Wing patterning genes of Nilaparvata lugens identification by transcriptome analysis, and their differential expression profile in wing pads between brachypterous and macropterous morphs

The brown planthopper, Nilaparvata lugens is an economically important pest on rice plants. This species produces macropterous and brachypterous morphs in response to environmental cues, which makes it very dififcult to control. The molecular basis of wing patterning in N. lugens is stil unknown. It is necessary to identify wing patterning genes of N. lugens, and also to clarify the expression differences of wing patterning genes between macropterous and brachypter-ous morphs. High-throughput deep sequencing of transcriptome of N. lugens wing pad yielded 116 744 580 raw reads and 113 042 700 clean reads. Al the reads were assembled into 55 963 unigenes with an average length of 804 bp. With the E-value cut-off of 1.0E–5,18 359 and 2 883 unigens had hits in NCBI-NR (NCBI non-redundant protein sequences) and NCBI-NT (NCBI nucleotide sequences) databases, respectively. A total of 16 502 unigenes were assigned to GO (gene ontology) classiifcation, 9 709 ungenes were grouped into 26 COG (cluster of orthologous groups of proteins) classiifcations, and 6 724 unigenes were assigned to different KEGG (Kyoto encyclopedia of genes and genomes) path-ways. In total, 56 unigenes which are homologous to wing patterning genes of Drosophila melanogaster or Tribolium castaneum were identiifed. Out of the 56 unigenes, 24 unigenes were selected, and their expression levels across the ifve nymphal stages between macropterous strain and brachypterous strain were examined by qRT-PCR. Two-way ANOVA analysis showed that development stage had signiifcant effects on the expression level of al the 24 genes (P<0.05). The expression levels of 8 genes (Nlen, Nlhh, Nlsal, NlAbd-A, Nlwg, Nlvg, Nlexd and NlUbx) were signiifcantly affected by wing morph. This is the ifrst transcriptome analysis of wing pads of hemimetabolous insect, N. lugens. The identiifed wing patterning genes would be useful resource for future exploration of molecular basis of wing development. The 8 differential y expressed wing patterning genes between macropterous strain and brachypterous strain would contribute to explain molecular mechanism of wing-morph differentiation in N. lugens.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human

ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

Genetic basis of negative heterosis for growth traits in chickens revealed by genome-wide gene expression pattern analysis

Background:Heterosis is an important biological phenomenon that has been extensively utilized in agricultural breeding.However,negative heterosis is also pervasively observed in nature,which can cause unfavorable impacts on production performance.Compared with systematic studies of positive heterosis,the phenomenon of negative heterosis has been largely ignored in genetic studies and breeding programs,and the genetic mechanism of this phenomenon has not been thoroughly elucidated to date.Here,we used chickens,the most common agricultural animals worldwide,to determine the genetic and molecular mechanisms of negative heterosis.Results:We performed reciprocal crossing experiments with two distinct chicken lines and found that the body weight presented widely negative heterosis in the early growth of chickens.Negative heterosis of carcass traits was more common than positive heterosis,especially breast muscle mass,which was over−40%in reciprocal progenies.Genome-wide gene expression pattern analyses of breast muscle tissues revealed that nonadditivity,including dominance and overdominace,was the major gene inheritance pattern.Nonadditive genes,including a substantial number of genes encoding ATPase and NADH dehydrogenase,accounted for more than 68%of differentially expressed genes in reciprocal crosses(4257 of 5587 and 3617 of 5243,respectively).Moreover,nonadditive genes were significantly associated with the biological process of oxidative phosphorylation,which is the major metabolic pathway for energy release and animal growth and development.The detection of ATP content and ATPase activity for purebred and crossbred progenies further confirmed that chickens with lower muscle yield had lower ATP concentrations but higher hydrolysis activity,which supported the important role of oxidative phosphorylation in negative heterosis for growth traits in chickens.Conclusions:These findings revealed that nonadditive genes and their related oxidative phosphorylation were the major genetic and molecular factors in the negative heterosis of growth in chickens,which would be beneficial to future breeding strategies.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Study on gene expression patterns and functional pathways of peripheral blood monocytes reveals potential molecular mechanism of surgical treatment for periodontitis

