金针菇成熟期和伸长期菌盖的转录组与蛋白组比较分析

菌盖是大型真菌的重要组成部分,也是其产生有性孢子的部位,但是其发育机制仍不明确。本研究以金针菇Flammulina filiformis为材料,采用转录组和蛋白组联合分析的方法,比较分析了金针菇成熟期和伸长期菌盖的差异基因与蛋白,并对其进行GO (gene ontology)功能聚类分析、KEGG (Kyoto encyclopedia of genes and genomes)富集分析和蛋白互作网络分析。本研究筛选到差异表达基因有1 391个,差异表达蛋白147个,均以上调表达为主。GO功能聚类分析结果表明,催化活性(catalytic activity)条目富集基因最多,其次是细胞组分(cell part)、细胞过程(cellular process)和细胞器(organelle)。KEGG富集分析结果表明,差异表达基因和蛋白主要富集在碳水化合物代谢通路(carbohydrate metabolism)和氨基酸代谢通路(amino acid metabolism)等。本研究选取了9个关键的差异表达基因,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)对其表达量进行了验证。RT-qPCR验证结果与转录组测序结果相一致。蛋白互作网络分析表明,水解酶类、结构域类和转录调节类蛋白为互作网络的主要结点。本研究联合转录组、蛋白组测序数据,通过分析差异基因与蛋白,为深入了解金针菇菌盖发育机制提供数据参考。     

金针菇菌柄发育的转录组与蛋白组分析

菌柄是金针菇等食用菌的主要商品部位,但其生长机制仍不明确。本研究对金针菇伸长期和成熟期菌柄进行了转录组联合蛋白组分析,结果显示,两样本显著性差异表达基因和蛋白分别为721个和61个,均以上调表达为主。GO（gene ontology）功能聚类分析表明：有72.41%的差异表达基因富集在催化活性（catalytic activity）条目下。细胞组分（cell part）和绑定结合（binding）条目同时富集了较多的差异表达基因和蛋白。KEGG通路富集分析显示：碳水化合物代谢通路（carbohydrate metabolism）和氨基酸代谢通路（amino acid metabolism）富集的差异表达基因较多。差异表达蛋白富集较多的通路是单环菌素生物合成（monobactam biosynthesis,ko00261）、链霉素生物合成（streptomycin biosynthesis,ko00521）和有机含硒化合物代谢（selenocompound metabolism,ko00450）等。内质网蛋白质加工（protein processing in endoplasmic reticulum,ko04141）和MAPK信号通路（MAPK signaling pathway-yeast,ko04011）在转录组和蛋白组的KEGG富集分析中均为差异通路。本研究联合转录组和蛋白组数据筛选了40个金针菇菌柄发育中差异表达基因,为深入研究揭示食用菌菌柄发育过程提供候选基因。     

Combined transcriptomic and proteomic analysis of developmental features in the immune system of Plutella xylostella during larva-to-adult metamorphosis

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is considered one of the most destructive worldwide agricultural pests and has developed various defence mechanisms to fight against the available pesticides. Understanding the host-defence system of P. xylostella is vital for developing biocontrol-based pest management strategies. Although there are several studies on P. xylostella, little is known about the changes in the immune system during the larva-to-adult metamorphosis. RNA-seq and iTRAQ investigations of P. xylostella from 2-day-old fourth instar larvae (L4D2), pupa (P0), and adult (A0) were done to understand these alterations at a molecular level. A total of 412/ 584 up-regulated and 1430/ 757 down-regulated genes/proteins between larva and pupa, 813/ 589 up-regulated and 1206/ 846 down-regulated genes/proteins between pupa and adult were identified. It was shown that the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) expression were up-regulated during the pupation and emergence of metamorphosis. The pathway enrichment analysis demonstrated that DEGs and DEPs were mainly associated with the energy generation and metabolism and innate immunity of the insect. The expression of immune-related and developmental-related genes were significantly different during the developmental process of P. xylostella. Moreover, the expression of four focused genes, i.e., serine proteinase inhibitor (Serpin-15), prophenoloxidase activating proteinase 1 (PAP-1) and 3a (PAP-3a), Gram-negative bacteria-binding protein (GNBP-6), was different in developmental stages and after Bacillus thuringiensis HD73 and Metarhizium anisopliae infection. The phenoloxidase (PO) activity in plasma was also significantly up-regulated during the pathogen infection. Recombinant proteins PAP-1, PAP-3a, GNBP-6 could significantly trigger the PO activity in vitro, Serpin-15 could suppress the PO activity. Taken together, these results indicate that Serpin-15, PAP-1, PAP-3a, and GNBP-6 might have the potential for co-regulation of immunity and development in P. xylostella. In conclusion, this study provided the immune system dynamics in the developmental process of P. xylostella and identified four candidate genes that can serve as potential targets for pest control strategies.     

