广西麻鸡FOXL2基因的克隆、序列分析及在高低产蛋量卵巢中的表达

为了研究鸡FOXL2基因的结构和功能,本研究克隆了广西麻鸡FOXL2基因编码区序列,分析了其突变位点及与其他物种的同源性和进化距离,以及在高产组和低产组母鸡卵巢组织中的表达水平.结果 表明:广西麻鸡FOXL2基因的编码区长度为918 bp,共编码305个氨基酸,存在一处错义突变和两处同义突变.通过物种间的同源性分析显示,广西麻鸡FOXL2基因与原鸡(Gallus gallus)、雉鸡(Phasianus colchicus)、绿头鸭(Anas platyrhynchos)、野鸽(Columba livia)、人(Homo sapiens)、小鼠(Mus musculus)的同源性分别为99.7％、98.8％、95.1％、92.0％、75.7％、75.5％.通过种间进化树分析表明,广西麻鸡与原鸡(Gallus gallus)的亲缘关系最近,与小鼠(Mus musculus)的亲缘关系最远.FOXL2在高低产蛋量鸡卵巢中的表达水平结果显示:FOXL2基因在高产蛋组中的表达量显著高于低产蛋组中的表达量(P＜0.05).该研究结果提示鸡FOXL2的序列相对较保守,对卵巢功能的维持和提高产蛋量具有功能性作用.     

引入新型异戊二烯醇利用途径促进解脂耶氏酵母中β-胡萝卜素的合成*

目的:微生物体内异戊二烯类化合物的前体物异戊烯焦磷酸酯的天然合成路径受到严格的代谢调控,因此限制了异戊二烯类化合物的高效生物合成,而新型异戊二烯醇利用途径独立于生物体内源性代谢路径,通过在微生物中引入IUP能够进行异戊烯焦磷酸酯的大量合成,从而促进异戊二烯类化合物的大量合成。方法:在油脂酵母解脂耶氏酵母中引入IUP,强化异戊烯焦磷酸酯生物合成,促进β-胡萝卜素的高效积累。结果:通过生物信息学的方法预测IUP中两个关键蛋白酿酒酵母来源的胆碱激酶ScCK和拟南芥来源的异戊烯磷酸激酶AtIPK,均为酸性亲水性蛋白,无跨膜区和信号肽,二者都具有疏松不稳定的结构特征,显著富集于磷酸类物质的合成通路中。在解脂耶氏酵母中利用同源重组技术引入外源β-胡萝卜素合成关键基因carRP和carB,强化甲羟戊酸途径的关键基因thmgR和ggs1,使工程菌株中积累2.68 mg/L β-胡萝卜素。通过Cre-loxP系统回收基因组上的ura标签,再将IUP进一步整合到工程菌株染色体上。当培养基中含有20 mM异戊二烯醇作为底物、碳氮比为4/3且发酵96 h后,重组解脂耶氏酵母中β-胡萝卜素的产量提高到410.2 mg/L,较原始工程菌的产量提高了近200倍。结论:IUP能够促进解脂耶氏酵母中β-胡萝卜素的高效积累,为利用IUP开展β-胡萝卜素和其他异戊二烯类化合物的高效生物合成提供新思路。     

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.     

Autophagy in plants: Physiological roles and post‐translational regulation

In eukaryotes, autophagy helps maintain cellular homeostasis by degrading and recycling cytoplasmic materials via a tightly regulated pathway.Over the past few decades, significant progress has been made towards understanding the physiological functions and molecular regulation of autophagy in plant cells. Increasing evidence indicates that autophagy is essential for plant responses to several developmental and environmental cues, functioning in diverse processes such as senescence, male fertility, root meristem maintenance, responses to nutrient starvation,and biotic and abiotic stress. Recent studies have demonstrated that, similar to nonplant systems,the modulation of core proteins in the plant autophagy machinery by posttranslational modifications such as phosphorylation, ubiquitination,lipidation, S-sulfhydration, S-nitrosylation, and acetylation is widely involved in the initiation and progression of autophagy. Here, we provide an overview of the physiological roles and posttranslational regulation of autophagy in plants.     

Targeted sequencing and integrative analysis of 3,195 Chinese patients with neurodevelopmental disorders prioritized 26 novel candidate genes

Neurodevelopmental disorders(NDDs) are a set of complex disorders characterized by diverse and cooccurring clinical symptoms. The genetic contribution in patients with NDDs remains largely unknown.Here, we sequence 519 NDD-related genes in 3,195 Chinese probands with neurodevelopmental phenotypes and identify 2,522 putative functional mutations consisting of 137 de novo mutations(DNMs) in 86 genes and 2,385 rare inherited mutations(RIMs) with 22 X-linked hemizygotes in 13 genes, 2 homozygous mutations in 2 genes and 23 compound heterozygous mutations in 10 genes. Furthermore, the DNMs of16,807 probands with NDDs are retrieved from public datasets and combine in an integrated analysis with the mutation data of our Chinese NDD probands by taking 3,582 in-house controls of Chinese origin as background. We prioritize 26 novel candidate genes. Notably, six of these genes d ITSN1, UBR3, CADM1,RYR3, FLNA, and PLXNA3 d preferably contribute to autism spectrum disorders(ASDs), as demonstrated by high co-expression and/or interaction with ASD genes confirmed via rescue experiments in a mouse model. Importantly, these genes are differentially expressed in the ASD cortex in a significant manner and involved in ASD-associated networks. Together, our study expands the genetic spectrum of Chinese NDDs,further facilitating both basic and translational research.     

Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.     

PLCγ1 inhibition-driven autophagy of IL-1β-treated chondrocyte confers cartilage protection against osteoarthritis,involving AMPK,Erk and Akt

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1β for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.     

Digestion-Promoting Effects and Mechanisms of Dashanzha Pill Based on Raw and Charred Crataegi Fructus

Emerging evidence suggests that a high-fat diet (HFD) can influence endoplasmic reticulum (ER) stress and gut microbiota. Crataegi Fructus is a traditional Chinese herb widely used in formulas for dyspepsia, with Dashanzha Pill composed of raw Crataegi Fructus (DR) being a representative drug. Processing products of Crataegi Fructus, however, have a stronger pro-digestive effect, and we hypothesized that Dashanzha Pill composed of charred Crataegi Fructus (DC) is more effective. We found that the contents of glucose 1-phosphate and luteolin in DR and DC were substantially different via ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry. DC outperformed DR in improving histopathological changes, increasing gastrin and motilin, and decreasing vasoactive intestinal peptides in rats with HFD induced dyspepsia. Fecal microbiota analysis revealed that DC could restore the disturbed intestinal microbiota composition, including that of Bacteroides, Akkermansia, and Intestinimonas to normal levels. Furthermore, DC significantly reduced the mRNA and protein levels of glucose-regulated protein 78, protein kinase R-like ER kinase, and eukaryotic initiation factor 2α. Taken together, DC outperformed DR in relieving dyspepsia by regulating gut microbiota and alleviating ER stress.