BACKGROUND Periodontitis is a chronic inflammation of periodontal supporting tissue caused by local factors. Periodontal surgery can change the gene expression of peripheral blood mononuclear cells. However, little is known about the potential mechanism of surgical treatment for periodontitis. AIM To explore the potential molecular mechanism of surgical treatment for periodontitis. METHODS First, based on the expression profiles of genes related to surgical treatment for periodontitis, a set of expression disorder modules related to surgical treatment for periodontitis were obtained by enrichment analysis. Subsequently, based on crosstalk analysis, we proved that there was a significant crosstalk relationship between module 3 and module 5. Finally, based on predictive analysis of multidimensional regulators, we identified a series of regulatory factors, such as endogenous genes, non-coding RNAs (ncRNAs), and transcription factors, which have potential regulatory effects on periodontitis. RESULTS A total of 337 genes related to surgical treatment for periodontitis were obtained, and 3896 genes related to periodontitis were amplified. Eight expression modules of periodontitis were obtained, involving the aggregation of 2672 gene modules. These modules are mainly involved in G-protein coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger, and adenylate cyclasemodulating G-protein coupled receptor signaling pathway. In addition, eight endogenous genes (including EGF, RPS27A, and GNB3) were screened by network connectivity analysis. Finally, based on this set of potential dysfunction modules, 94 transcription factors (including NFKB1, SP1, and STAT3) and 1198 ncRNAs (including MALAT1, CRNDE, and ANCR) were revealed. These core regulators are thought to be involved in the potential molecular mechanism of periodontitis after surgical treatment. CONCLUSION Based on the results of this study, we can show biologists and pharmacists a new idea to reveal the potential molecular mechanism of surgical treatment for periodontitis, and provide valuable reference for follow-up treatment programs.

Molecular Characterization and Expression Pattern of Rheb Gene in Inner Mongolia Cashmere Goat(Capra hircus)

As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between Rheb and mTOR in Inner Mongolian Cashmere goat （Capra hircus） cells, Ras homolog enriched in brain （Rheb） gene eDNA was amplified by RT-PCR. It is 555 bp in length and includes the complete ORF encoding 184 amino acids （GenBank accession no. HM569224）. The full eDNA nucleotide sequence has a 99% identity with that of sheep, 98% with cattle and 93% with human while their amino acids sequence shares identity with 98, 97 and 97% of them, correspondingly. The bioinformatics analysis showed that Rheb has a Ras family domain, two casein kinase II phosphorylation sites, two ATP/GTP-binding sites motifA （P-loop）, a prenyl group binding site （CAAX box）. Tissue-specific expression analysis performed by semi- quantitative RT-PCR. The Rheb gene was expressed in all the tested tissues and the highest level ofmRNA accumulation was detected in brain, suggesting that Rheb played an important role in goat cells.

Cloning, Expression Pattern Analysis and Subcellular Localization of Resveratrol Synthase Gene in Peanut (<i>Arachis hypogaea</i>L.)

Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.

Global pattern of pairwise relationship in genetic network

In recent times genetic network analysis has been found to be useful in the study of gene-gene interactions, and the study of gene-gene correlations is a special analysis of the network. There are many methods for this goal. Most of the existing methods model the relationship between each gene and the set of genes under study. These methods work well in applications, but there are often issues such as non-uniqueness of solution and/or computational difficulties, and interpretation of results. Here we study this problem from a different point of view: given a measure of pair wise gene-gene relationship, we use the technique of pattern image restoration to infer the optimal network pair wise relationships. In this method, the solution always exists and is unique, and the results are easy to interpret in the global sense and are computationally simple. The regulatory relationships among the genes are inferred according to the principle that neighboring genes tend to share some common features. The network is updated iteratively until convergence, each iteration monotonously reduces entropy and variance of the network, so the limit network represents the clearest picture of the regulatory relationships among the genes provided by the data and recoverable by the model. The method is illustrated with a simulated data and applied to real data sets.