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.     

RNA-seq analysis reveals a key role of brassinolide-regulated pathways in NaCl-stressed cotton

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.     

A high‐confidence reference dataset of differentially expressed proteins in elongating cotton fiber cells

In our previous study, we used a comparative proteomic approach based on 2DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post‐anthesis. Of them, only 106 differentially expressed proteins (DEPs) were identified by MS due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high‐resolution MS with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high‐confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.     

巴氏蘑菇子实体不同发育阶段的转录组分析

为探讨巴氏蘑菇子实体不同发育阶段基因的表达情况,本研究对巴氏蘑菇子实体不同发育时期（原基、采收期和开伞期）进行转录组测序,以本实验室已获得的巴氏蘑菇JA菌株的不育单孢菌株JA-15036基因组为参考基因组研究原基与采收期及开伞期样本间差异表达基因,并对差异表达基因进行了GO功能和Pathway富集分析。GO功能分析结果显示,差异表达基因主要富集在跨膜转运、碳水化合物代谢途径和膜组分,它们协同调控为子实体生长发育提供稳定的内环境。KEGG富集分析结果表明,原基期上调的差异表达基因主要富集在核糖体蛋白和DNA复制,表明原基期细胞代谢旺盛,其中核糖体蛋白基因上调为后期蛋白质合成提供重要场所;采收期和开伞期子实体时期差异表达基因主要富集在碳水化合物代谢、脂肪酸降解和氨基酸代谢等途径,为巴氏蘑菇子实体的生长发育与成熟提供营养与能量。     

卵巢成熟期和退化期星斑川鲽脑垂体和下丘脑组织的转录组比较分析

为了解星斑川鲽（Starry Flounder,Platichthys stellatus）脑组织基因表达与卵巢发育调控的关系,发掘相关功能基因,研究提取卵巢成熟期和退化期星斑川鲽雌鱼脑垂体和下丘脑组织总RNA,运用HiSeq 2000高通量测序技术进行转录组测序分析。测序结果经拼接组装后共获得30640条Unigenes,Blast同源性比对显示,其中24128条Unigenes获得注释;经eggNog功能注释后29137条Unigenes序列分为26类,分别涉及信号转导、翻译机制等生理生化过程。KEGG pathway数据比对显示,卵巢成熟期涉及98种代谢途径,135条序列表达上调;卵巢退化期涉及192种代谢途径,648条序列表达上调。Unigenes表达量及表达差异分析表明在卵巢成熟期334条序列表达上调,卵巢退化期987条序列表达上调。获得功能注释的Unigene中,408条涉及生殖调控和内分泌调控,参与生殖调控的信号分子有1508条。试验采用实时定量PCR研究了涉及生殖调控和内分泌调控基因促性腺激素释放激素（GnRH）、神经激肽B（NKB）、促性腺激素（GtH）、促滤泡激素（FSH）、肿瘤转移抑制因子（Kiss）以及催乳素释放肽受体（PrRPR）在卵巢两个不同发育时期脑垂体和下丘脑的表达情况,结果表明除FSH外,其余均在卵巢退化期时期脑组织中表达升高,与转录组测序结果趋势一致。