木耳发酵液中多糖的形成具有显著促进作用;刺五加、五味子的添加量分别为1、6 g/L对黑木耳菌丝体中腺苷的形成具有显著促进作用;在黑木耳发酵过程中,刺五加活性成分紫丁香苷及刺五加苷E降低到无法检测;五味子中木脂素成分存在增减变化。黑木耳发酵过程中存在活性成分的动态变化。     

转录组解析外源ABA对玉米脱水速率的影响

为了探明迪卡517、郑单1002喷施外源ABA后脱水速率变化的分子机制,发掘影响脱水速率的关键差异表达基因及代谢通路,利用Illumina HiSeq~(TM)2500测序仪分别对喷施ABA前后迪卡517(脱水速率较快)和郑单1002(脱水速率较慢)的穗位叶进行高通量转录组测序。原始序列经过质量控制、基因组比对、测序饱和度、基因覆盖度及冗余序列分析后进行基因功能注释。结果显示,迪卡517外源ABA处理与正常生长条件对比组检测到1 732个差异表达基因,其中1 251个为上调表达基因,481个为下调表达基因;郑单1002外源ABA处理与正常生长条件对比组检测到52个差异表达基因,其中24个为上调表达基因;迪卡517外源ABA处理与郑单1002外源ABA处理对比组检测到3 867个差异表达基因,其中1 946个为上调表达基因。不同对比组中筛选到差异表达基因GO分类情况、COG基因产物分类、KEGG代谢通路分析不同。转录因子分析共获得多个重要的转录因子家族,主要有AGC蛋白激酶家族、AP2/ERF转录因子家族、ARID转录因子、AUX/IAA转录因子家族、Alfin-like转录因子、Aur转录因子家族、B3转录因子家族、BBR-BPC转录因子家族、BES1转录因子、BUB转录因子、C2C2-CO-like转录因子等。Zm00001d035000、Zm00001d042063、Zm00001d045314等共同差异表达基因均在迪卡517和郑单1002中表达。对所有检测到的差异表达基因GO分类分析获得73个差异表达基因与水分代谢相关,推测这些基因作为外源ABA调控的下游基因起作用。     

断营养影响青梗菜硝酸盐含量的转录组学分析

为降低青梗菜硝酸盐含量，在采收前对其进行断营养处理。生理分析发现，断营养5 d青梗菜产量无明显变化，但根、叶柄和叶片的硝酸盐含量分别降低了49.77%,23.90%,33.39%。转录组比较发现，断营养5 d青梗菜根、叶柄和叶片分别鉴定出301,2 270,2 271个差异表达基因(DEGs)。基因本体论(GO)分析表明，这些DEGs均主要富集在氮(N)代谢、碳(C)代谢和活性氧(ROS)代谢过程中。在N代谢过程中，根、叶柄和叶片硝酸盐摄取转运蛋白NPF7.2基因下调表达；叶柄和叶片NPF7.2的上游调控因子ERF104下调表达；叶片硝酸盐再转运蛋白NPF2.13和NPF1.1基因上调表达；根和叶片硝酸盐同化关键酶谷氨酰胺合成酶(GS)基因上调表达。在C代谢过程中，叶柄和叶片蔗糖合成关键酶磷酸蔗糖合酶(SPS)基因上调表达。在ROS代谢过程中，根的超氧化物歧化酶(SOD)基因和抗坏血酸过氧化物酶(APX)基因上调表达；叶柄和叶片细胞色素P450、过氧化物酶体和脂肪氧化酶下调表达；叶片过氧化氢酶(CAT)基因和过氧化物酶(POD)基因上调表达。综上，在青梗菜采收前5 d进行断营养处理，不...     