西南少数民族八角纹设计文化基因“数字化”表达与传递机理研究

八角纹在西南各少数民族染织纹样中的传递已有几千年的历史。类比生物基因“数字对位传递”所具有的高度精确性,如果对染织纹样中的八角纹进行“显微镜式的观察”,可以发现,八角纹隐含着“几何式”与“模件式”两种设计文化基因。其产生与复制的过程,是按照均衡比例的“数字化”机理进行表达与传递;指令信息的明确,使得传递后的设计文化基因具有高度的保真性、多产性与长寿性。本文研究表明,在西南少数民族八角纹等纹样的设计与制作过程中,传递“如何制作”(机理)比传递“制作什么”(纹样)更具有效性。

基于层次分析与二十四节气动态图案文化基因的设计研究

目的运用动态图案的独特性质和优势,提升视觉体验,超越传统的二维界限。采用创新、生动且引人入胜的数字化方法展示内容,增强中华传统文化对公众的吸引力,促进非物质文化遗产在数字时代的繁荣发展。方法利用层次分析法的相关理论,对二十四节气的物候特征、农事活动特征等相关文化因子进行分析,提炼出节气的文化的典型基因,并将其转换为设计元素,从而构建二十四节气动态图案设计。结果将层次分析法和符号学理论运用到二十四节气动态图案设计中,确定影响二十四节气动态图案设计的典型因子,成功完成了该研究的创新设计方向以及具体方案。结论使用层次分析法和符号学理论进行二十四节气动态图案设计的创新研究,能够清晰了解设计中的典型因子,并进行设计定义和方向决策,顺利完成了研究目标,提升了二十四节气文化新的时代内涵。

苦瓜McMLO11基因克隆及其表达模式分析

【目的】克隆苦瓜McMLO11基因并研究其在白粉病和非生物胁迫下的表达规律,为阐明其在逆境调控中的作用提供参考。【方法】从苦瓜叶片中克隆McMLO11基因,利用生物信息学对其分子特征进行分析,并采用实时荧光定量PCR(qRT-PCR)方法,检测McMLO11基因在苦瓜不同组织(茎、卷须、老叶、嫩叶、根)及白粉菌侵染和盐、干旱胁迫下苦瓜真叶中的表达模式。【结果】苦瓜McMLO11基因含有1个1662 bp的开放阅读框(ORF),共编码553个氨基酸;McMLO11属于MLO基因家族,包含典型的Mlo超家族保守结构域。McMLO11蛋白的相对分子质量为63.83 ku,理论等电点为8.53,含7个跨膜域,无信号肽,属于不稳定蛋白;其二级结构主要由无规则卷曲和α-螺旋组成。系统进化树分析表明,McMLO11与黄瓜、南瓜、冬瓜等瓜类的MLO同源性较高。qRT-PCR检测发现,Mc-MLO11基因在苦瓜不同组织中的相对表达量依次表现为茎>卷须>老叶>根>嫩叶;与对照相比,白粉菌侵染后苦瓜叶片中的McMLO11表达量升高,至接种24 h达到峰值,推测其在白粉菌侵染苦瓜的早期产生应答反应;与对照相比,在盐胁迫6 h和干旱胁迫12 h时苦瓜叶片中的McMLO11相对表达量达到峰值,分别为对照的56.7和9.07倍,提示McMLO11基因可积极响应干旱和盐诱导。【结论】成功克隆了苦瓜McMLO11基因,其表达具有组织特异性,该基因可能参与白粉菌胁迫和多种非生物胁迫的调控过程。

野大麦肌动蛋白基因片段的克隆及其表达特征分析

野大麦(Hordeum brevisubulatum)是重要的野生禾本科牧草,具有多种抗逆性。为深入挖掘野大麦的抗逆基因,本研究以RT-PCR法克隆了野大麦肌动蛋白基因(Actin,ACT)的核心片段。采用BLAST序列比对,qRT-PCR实时定量方法对其序列特征和表达特性进行了分析。结果表明,野大麦ACT的基因片段长518 bp,编码171个氨基酸。BLAST分析表明,野大麦的肌动蛋白基因片段与大麦(Hordeum vulgare)ACT基因核苷酸序列的一致性达97%,与ACT蛋白氨基酸序列的同源性达91%,并将其命名为HbACT。与11种相关植物进行ACT氨基酸序列比对发现,HbACT含152个保守氨基酸和19个非保守氨基酸,说明HbACT蛋白具有高度保守性。qRT-PCR分析表明,在不同盐浓度处理条件下,不同处理时间野大麦各器官的HbACT的Ct平均值为19.30,地上部的变异系数为3.5,峰度系数为0.262;地下部的变异系数为5.3,峰度系数为0.409,均属于尖峰分布,Ct值的稳定系数均小于1.7。综上所述,HbACT基因表达稳定,可作为内参基因用于研究野大麦功能基因的表达模式分析。

