滴度及注射方式对2型腺相关病毒载体表达效果的影响

由于腺相关病毒载体(adeno-associated viral vector,AAV)本身的侵染不具有神经元特异性,其在神经系统相关研究中会存在侵染范围不符合试验要求的情况,因此本试验拟研究不同滴度和注射方式对病毒侵染范围的影响。结果显示,在注射较高滴度的AAV2-CMV-EGFP(1.3×10¹¹mg/L)3周后,其侵染范围可由延髓中缝苍白核(raphe pallidus nucleus,RPa)区域延伸至下丘脑室旁核(paraventricular nucleus,PVN)区域;较低滴度(1.3×10⁸mg/L)结合压力注射的方式可以在一定程度上限制AAV的侵染范围,并且载体携带的EGFP荧光基团在637 nm波段不具有明显的假阳性信号。     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

Control of insect pests using trunk injection in a newly established apple orchard

We evaluated the potential for using preplant trunk injections of emamectin benzoate in nonbearing apple trees. Trees were evaluated for pest injury and emamectin residues throughout the planting season and into the following year. Injections into the trunk best delivered emamectin benzoate to the canopy compared with injections into the taproot, and the higher rate reduced insect pests more than the lower rate. In the following year, differences in insect control between trunk and root injections were less pronounced, but the higher rate of emamectin benzoate persisted longer and better reduced pests relative to the other treatments.     

Modulation of plant root traits by nitrogen and phosphate: transporters,long-distance signaling proteins and peptides,and potential artificial traps

As sessile organisms, plants rely on their roots for anchorage and uptake of water and nutrients. Plant root is an organ showing extensive morphological and metabolic plasticity in response to diverse environmental stimuli including nitrogen (N) and phosphorus (P) nutrition/stresses. N and P are two essential macronutrients serving as not only cell structural components but also local and systemic signals triggering root acclimatory responses. Here, we mainly focused on the current advances on root responses to N and P nutrition/stresses regarding transporters as well as long-distance mobile proteins and peptides, which largely represent local and systemic regulators, respectively. Moreover, we exemplified some of the potential pitfalls in experimental design, which has been routinely adopted for decades. These commonly accepted methods may help researchers gain fundamental mechanistic insights into plant intrinsic responses, yet the output might lack strong relevance to the real situation in the context of natural and agricultural ecosystems. On this basis, we further discuss the established—and yet to be validated—improvements in experimental design, aiming at interpreting the data obtained under laboratory conditions in a more practical view.     

蛙皮素样肽家族及其受体氨基酸序列信息学比较分析

【目的】研究反刍动物蛙皮素样肽家族多肽及其受体的保守性。为不同动物,特别是反刍动物这2种蛙皮素样多肽及其受体蛋白抗原设计和活性多肽筛选等研究提供参考。【方法】采用生物信息分析学的方法,通过UniProt数据库比较牛的蛙皮素样肽家族胃泌素释放肽(Gastrin-releasing peptide, GRP)和神经介素B(Neuromedin B, NMB)2种多肽及其特异性受体GRP-R和NMB-R与其他动物氨基酸序列的差异性。【结果】13种动物成熟GRP多肽C-端的8个氨基酸序列完全相同,牛GRP氨基酸序列与绵羊、猪和豚鼠最高,分别为88.7%、77.8%和77.8%,与其他动物相似性低于74.1%。10种动物成熟NMB多肽C-端10个氨基酸序列完全相同。牛GRP-R与狗、马和猪等相似性最高,分别为96.1%、94.3%和94.0%,牛NMB-R与猪、人和马相似性最高,分别为92.3%、91.5%和91.0%;牛GRP-R与NMB-R相似性仅为62.2%。【结论】GRP和NMB的C-端氨基酸序列在不同动物之间均具有高度的保守性,而N-端为变化区域,且GRP-R和NMB-R氨基酸序列在动物之间保守性较高。     

Embryo-mediated genome editing for accelerated genetic improvement of livestock

Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomically-selected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.     

