Viperin (Virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is a broad-spectrum antiviral protein widely involved in vertebrate innate immune regulation. This study cloned the 5' flanking promoter region (2010 bp) of the viperin gene (named as LcViperin) in large yellow croaker (Larimichthys crocea) and analyzed its promoter characteristics through bioinformatics and dual-luciferase reporter assays. Results indicated that the LcViperin promoter region contains two TATA boxes and several critical transcription factor binding sites, including interferon-stimulated response elements (ISREs), GATA-binding factor 1 (GATA1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon regulatory factor 7 (IRF7), and signal transduction and activator of transcription 1 (STAT1). Promoter region truncation experiments in HEK 293 T cells further confirmed that the core promoter region is located between -331 and - 121 bp. LcViperin overexpression significantly activated IRF3, IRF7, and IFN1 promoters in a dose-dependent manner. Domain deletion experiments demonstrated that the N-terminal and SAM (S-adenosyl-L-methionine) domains play key roles in promoter activation. Additionally, co-transfection with LcViperin and IRAK1 (interleukin-1 receptor-associated kinase 1, named as LcIRAK1) significantly enhanced IRF3, IRF7, and IFN1 promoter activity. This study reveals the regulatory characteristics of the large yellow croaker viperin promoter and its role in the interferon signaling pathway, providing a theoretical basis for understanding its immune regulatory mechanism and improving disease resistance in large yellow croaker. It also offers a scientific basis for understanding viperin regulation and its application in disease prevention in aquaculture.

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), also known as RSAD2 (radical SAM domain-containing 2), is one of the interferon-stimulated genes and plays an important role in cellular antiviral response. In this study, the viperin gene in large yellow croaker (Larimichthys crocea), designated LcViperin, was identified and characterized by investigating its tissue expression pattern and transcriptional response to viral infection and analyzing the differentially expressed genes (DEGs) upon overexpression of LcViperin in LCM10 cells revealed by RNA-seq transcriptome profiling. The results showed that LcViperin was highly expressed in the blood and organs of the immune system such as spleen and kidney, and the LcViperin protein was localized to the endoplasmic reticulum. The transcript level of LcViperin can be upregulated by large yellow croaker iridovirus (LYCIV) infection and the simulated viral infection of poly (I: C). GO and KEGG analysis of the DEGs demonstrated that the overexpression of LcViperin activated macrophage endocytosis pathway and other pathways to eliminate the viral particles. Flow cytometry analysis indicated an increase in macrophage phagocytotic activity and an induced respiratory burst in LCM10 cells upon exposure to LcViperin protein. The transcriptome analysis results would help to understand the host immune response and the potential mechanism of fish Viperin protein in response to pathogen stimulation.

Cytoplasmic male sterility (CMS) is critical in maximizing crop yield and quality by utilizing tobacco heterosis. However, the mechanism of tobacco CMS formation remains unknown. Using paraffin section observation, transcriptome sequencing, and TMT proteomic analysis, this study describes the differences in expression profiles in morphology, transcription, and translation between the sua-CMS tobacco line (MSYY87) and its corresponding maintainer line (YY87). According to the microspore morphology, MSYY87 began to exhibit abnormal microspore development during the early stages of germination and differentiation (androgynous primordium differentiation stage). According to transcriptomic and proteomic analyses, 17 genes/proteins involved in lipid transport/binding and phenylpropane metabolism were significantly down-regulated at both the mRNA and protein levels. Through further analysis, we identified some key genes that may be involved in tobacco male sterility, including β-GLU related to energy metabolism, 4CL and bHLHs related to anther wall formation, nsLTPs related to pollen germination and anther cuticle, and bHLHs related to pollen tapetum degradation. We speculate that the down-regulation of these genes affects the normal physiological metabolism, making tobacco plants show male sterility. SIGNIFICANCE: Cytoplasmic male sterility (CMS) plays a vital role in utilizing tobacco heterosis and enhancing crop yield and quality. We observed paraffin sections and conducted transcriptome sequencing and mitochondrial proteomics to examine the tobacco CMS line Yunyan 87 (MSYY87) and its maintainer line Yunyan 87 (YY87). The down-regulation expression of β-GLU resulted in insufficient ATP supply, which resulted in disordered energy metabolism. The down-regulation expression of 4CL, nsLTPs and bHLHs may affect the formation of anther wall and anther cuticle, pollen germination, as well as the degradation of pollen tapetum. These various abnormal physiological processes, the male sterility of tobacco is finally caused. The findings shed light on the molecular mechanisms of tobacco CMS and serve as a model for fertility research in other flowering plants.