洮绣纹样文化意象基因提取及其应用

为保护和传承洮绣文化,促进洮绣民间艺术与现代设计的有机融合,提出洮绣纹样文化意象基因提取与应用方法,从意象风格角度探索洮绣纹样的创新设计路径。首先,建立洮绣纹样样本库,并收集洮绣纹样的造型意象词汇与色彩意象词汇,通过语义差分法进行意象值评价,从而归纳各意象维度的典型样本;其次,分析典型样本,结合形态分析法、解构重组、图谱分析法提取造型因子和色彩因子,构建契合用户感知意象的文化基因库;再次,通过用户满意度建立目标产品与文化意象基因的关联机制,并利用形状文法进行造型推演;最后,输出融合洮绣文化内涵的产品创新设计方案。基于上述流程,以丝巾为案例开展设计实践,所得方案能够展现出洮绣纹样的特征,该流程适用于洮绣纹样的创新设计,具有可行性和有效性,可为其他刺绣纹样创新设计提供参考。

樟树WRKY转录因子分析

WRKY基因家族是一种重要的转录因子,参与植物生长发育及激素调节通路,可以正向或负向调节其他基因的表达。为了研究樟树WRKY基因家族的功能,更好地了解其对萜类物质的调控机制,研究基于已获得的樟树全基因组及不同化学型转录组数据,采用生物信息学方法对樟树WRKY转录因子进行鉴定与分析。结果表明:TPS关键基因转录水平的表达调控可能受樟树WRKY转录因子的影响。使用HMMER 3.0软件对樟树蛋白序列进行WRKY基因的检索比对,分离出61条候选CcWRKY序列。根据WRKY保守域数目和锌指结构类型,WRKY蛋白被分为Group I、GroupⅡ、GroupⅢ3种类型,其中GroupⅡ型WRKY基因依据其结构特征可进一步分为5个亚组:Ⅱa、Ⅱb、Ⅱc、Ⅱd和Ⅱe。对CcWRKY蛋白进行WRKY保守域分析,发现Ⅱ型蛋白上的3条WRKYGQK七肽系列结构上发生了突变。对CcWRKY基因的结构和表达模式进行分析,结果表明其结构可以分为不含内含子、含有1~6个内含子7种。所有CcWRKY基因在5种化学类型转录谱中均有表达,其中CCA009569.1、CCA002757.1、CCA027442.1在芳樟中的表达量远高于其他4种化学型,推测其可能参与芳樟醇的合成调控途径。

灰毡毛忍冬bZIP25基因的克隆及其表达模式分析

目的克隆灰毡毛忍冬bZIP25基因序列全长,并进行生信分析及表达模式分析,以初步探索其在灰毡毛忍冬中的生物学功能。方法通过逆转录PCR技术克隆bZIP25基因的序列全长,采用生物信息学分析方法对bZIP25及其编码的蛋白进行分析。运用实时荧光定量PCR(qRT-PCR)技术测定其在茎、叶及七个花期花中的表达水平。结果克隆得到LmbZIP25基因(OR551766),其编码191个氨基酸,具有典型的bZIP家族结构,在bZIP结构域中与其他植物同源性较高。LmbZIP25基因具有组织特异性,在茎中的表达量显著高于花和叶,在七个花期中黄色花蕾期的表达量最高。结论克隆得到LmbZIP25基因全长,分析其在不同器官和不同花期的表达差异,为进一步探索其在灰毡毛忍冬中花发育中的功能奠定了研究基础。