灭多威胁迫下罗非鱼精巢转录组变化及信号通路分析

应用高通量测序技术(RNA-seq),获得显著差异表达基因369条,160条基因显著下调,209条基因显著上调。其中显著差异表达基因在ECM与受体相互作用信号通路和MAPK信号通路中富集明显。分析发现,灭多威对精巢组织细胞粘附性造成影响,诱导ECM与受体相互作用中相关基因表达显著上调,如层粘连蛋白Laminins、整合素ɑ6、β1等,以稳定精巢组织结构；JNK途径中MAP3K、MKK4、JNK显著表达上调,增强录因子AP-1,促进 P53、Bax等促凋亡蛋白的表达；MAPK信号通路ERK途径中相关生长因子表达受到影响,从而可能对罗非鱼精巢组织生殖细胞增殖产生影响。     

外源ABA对低温胁迫下玉米幼苗内源激素含量及Asr1基因表达的调节

脱落酸(ABA)是低温逆境下的重要信号因子,为了探讨外源ABA对低温胁迫下玉米幼苗的生长调节作用,以耐低温玉米品种久龙5号为试验材料,采用不同浓度(5、15、25、35 mg L–1)ABA于玉米三叶一心时喷雾于叶片,并进行低温梯次处理。分析处理后玉米叶片相对电导率、抗氧化酶活性及内源激素ABA、IAA的含量变化,并采用Real-time PCR明确Asr1基因表达水平变化。结果表明,低温胁迫下不同浓度外源ABA处理的玉米叶片相对电导率整体呈上升趋势,SOD和POD活性加强,15 mg L–1和25 mg L–1ABA处理的SOD活性均显著高于未应用ABA处理,玉米內源ABA和IAA合成水平上升,应用ABA后Asr1基因相对表达水平上调,其中5、15和25 mg L–1浓度处理基因表达上调显著。相关分析表明,ABA含量与Asrl基因相对表达量、SOD活性均表现为极显著正相关,与POD活性显著正相关。说明Asr1基因表达受ABA的介导调控,Asr1基因表达量的提升,也促进了内源ABA的合成,抗氧化酶活性加强,提升了应用ABA后玉米的抗低温能力。但外源ABA的介导调控具有一定浓度效应,表现为低促高抑。     

奶山羊ATGL基因启动子鉴定与转录活性分析

脂肪甘油三酯水解酶(ATGL)是脂肪水解过程中的主要限速酶，是调控细胞中甘油三酯和脂肪酸之间平衡的关键因子。为了分析奶山羊ATGL基因启动子的结构及转录调控机制，以奶山羊全血DNA为模板，通过PCR技术扩增出ATGL基因5′侧翼序列并进行生物信息学分析；构建了5个不同长度的启动子缺失片段报告基因载体，通过转染奶山羊乳腺上皮细胞并检测其活性，确定其核心区域；分别用PPARG激动剂罗格列酮和SREBP1激动剂T0901317处理细胞，检测其对ATGL启动子活性的影响。结果表明，克隆得到奶山羊ATGL基因5′侧翼序列2 721 bp,包含转录起始位点上游2 024 bp。经在线软件分析发现，ATGL启动子含有真核生物启动子元件TATA-box和CpG岛；对转录因子结合位点预测发现，其上含有FOXO、SREBF1、PPARG、C/EBPα和E2F1等结合位点。启动子活性分析结果表明，ATGL启动子核心区域位于转录起始位点上游-256—+1位，且在-527—-256位存在负调控元件。用罗格列酮和T0901317处理细胞后发现，二者均能显著上调ATGL启动子活性，且罗格列酮作用的区域为-527—-...     

马铃薯StERF5转录因子酵母双杂交诱饵载体的构建及鉴定

为了筛选与马铃薯StERF5转录因子相互作用的宿主蛋白,构建其诱饵载体,本研究利用PCR方法扩增得到马铃薯StERF5基因的编码序列,克隆至载体p MD18-T,验证正确后插入到酵母双杂交系统诱饵表达载体pGBKT7,经双酶切、PCR反应及测序验证其正确后,利用PEG/LiAc法将构建好的重组诱饵载体pGBKT7-StERF5及空载体pGBKT7分别转化到酵母Y2HGlod感受态细胞中,检测其对酵母菌株是否有毒性作用和自激活活性。结果表明:扩增得到StERF5基因,成功构建了诱饵表达载体pGBKT7-StERF5,此诱饵载体对酵母宿主细胞无毒性且不具自主激活报告基因的功能。研究结果可为进一步利用酵母双杂交技术筛选与ERF5基因互作的宿主蛋白及功能研究提供科学依据。     