In order to support the optimal cultivation technology of the New Flue-cured Tobacco Variety Yunyan 121 and promote the popularization and application of the variety, Weining County, a representative tobacco area in western Guizhou, which is suitable for the cultivation of Yunyan 121, was selected. Three factors of nitrogen application rate, density and number of leaves were set. The effects of different nitrogen application rate, density and number of leaves on the yield and quality of Yunyan 121 were analyzed by using quadratic saturated D-optimal design, index sum method and regression analysis, The optimum combination of nitrogen application rate, density and leaf number of Yunyan 121 was found out. The results showed that the yield and quality of Yunyan 121 were significantly affected by different nitrogen application rate, density and leaf number. When the nitrogen application rate was 98.80 kg/hm~2, the density was 15 247 plants/hm~2 and the leaf number was 16 pieces/plant, Yunyan 121 had the highest yield per mu（1 941.53 kg/hm~2）; When the nitrogen application rate was109.9 kg/hm~2, the density was 14 993 plants/hm~2 and the number of leaves left was 16 pieces/plant, Yunyan 121 had the highest output value per mu（53 286.36 yuan/hm~2）; When the nitrogen application rate was 85.72 kg/hm~2, the density was 17 571 plants/hm~2 and the number of leaves left was 19 pieces/plant, Yunyan 121 had the highest comprehensive quality value（92.1）. According to the yield, output value and comprehensive quality value of tobacco leaves, Yunyan 121 is planted in the Western tobacco area of Guizhou. When the nitrogen application rate is 98.2 kg/hm~2, the density is 15 937 plants/hm~2 and the number of leaves left is 17 pieces/plant, the yield of tobacco leaves can be1 922.64 kg/hm~2, the output value is 52 258.92 yuan/hm~2 and the comprehensive quality value is 89.78, which can give consideration to the yield and quality of tobacco leaves.

Heterosis has greatly improved the yield and quality of crops. However, previous studies often focused on improving the yield and quality of the shoot system, while research on the root system was neglected. We determined the root numbers of 12 F 1 hybrids, all of which showed strong heterosis, indicating that tobacco F 1 hybrids have general heterosis. To understand its molecular mechanism, we selected two hybrids with strong heterosis, GJ (G70 × Jiucaiping No.2) and KJ (K326 × Jiucaiping No.2), and their parents for transcriptome analysis. There were 84.22% and 90.25% of the differentially expressed genes were overdominantly expressed. The enrichment analysis of these overdominantly expressed genes showed that “Plant hormone signal transduction”, “Phenylpropanoid biosynthesis”, “MAPK signaling pathway - plant”, and “Starch and sucrose metabolism” pathways were associated with root development. We focused on the analysis of the biosynthetic pathways of auxin(AUX), cytokinins(CTK), abscisic acid(ABA), ethylene(ET), and salicylic acid(SA), suggesting that overdominant expression of these hormone signaling pathway genes may enhance root development in hybrids. In addition, Nitab4.5_0011528g0020、Nitab4.5_0003282g0020、Nitab4.5_0004384g0070 may be the genes involved in root growth. Genome-wide comparative transcriptome analysis enhanced our understanding of the regulatory network of tobacco root development and provided new ideas for studying the molecular mechanisms of tobacco root development.