固本与造意:梁平传统蓝印花布纹样的当代表达

目的为梁平蓝印花布的保护与传承提供新的思路和方法,为“非遗”的创造性转化和创新性发展增添动力。方法采用文献资料、走访调查、DFA分析、计算机辅助设计等方法,对梁平蓝印花布传统纹样进行研究。首先,基于文化基因理论,对梁平蓝印花布传统纹样进行基因识别和提取;然后,利用形状文法对提取的形态基因进行推演,形成新的纹样图形;最后,将新纹样图形应用到文创产品设计上。结论固本与造意是梁平蓝印花布纹样保护与传承的基本思路,梁平蓝印花布纹样的文化基因是由纹样的形态基因、结构基因、色彩基因和意蕴基因共同构成的。以纹样的形态基因为基点,以结构基因、色彩基因和意蕴基因为约束条件,借助形状文法,可以推演出多种纹样图案,进而设计出传统与现代相结合的创新型文创产品。

荔枝蒂蛀虫海藻糖酶基因克隆及生物信息学分析

【目的】海藻糖酶(Trehalase,Tre)是昆虫体内海藻糖代谢的关键酶,通过专一性地将海藻糖分解为葡萄糖,在昆虫能量代谢和生长发育中发挥着重要的作用。旨在克隆荔枝蒂蛀虫(Conopomorpha sinensis Bradley)可溶型海藻糖酶基因(CsTre1)和膜结合型海藻糖酶基因(CsTre2),探讨其在荔枝蒂蛀虫不同发育阶段和不同组织中的表达模式,解析这2个基因及其酶蛋白的分子特征。【方法】利用荔枝蒂蛀虫转录组数据和RACE技术,克隆CsTre1和CsTre2的全长cDNA序列,并应用ORF Finder、ProtParam、SignalP 4.1、ProtScale、NetPhos2.0 Server和IQ-TREE等软件对其进行生物信息学分析;采用实时荧光定量PCR(RT-qPCR)分析CsTre1和CsTre2在荔枝蒂蛀虫不同发育阶段及成虫不同组织的mRNA表达模式。【结果】CsTre1的开放阅读框(ORF)长1701 bp,编码566个氨基酸,蛋白分子量为64.53 kD。CsTre2的ORF长1821 bp,编码606个氨基酸,蛋白分子量为69.08 kD。信号肽预测分析表明,CsTre1和CsTre2前端均有1个信号肽,其位置分别为1-16和1-17。蛋白二级结构分析结果显示,二者均主要由α-螺旋和无规则卷曲组成,CsTre1有24个Ser、15个Tyr、10个Thr可能成为蛋白激酶的结合位点,而CsTre2有27个Ser、10个Tyr、13个Thr可能成为蛋白激酶的结合位点。RT-qPCR结果显示,CsTre在荔枝蒂蛀虫的蛹和成虫期均有表达。在成虫期中,CsTre1的表达水平远高于CsTre2,且CsTre1在雄成虫第2 d和第5 d的表达水平陡然下降,在雌成虫中则保持稳定高表达。【结论】该研究成功克隆了荔枝蒂蛀虫的2个海藻糖酶基因,其分子特征及表达模式结果表明,CsTre1可能是荔枝蒂蛀虫主要调控海藻糖代谢的基因。研究结果可为阐明海藻糖酶基因的功能提供重要线索,为开展害虫防治策略的研究奠定基础。

凡纳对虾高低繁殖力群体卵巢组织学观察及相关候选基因的表达分析

在生产中,凡纳对虾(Penaeus vannamei)雌虾的繁殖性能表现出巨大差异。本研究以一个生产周期中雌虾产卵频次为繁殖力指标性状,以高繁殖力和低繁殖力雌虾为研究对象,对雌虾不同卵巢发育阶段(增殖期、小生长期、大生长期和成熟期)进行组织学观察。针对在前期选择清除分析中筛选到的繁殖候选基因TRX2、PARD3、PLCβ4和RERE,在高、低繁殖力雌虾不同卵巢发育阶段,采用实时荧光定量PCR方法,首次分析比较这些基因在卵巢、眼柄组织中的表达规律。组织观察结果显示,低繁殖力雌虾卵巢在整个生产周期中无法发育至成熟期,高繁殖力雌虾卵巢在卵黄颗粒的形成和积累、皮质棒形成等关键过程的速度要快于低繁殖力雌虾。基因表达检测结果显示,在卵巢小生长期和大生长期,TRX2、PLCβ4和RERE基因在高繁殖力组卵巢的表达量均显著高于低繁殖力组卵巢(P<0.05),4个基因在高繁殖力组成熟期卵巢中均维持较高的表达水平;在不同时期的眼柄组织中,低繁殖力组中TRX2、PARD3、PLCβ4和RERE基因的表达量均高于高繁殖力组。研究表明,上述4种基因可能在凡纳对虾卵巢发育过程中发挥重要作用。以上结果为深入研究凡纳对虾雌虾繁殖力产生差异的分子机制以及分子辅助育种提供了重要参考。