基于nanoUPLC-MS/MS的螺旋藻游离肽组成分析

为了研究螺旋藻中的多肽成分,以钝顶螺旋藻为原料,采用超滤法提取螺旋藻中的游离肽,利用nanoUPLC-MS/MS和PEAKS Studio分析谱图信息,结合NCBI数据库进行比对和从头测序分析,获得游离肽的结构组成和百分含量信息。结果表明,利用数据库比对法和从头测序法,分别得到可信的游离肽4 485个和20 597个,匹配到的蛋白质有1 036种。数据库比对结果中的游离肽主要为七肽到二十一肽,少量为二十一肽以上;从头测序结果中游离肽主要为二肽到十六肽,少量为十六肽以上。数据库比对结果中百分含量最高的游离肽是十肽,为15.95%;从头测序结果中百分含量最高的是五肽,达到24.09%。本研究结果为螺旋藻蛋白资源的进一步开发和利用提供了依据。     

Amino acid absorption and protein synthesis responses of turbot Scophthalmus maximus to lysine and leucine in free,dipeptide and tripeptide forms

The study aimed to investigate the effects of crystalline Lys and Leu, Lys‐Leu dipeptide and Lys‐Leu‐Lys/Leu‐Lys‐Leu tripeptide, on growth, postprandial Lys and Leu concentrations, gene expression of peptide and amino acid (AA) transporters and related gene expression of protein synthesis pathway in turbot. Three diets (CAA, Di and Tri) contained Lys and Leu as free (Lys and Leu), dipeptide (Lys‐Leu) and tripeptide (Lys‐Leu‐Lys/Leu‐Lys‐Leu) forms, respectively. Improved growth was observed in the Di group compared with the CAA group. For peptide and AAs transporters, PepT1, B⁰AT1 and y⁺LAT2 mRNA levels were affected in proximal intestine by dietary treatments. Free Lys and Leu concentrations in the CAA group were significantly higher than that of the Di and Tri groups at 6 hr postfeeding in serum and at 2, 6 and 24 hr postfeeding in muscle. For protein synthesis pathway in muscle, Akt2, TOR and S6k1 gene expression were the highest in the Di group and the lowest in the CAA group, whereas MuRF1 relative expression was the highest in the Tri group. In conclusion, dietary Lys‐Leu dipeptide was utilized more efficiently than free Lys and Leu or Lys‐Leu‐Lys/Leu‐Lys‐Leu tripeptide for turbot by regulating AAs transport, postprandial AAs concentration and the synthesis of muscle proteins.     

可可毛色二孢全基因组分泌蛋白的预测及分析

【目的】可可毛色二孢（Lasiodiplodia theobromae）是一种世界性分布的重要植物病原真菌,可引起严重的葡萄溃疡病（Botryosphaeria dieback）,影响果木品质并造成巨大的经济损失。本研究预测并分析可可毛色二孢基因组范围内的分泌蛋白,并明确其基本特征,为该病菌分泌蛋白致病机理的研究打下基础。【方法】依据已公布的可可毛色二孢全基因组序列,利用信号肽预测软件SignalP v5.0、跨膜结构分析软件TMHMM v2.0、细胞器定位分析软件ProtComp v9.0、GPI锚定预测软件big-PI Fungal Predictor和亚细胞器定位分析软件TargetP v2.0生物信息学软件对该菌中的典型分泌蛋白进行筛选。对分泌蛋白N端信号肽的长度、氨基酸使用频率及其切割位点进行统计分析。依据蛋白序列的同源性,应用BLASTP程序对分泌组蛋白进行功能注释分析,预测其生物学功能。采用蔗糖酶缺陷的酵母分泌系统,对所选分泌蛋白的信号肽进行活性检测。利用qRT-PCR方法检测所选分泌蛋白基因在可可毛色二孢侵染葡萄中的表达情况。【结果】在可可毛色二孢全基因组编码蛋白中共筛选获得552个潜在的具有典型信号肽的分泌蛋白,占全基因组预测蛋白总数的4.3%,其编码蛋白长度集中于101—400 aa。信号肽统计分析表明,其信号肽长度以18—20 aa的序列最为集中,信号肽长度为20 aa的蛋白数量最多。信号肽中使用频率最高的氨基酸为丙氨酸;非极性、疏水的氨基酸使用频率最高,占氨基酸总数的60.2%。其信号肽的-3至-1位置上的氨基酸相对保守,切割位点属于A-X-A类型,可被Sp I型信号肽酶识别并切割。336个分泌蛋白具有功能注释,其功能较多集中于细胞壁降解有关的酶类以及致病相关蛋白,并且这些蛋白在分子量、等电点、脂肪族氨基酸指数等方面均存在差异。通过蔗糖酶缺陷的酵母分泌系统证实,挑选的9个分泌蛋白信号肽均具有分泌活性。qRT-PCR检测结果表明,所选分泌蛋白基因在该病菌侵染初期的表达发生变化。【结论】利用生物信息学分析技术从可可毛色二孢全基因组中共预测获得552个经典分泌蛋白。其信号肽氨基酸长度分布广泛,氨基酸组成中非极性、疏水的氨基酸使用频率最高。功能注释主要集中在细胞壁组分降解相关的酶类、致病侵染相关的坏死诱导相关蛋白以及几丁质结合蛋白等。