Potassium (K+) is essential for crop growth. Increasing the K+ content can often directly promote the improvement of crop yield and quality. Heterosis plays an important role in genetic improvement and leads to genetic gains. We found that the K+ content of tobacco showed significant heterosis, which is highly significant for cultivating tobacco varieties with high K+ content. However, the mechanism by which K+ content heterosis occurs in tobacco leaves is not clear. In this study, a comprehensive comparative transcriptome sequencing analysis of root samples from the hybrid G70 × GDH11 and its parental inbred lines G70 and GDH11 was performed to elucidate the importance of the root uptake capacity of K+ in the formation of heterosis. The results showed that 29.53% and 60.49% of the differentially expressed genes (DEGs) exhibited dominant and over-dominant expression patterns, respectively. These non-additive upregulated DEGs were significantly enriched in GO terms, such as metal ion transport and reaction, ion balance and homeostasis, ion channel activity, root meristem growth, and regulation of root hairs. The KEGG annotation results indicated that these genes were mainly involved in the pathways such as energy metabolism, carbohydrate formation, amino acid metabolism, and signal transduction. Further analysis showed that probable potassium transporter 17 (NtKT17) and potassium transporter 5-like (NtKT5), associated with potassium ion absorption, glutamate receptor 2.2-like and glutamate receptor 2.8-like, associated with ion channel activity, LOC107782957, protein detoxification 42-like, and probable glutamate carboxypeptidase 2, associated with root configuration, showed a significantly higher expression in the hybrids. These results indicated that the over-dominant expression pattern of DEGs played a key role in the heterosis of K+ content in tobacco leaves, and the overexpression of the genes related to K+ uptake, transport, and root development in hybrids helped to improve the K+ content of plants, thus showing the phenomenon of heterosis.

含梗率是烟叶的重要经济性状和物理特性指标,其测定方法过程繁琐、时间长,在品种选育中难以应用于大量品系鉴定和选择.为快速地测定含梗率,本研究设置杀青和烘烤干燥方式、叶梗分离和不分离处理以及0、5％、10％、15％和20％共5种烤后烟叶平衡含水率处理,测定不同品种间各处理的烟叶含梗率.结果表明,不同品种间烟叶含梗率相差极显著,烟草叶片梗的生长发育受遗传控制,说明选育含梗率低的品种是降低烟叶含梗率的有效途径;采用烘烤干燥方式的烟叶含梗率极显著高于杀青干燥方式,两种干燥方式间的烟叶含梗率差异极显著,相关系数为0.98,回归方程为y=1.615 9x+5.203 9;叶梗分离和不分离干燥后的烟叶含梗率差异极显著,叶梗分离后杀青干燥与不分离烘烤的烟叶含梗率的差异达到极显著水平,相关系数为0.93,回归方程为y=1.2299x-2.0705;当含水率在0～20％时,烟叶含梗率测定结果差异不显著.因此,在研究烟叶含梗率的遗传和进行新品系烟叶含梗率鉴定选择时,可采用将成熟烟叶的叶梗分离后进行杀青干燥,冷却后即可测定烟叶含梗率,根据测定值与对照品种烟叶含梗率的高低进行取舍,或采用回归方程预测该品系烘烤后的烟叶含梗率.成熟烟叶叶梗分离后杀青干燥测定烟叶含梗率的方法能提早鉴定时间、提高鉴定效率,可及时、快速、准确地对育种材料烟叶含梗率进行鉴定选择.

Nicotine is a unique alkaloid present in tobacco that is widely used in cigarettes and in the agricultural, chemical, and pharmaceutical industries. However, the research on nicotine is mostly limited to its synthesis pathways, and only a few studies have explored the effects of other metabolic pathways on nicotine precursors. Regulating the nicotine content in tobacco can greatly promoting the application of nicotine in other fields. In this study, we performed global data-independent acquisition proteomics analysis of four tobacco varieties. Of the four varieties, one had high nicotine content and three had a low nicotine content. A total of 31,259 distinct peptides and 6,018 proteins across two samples were identified. A total of 45 differentially expressed proteins (DEPs) co-existed in the three comparison groups and were mainly involved in the transport and metallic processes of the substances. Most DEPs were enriched in the biosynthesis of secondary metals, glutathione metabolism, carbon metabolism, and glycolysis/gluconeogenesis. In addition, the weighted gene co-expression network analysis identified an expression module closely related to the nicotine content (Brown, r = 0.74, P = 0.006). Gene Ontology annotation and Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that the module proteins were mainly involved in the synthesis and metabolism of nicotine precursors such as arginine, ornithine aspartate, proline, and glutathione. The increased levels of these precursors lead to the synthesis and accumulation of nicotine in plants. More importantly, these proteins regulate nicotine synthesis by affecting the formation of putrescine, which is the core intermediate product in nicotine anabolism. Our results provide a reference for tobacco variety selection with a suitable nicotine content and regulation of the nicotine content. Additionally, the results highlight the importance of other precursor metabolism in nicotine synthesis.