东方蜜蜂微孢子虫nce-miR-11248及其靶基因SGT1的生物信息学和表达谱研究

【目的】对前期鉴定所获得的东方蜜蜂微孢子虫的nce-miR-11248进行表达和序列验证,预测、分析其靶基因,并检测nce-miR-11248及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中的表达谱,为进一步开展nce-miR-11248调控东方蜜蜂微孢子虫侵染的功能及作用机制研究提供参考。【方法】利用Stem-loop RT-PCR和Sanger测序验证nce-miR-11248的真实性;利用相关生物信息学软件预测nce-miR-11248的靶基因,并分析其编码蛋白的分子特性;使用MEME和TBtools软件预测SGT1蛋白的保守基序和结构域,通过Mega 11.0软件进行氨基酸序列多重比对,采用邻接法构建系统进化树。采集东方蜜蜂微孢子虫孢子侵染1,2,4,6和8 d的意蜂工蜂中肠组织,采用RT-qPCR检测nce-miR-11248及其靶基因SGT1的表达谱。【结果】nce-miR-11248在东方蜜蜂微孢子虫孢子中真实存在,其序列长度为25 bp。nce-miR-11248的靶基因为SGT1。SGT1蛋白分子式为C_(765)H_(1213)N_(195)O_(241)S_(4),分子质量约为17.10 ku,脂溶系数为82.67,等电点为5.50,亲水系数为-0.709,不含典型的信号肽和跨膜结构域,可同时定位于细胞核、线粒体和过氧化物酶体。东方蜜蜂微孢子虫、家蚕微孢子虫、泛胞虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的SGT1中均含有4个保守基序(Motif 1、Motif 2、Motif 3和Motif 4)和1个结构域(SGT1超家族结构域)。东方蜜蜂微孢子虫、家蚕微孢子虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的SGT1聚为一支,且东方蜜蜂微孢子虫与家蚕微孢子虫的SGT1进化距离最近。相较于侵染东方蜜蜂微孢子虫后1 d,侵染后2,4,6和8 d nce-miR-11248的表达量均显著下调(P<0.05);侵染后2,4,6和8 d SGT1的表达量均下调,其中侵染后4,6和8 d的表达量与2 d的差异达显著水平(P<0.05)。【结论】nce-miR-11248在东方蜜蜂微孢子虫孢子中真实存在;东方蜜蜂微孢子虫与家蚕微孢子虫的SGT1亲缘关系最近;随着侵染时间的延长,nce-miR-11248及其靶基因SGT1在东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中的表达量均呈持续下降;nce-miR-11248和SGT1是潜在的参与调控微孢子虫侵染过程的因子。

普通烟草GRAS基因家族鉴定及表达模式分析

GRAS转录因子是植物中特有的转录因子,在植物的生长发育、信号转导、生物和非生物胁迫响应中均起着重要作用。本研究利用生物信息学手段从普通烟草基因组中鉴定得到了95个烟草GRAS基因,其中有50个GRAS家族成员不均匀地分布在20条染色体上。从系统进化、共线性和表达模式等方面对烟草GRAS基因家族成员进行了深入分析。结果表明,烟草GRAS基因可划分为8个亚家族,共有26个烟草GRAS成员源自全基因组复制事件,与拟南芥GRAS基因之间预测到16个共线性基因对。GRAS基因家族成员的表达具有一定的组织特异性,其启动子含有多个与生长发育、信号转导和胁迫相关的顺式作用元件,且部分成员响应盐和干旱胁迫,暗示其可能参与烟草胁迫响应等信号传导过程。本研究将为深入研究烟草GRAS家族基因的生物学功能奠定理论基础。