丙环唑对玉米幼苗生长的调控及其相关机制

丙环唑(propiconazole,简称Pcz)作为一种杀菌剂被广泛应用于作物生产,它同时具有调节作物生长发育的作用,但关于丙环唑在玉米上的应用研究较少。本研究以玉米品种郑单958为材料,研究丙环唑(Pcz)对玉米苗期植株生长、细胞形态和激素信号的影响。结果表明,Pcz处理显著抑制玉米中胚轴与胚芽鞘的生长,降低株高,缩短叶片和叶鞘长度,减小叶夹角,同时显著抑制叶片和叶鞘细胞的纵向伸长,促使叶枕细胞排列由疏松变为紧密;Pcz处理显著降低玉米中赤霉素(GA1和GA3)的含量,下调GA合成酶基因GA3ox1基因的表达,上调GA钝化酶基因GA2ox5和GA2ox8表达,而GA合成酶基因GA20ox1的表达呈现先上调后下调模式;Pcz处理显著降低油菜素内酯(BR)含量,但BR合成基因CPD和DWF4的表达上调,可能是由于反馈调节。此外,Pcz处理下调扩张蛋白基因EXPA4、EXPA5和木葡聚糖内糖基转移酶/水解酶基因XTH1、XET1的表达。综上所述,Pcz处理调节GA和BR信号转导途径,抑制GA和BR在植株内积累,调控扩张蛋白、木葡聚糖内糖基转移酶/水解酶基因表达,操纵细胞生长,有效调控株型。     

Inhibitory Effects of Polyphenols from Pinus koraiensis Seed Scale on the Proliferation of Human Cancer Cells in Vitro

[Objective] To research on the inhibitory effects of polyphenols from Pinus koraiensis seed scale on the proliferation of human cancer cells in vitro. [Methods] Polyphenols from Pinus koraiensis seed scale were prepared into extracting solutions at different concentrations. Suspension cultures of five tumor cells were processed,which were lung carcinoma cell A549,human bone marrow neuroblastoma cell SH-SY5 Y,human skin cancer cell A375,human hepatocarcinoma cell HepG-2 and human ovarian cancer cell SKOV3. Inhibitory rate of cell proliferation in vitro was detected by MTT method. [Results] P. koraiensis polyphenols had relatively good inhibitory effects on A549,showing certain correlation with time or concentration. The inhibitory rare was the optimal when the concentration of P. koraiensis polyphenols was0. 4 mg / mL. Under this concentration,the inhibitory rate of extracting solution of P. koraiensis polyphenols reached 55% on lung carcinoma cell A549. P. koraiensis polyphenols showed no significant inhibitory effects on SH-SY5 Y,A375,HepG 2 or SKOV3. [Conclusions] P. koraiensis polyphenols had inhibitory effects on lung carcinoma cells.     

SSCP Marker Development and Analysis of Candidate Restorer Gene Rf1 for Cotton Cytoplasmic Male Sterility

[Objective] Locating the cotton cytoplasmic male sterility (CMS) restorer gene Rf₁ is important for investigating restorer gene mechanisms and improving restorer lines. In our previous study, a gene cluster, with nine Pentatricopeptide repeat(PPR) genes and nine other genes, was found within the 160-kb Rf₁ target region in Scaffold 333. The objective here was to improve the density of Rf1-linked markers in the target region and determine the expression profiles of candidate genes. [Method] Using the sequences of the 18 genes, we designed 155 single-strand conformation polymorphism (SSCP) primers covering all of the gene sequences to identify the polymorphic SSCP markers between the fertile and sterile pools. Additionally, real-time polymerase chain reaction(PCR) was performed to analyze the expression profiles of eight candidate genes in the four developmental stages of buds of sterile, maintainer and restoring lines, respectively. [Result] In total, 15 polymorphic primers were identified. A genotype analysis of the F₂ population was conducted using the 15 primers and 3 other polymorphic simple sequence repeats (SSR) markers. The markers were distributed in a 4.8 cM range. In addition, owing to the influence of sterile cytoplasm or restorer genes, most of the genes showed different expression patterns in the four developmental stages of the three lines' buds. [Conclusion] SSCP markers tightly linked to Rf₁ were identified and the expression profiles of candidate genes were determined. This study provides a basis for the further fine mapping of restorer genes and for candidate gene screening.     