Background Potassium(K + ) plays a vital role in improving the quality of tobacco leaves. However, how to improve the potassium content of tobacco leaves has always been a difficult problem in tobacco planting. K + content in tobacco hybrid is characterized by heterosis, which can improve the quality of tobacco leaves, but its underlying molecular genetic mechanisms remain unclear. Results Through a two-year field experiment, G70×GDH11 with strong heterosis and K326×GDH11 with weak heterosis were screened out. Transcriptome analyses revealed that 80.89% and 57.28% of the differentially expressed genes (DEGs) in the strong and weak heterosis combinations exhibited an overdominant expression pattern, respectively. The genes that up-regulated the overdominant expression in the strong heterosis hybrids were significantly enriched in the ion homeostasis. Genes involved in K + transport (KAT1/2, GORK, AKT2, and KEA3 ), activity regulation complex ( CBL-CIPK5/6 ), and vacuole ( TPKs ) genes were overdominant expressed in strong heterosis hybrids, which contributed to K + homeostasis and heterosis in tobacco leaves. Conclusions K + homeostasis and accumulation in tobacco hybrids were collectively improved. The overdominant expression of K + transport and homeostasis-related genes conducted a crucial role in the heterosis of K + content in tobacco leaves.

钾含量是烟叶重要的品质指标之一,研究烟叶钾含量杂种优势形成的原因,对高钾含量杂交种的选育具有重要意义.本研究选用钾含量差异较大的12个烟草品种(系)为亲本,按照NCⅡ遗传交配设计组配出35个杂交组合,分析了烟叶钾含量杂种优势的表现,并探讨了烟叶钾含量杂种优势相关基因表达差异.结果表明:烟叶钾含量主要受非加性效应基因的影响,具有明显的杂种优势,中亲优势变幅为-38.06％～29.74％.AKT1、NtKC1和NtTPK1基因在烟叶中的中亲相对表达量与烟叶钾含量杂种优势显著相关,相关系数分别为0.86、0.84和0.86;AKT1和KAT1基因在根系中的中亲相对表达量与烟叶钾含量杂种优势的相关性达到了显著水平,相关系数分别为0.80和0.77.与亲本相比,杂种优势相关基因AKT1和NtKC1在烟叶和根系中均呈正向超显性表达模式,tTPK1在烟叶和根系中均呈正向显性表达模式,KAT1基因在烟叶中呈正向显性表达模式,在根系中呈正向超显性表达模式.说明内流型钾离子通道基因AKT1、NtKC1、KAT1和钾离子转运蛋白基因NtTPK1在杂交种根系和叶片中的显性和超显性表达,在杂交种烟叶钾含量优势的形成中起了关键作用.