黄麻链霉菌NF0919菌株发酵提取液HPLC检测分析及稳定性研究

采用HPLC法检测分析了黄麻链霉菌NF0919菌株发酵提取液的主要有效成分,以确定该发酵液的HPLC检测方法,采用菌丝生长速率法对不同处理条件下的发酵提取液进行抑菌活性测定,以确定该发酵提取液的稳定性。结果表明,在采用C18柱,波长305nm,甲醇与水梯度洗脱的条件下,所检测到的发酵提取液物质成分最多;该菌株发酵提取液对热、碱、紫外线的稳定性较差,对酸、光照均较稳定,在冰箱贮藏条件下,该菌株的发酵提取液稳定性较强,具有一定的研究开发价值。     

Isolation and Characterization of GhLEA3 Gene from Upland Cotton and Its Expression in Response to Low Temperature Stress

[Objective] Based on the analysis of cotton GhLEA3 gene structure and its expression model in chilling stress, we investigated the resistance function of GhLEA3 in response to cold stress. [Method] We cloned GhLEA3 from upland cotton by homologous sequence cloning way. We analyzed the properties of the GhLEA3, and constructed phylogenetic tree by bioinformatics analysis. We constructed the transient expression vector 35S∷GhLEA3-GFP by In-Fusion connection technology and studied the subcellular localization of GhLEA3. The expression of GhLEA3 gene in leaves of TM-1 at three-leaf stage under low temperature stress treatments (4 ℃, 24 h) was performed. Agrobacterium tumefaciens mediated floral dipping method was used to transform the gene with expression vector 35S∷GhLEA3-GFP into Arabidopsis thaliana wild type. Low temperature germination experiment at 4 ℃ was conducted to verify germination ability of the transgenic Arabidopsis thaliana T₃ seed. The transgenic Arabidopsis seedlings were treated at 4 ℃(low temperature), and all the leaves were taken to measure their electrical conductivity. [Result] The coding sequence of GhLEA3 gene is 1 218 bp, which encodes 405 amino acids. GhLEA3 protein includes 4 functional domains of PF02987. Phylogenetic analysis showed that the similarity of amino acid sequences between upland cotton GhLEA3 and Arabidopsis thaliana AtLEA3 was 52.6%. GhLEA3 might be mainly localized in vacuoles and small vesicles. GhLEA3 was up-regulated in leaves after low temperature treatment. The germination rate of transgenic Arabidopsis thaliana of T₃ generation at low temperature was significantly higher than that of wild type, and its electrical conductivity of leaves after low temperature treatment was significantly lower than that of wild type. [Conclusion] GhLEA3 belonged to the member of LEA3 family. Phylogenetic analysis showed that GhLEA3 had the closest relationship with AtLEA3. GhLEA3 was induced by low temperature stress and may play an important role in improving cold resistance.     

木槿花挥发油化学成分的GC/MS分析

摘 要：[目的] 研究江西赣南红壤土产木槿花挥发油的化学成分。[方法] 采用国家药典法-水蒸汽蒸馏法提取其挥发油,利用GC-MS联用仪对木槿花挥发油的化学成分进行分离鉴定,用峰面积归一化法确定各成分的相对含量。[结果] 挥发油含量为0.1107g/100g,共分离出90种化合物。鉴定了其中的43个。[结论] 木槿花挥发油的主要化学成分中,Tridecanoic acid 59.08%、9,10-Octadecadienoic aicd(z,z)- 4.43%、Oleic acid 4.04%、Heneicosane 3.18%等的含量较高。为医学临床应用和自然资源的开发提供了科学参考。     

Effect of MORINDAE OFFICINALIS Extract on the Behavior and Zymology of Senile Dementia Model Rats

[Objective]To study the effect of MORINDAE OFFICINALIS extract on the behavior and zymology of senile dementia rat model. [Methods]Senile dementia rat model was prepared by intraperitoneal injection of D-galactose( 120 mg/kg) and sodium nitrite( 90 mg/kg) for 30 d. 60 rats were divided into 6 groups,normal control group,model group,nimodipine group,high-dosage group of aqueous extract of MORINDAE OFFICINALIS,medium-dosage group of aqueous extract of MORINDAE OFFICINALIS and low-dosage of aqueous extract of MORINDAE OFFICINALIS. The learning and memory ability was measured by rat step-down test. The activities of SOD,MDA and MAO-B were determined. The expression of MAO-B mRNA was determined by RT-PCR. [Results]After intraperitoneal injection of MORINDAE OFFICINALIS extract,the learning and memory abilities of model rats increased. The biochemical criterion of SOD,MDA and MAO-B was improved. The expression level of MAO-B mRNA decreased significantly. [Conclusions] Extract of MORINDAE OFFICINALIS could protect acute aging model rats at certain degree.     

蓝细菌血畿飞逝基因的克隆及其向甘蓝型油菜中的转化

血红蛋白存在于动物、植物、细菌、真菌、酵母、藻类、原生动物等生物体中。利用PCR方法克隆了蓝细菌Synechocystis sp. PCC 680的血红蛋白基因SLR2097,该基因属于“truncated”血红蛋白家族,大小为375 bp,编码123个氨基酸残基。数据库搜索的结果显示所有的血红蛋白都有一个保守基序,即‘F-[L]-x(4)-[G]-G-[T]-x(2)-[Y]-x-[G]-[R]-x-[M]-x(3)-H’, “truncated”血红蛋白家族也有该结构,表明它们的进化祖先相同。将获得的SLR2097连接到双元载体pCAMBIA1300-CaMV35S上,并转化甘蓝型油菜品种宁油12。转基因油菜表型分析表明,血红蛋白基因SLR2097在油菜中的表达促进油菜的生长并缩短发育期,引起早熟,这些表型对改良油菜的农艺性状有重要的应用价值。     

Regulatory Mechanism of the Photosynthetic Pathway in Dwarfed Upland Cotton (Gossypium hirsutum L.)

[Objective] Here, we examined the mechanism of the photosynthetic pathway in dwarfed upland cotton (Gossypium hirsutum L.). [Methods] The upland cotton dwarf line LA-1 and the near-isogenic line LH-1 were used as research materials. We screened for dwarf-related differentially expressed genes at protein and mRNA levels by applying the isobaric tags for relative and absolute quantitation method in combination with LC-MS/MS quantitative proteomic technology and quantitative reverse-polymerase chain reaction. The difference in the photosynthetic abilities between the two materials were detected using physiological and biochemical methods. [Results] An analysis on the differential expression of proteins encoded by photosynthetic system elements revealed that PsbO, PsaE, PsaH, PetF-1 and PetF-2 were down-regulated, while PetC and delta were up-regulated in LA-1. The expression trends of the mRNA levels were the same as at the protein levels, except for those of the delta gene. The net photosynthetic rate, stomatal conductance and transpiration rate in LA-1 were lower than those LH-1, but the intercellular CO₂ concentration was not significantly different after budding. This indicated that the non-stomatal factor led to a decreased net photosynthetic rate in LA-1. Chlorophyll fluorescence detection revealed that the actual photosynthetic efficiency and potential photochemical activity of photosystem II in LA-1 were significantly decreased, while non-photochemical quenching was significantly increased. [Conclusion] The dwarfism of LA-1 is related to the photosynthetic pathway, and the results lay a foundation for exploring key dwarf-related genes and the molecular basis for dwarfism in upland cotton.