[目的]探究烟草叶片数杂交优势表现,并分析烟草叶片数相关基因的差异表达情况及杂种优势形成的原因,为深入研究烟草叶片数的分子遗传基础和选育叶片数较多的杂交种提供理论依据.[方法]以叶片数差异较大的9个烟草品种(系)为亲本,按照NCⅡ遗传交配设计组配20个杂交组合,并测定亲本和杂交组合的叶片数,计算其杂种优势,从中筛选出强、弱优势组合,利用实时荧光定量PCR检测其叶片相关基因BRI1、BSK3、FLC、FPF1和PHYC的相对表达量.最后,对叶片数相关基因中亲表达优势间及其与叶片数中亲优势进行相关分析.[结果]9个亲本材料的叶片数为20.33～33.22片,以GDH94的叶片数最多,其次是南江三号和毕纳1号,三者间无显著差异(P＞0.05,下同),但GDH94显著高于其余6个亲本(P＜0.05,下同),表明供试亲本间的叶片数存在真实的遗传差异.20个杂交组合的叶片数存在明显差异,为20.89～31.33片,以GDH94×南江三号的叶片数最多,以NC82×青梗的叶片数最少,说明采用杂种优势育种方法可选育出烟草叶片数较多的杂交种.20个杂交组合叶片数的杂种优势差异较大,其中中亲优势为-14.71%～11.77%,表现为正向中亲优势和负向中亲优势的组合分别占25%和75%,其中,以K326×GDH88叶片数的正向中亲优势最强,为11.77%,以GDH94×湄潭大蛮烟叶片数的负向中亲优势最强,为-14.71%;NC82×南江三号叶片数的中亲优势最弱,为-0.22%,故选择K326×GDH88和GDH94×湄潭大蛮烟为强优势组合、NC82×南江三号为弱优势组合.不同叶片数相关基因的中亲表达优势之间存在一定的相关性,其中BRI1和BSK3基因的中亲表达优势之间存在显著正相关;FPF1和PHYC基因的中亲表达优势与叶片数杂种优势存在显著负相关.FPF1基因在正向强优势杂交组合K326×GDH88和弱优势组合NC82×南江三号中较其相应亲本下调表达,但负向强优势组合GDH94×湄潭大蛮烟较其亲本上调表达. PHYC基因在正向强优势组合K326×GDH88和弱优势组合NC82×南江三号中较其相应亲本下调表达,但在负向强优势组合GDH94×湄潭大蛮烟较其亲本上调表达.[结论]K326×GDH88组合的叶片数杂种优势最大,具有较大的高产潜力.FPF1和PHYC基因参与调控烟草叶片数杂种优势的形成,其下调表达是烟草叶片数性状杂种优势形成的分子基础,可指导亲本选配,提高烟草杂交选育效率.

杂种优势利用是提高作物产量和品质的一种重要育种方法,明确生态条件和打顶措施对杂种优势的影响,对提高烟草杂种优势研究利用效率有重要意义.本试验以烟叶烟碱含量杂种优势相差较大的3个杂交组合及其亲本为材料,选择生态条件差异较大的3个生态试验点,研究了不同生态环境和打顶措施对烟叶烟碱含量、烟叶烟碱含量杂种优势及其相关基因相对表达量的影响.结果表明,不同生态环境间的烟叶烟碱含量差异显著,烟株打顶前后烟叶烟碱含量发生了显著的变化,打顶处理的烟叶烟碱含量显著高于不打顶处理;不同生态环境、打顶和不打顶处理间的烟叶烟碱含量的中亲优势值差异不显著,而超亲优势值、超标优势值差异显著;不同生态环境、打顶和不打顶处理间,烟叶烟碱含量杂种优势相关的PM T、A O、QS基因的中亲优势表达量相差也不显著,与处理间中亲优势值的表现差异一致.结果表明,在选用中亲优势作为度量指标进行杂种优势的遗传研究中,可不考虑生态环境和打顶的影响,在一个试验点对双亲及F1采用相同的农艺措施就能获得较为准确的鉴定结果;而在选用超亲优势、超标优势作为度量指标进行杂交种应用潜力评价中,需在不同生态环境、不同农艺措施下进行鉴定.

[目的]分析数据非依赖性采集高分辨率色谱—质谱技术(DIA LC-MS)对根尖差异蛋白质样品检测的适用性,为深入研究烟草蛋白质组学提供技术支撑.[方法]以6个烟草品种(系)移栽后74 d的根尖组织为试验材料,采用DIA LC-MS法对裂解的蛋白质肽段进行扫描,从而分析该方法对根样组织的适用性.[结果]采用TCA/丙酮沉淀+SDT裂解法提取根尖总蛋白质,能得到横向条带平行性好,电泳条带清晰的蛋白质样品,样品等级评价均达到A级.构建的DDA数据库包含28042条肽段,共鉴定出7084个蛋白质.DIA数据的色谱峰平均数据点为7,平均的峰容量达260,主要肽段的保留时间(iRT)均被检测出,且整体较稳定,在Q值为0.01标准下的一致性评分(Cscore)为0.948,变异系数(CV)中值为11.5％(小于20.0％),相关系数大于0.9.DIA全息扫描模式下从6个烟草品种(系)的根尖组织中鉴定出的蛋白质数目为4149～5069个.[结论]DIA LC-MS分析鉴定的蛋白质范围广、数量大、可信度高,具有一定的可靠性和稳定性,各项质控指标均能达到试验分析要求,说明DIA LC-MS适用于烟草根尖蛋白质组样品的检测分析.