基于Golden Gate技术的齐整小核菌转录单元高效组装系统

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术,以mobius assembly为基础,分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体,从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体,4个用于单转录单元组装的Level 1载体,4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力,为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。     

Deciphering RNA m6A regulation in aging: Perspectives on current advances and future directions

N⁶-methyladenosine (m⁶A) is a dynamic and reversible RNA modification that has emerged as a crucial player in the life cycle of RNA, thus playing a pivotal role in various biological processes. In recent years, the potential involvement of RNA m⁶A modification in aging and age-related diseases has gained increasing attention, making it a promising target for understanding the molecular mechanisms underlying aging and developing new therapeutic strategies. This Perspective article will summarize the current advances in aging-related m⁶A regulation, highlighting the most significant findings and their implications for our understanding of cellular senescence and aging, and the potential for targeting RNA m⁶A regulation as a therapeutic strategy. We will also discuss the limitations and challenges in this field and provide insights into future research directions. By providing a comprehensive overview of the current state of the field, this Perspective article aims to facilitate further advances in our understanding of the molecular mechanisms underlying aging and to identify new therapeutic targets for aging-related diseases.     

Exercise enhancement by RGS14 disruption is mediated by brown adipose tissue

Enhanced exercise capacity is not only a feature of healthful aging, but also a therapy for aging patients and patients with cardiovascular disease. Disruption of the Regulator of G Protein Signaling 14 (RGS14) in mice extends healthful lifespan, mediated by increased brown adipose tissue (BAT). Accordingly, we determined whether RGS14 knockout (KO) mice exhibit enhanced exercise capacity and the role of BAT in mediating exercise capacity. Exercise was performed on a treadmill and exercise capacity was assessed by maximal running distance and work to exhaustion. Exercise capacity was measured in RGS14 KO mice and their wild types (WT), and also in WT mice with BAT transplantation from RGS14 KO mice or from other WT mice. RGS14 KO mice demonstrated 160 ± 9% increased maximal running distance and 154 ± 6% increased work to exhaustion, compared to WT mice. RGS14 KO BAT transplantation to WT mice, resulted in a reversal of phenotype, with the WT mice receiving the BAT transplant from RGS14 KO mice demonstrating 151 ± 5% increased maximal running distance and 158 ± 7% increased work to exhaustion, at three days after BAT transplantation, compared to RGS14 KO donors. BAT transplantation from WT to WT mice also resulted in increased exercise performance, but not at 3 days, but only at 8 weeks after transplantation. The BAT induced enhanced exercise capacity was mediated by (1) mitochondrial biogenesis and SIRT3; (2) antioxidant defense and the MEK/ERK pathway, and increased hindlimb perfusion. Thus, BAT mediates enhanced exercise capacity, a mechanism more powerful with RGS14 disruption.     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

壳聚糖酶结构与功能的研究进展

壳聚糖酶是一类对壳聚糖具有较高催化活性而几乎不水解几丁质的糖苷水解酶,其可将高分子量的壳聚糖转化为低分子量的功能性壳寡糖。近年来,对壳聚糖酶的相关研究取得了显著进展,因此,本文对其生化性质、晶体结构、催化机制和蛋白质工程改造进行总结和探讨,并对酶法制备壳寡糖纯品进行展望,这将加深研究者对壳聚糖酶作用机制的认识,推动壳聚糖酶的工业应用。     

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L⁻¹) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L⁻¹ sodium nitrophenol and 0.01 ~ 0.2 mg· L⁻¹ of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L⁻¹ sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L⁻¹ significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

     

Investigation of <i>GhFAT</i> Genes Related to Seed Oil Content and Fatty Acid Composition in <i>Gossypium hirsutum</i> L.

Fatty Acyl-ACP thioesterase (FAT) is a key enzyme controlling oil biosynthesis in plant seeds. FATs can be divided into two subfamilies, FATA and FATB according to their amino acid sequences and substrate specificity. The Upland cotton genome contains 20 GhFAT genes, amongst which 6 genes were of the GhFATA subfamily and 14 of the GhFATB subfamily. The 20 GhFAT genes are unevenly distributed on 14 chromosomes. The GhFATA genes have 5 or 7 exons and the GhFATB genes have 6 or 7 exons. All GhFAT proteins have the conserved Acyl-ACP_TE domain and PLN02370 super family, the typical characteristics of plant thioesterases. Analyses of the expression level of GhFATs and the compositions of fatty acid in 5–60 days-post-anthesis seeds showed that the ratio of saturated fatty acids to unsaturated fatty acids was consistent with the expression profile of GhFATB12, GhFATB3, and GhFATB10; the ratio of monounsaturated fatty acid to polyunsaturated fatty acids was consistent with the expression profile of GhFATA3. The oil contents of mature cottonseeds were positively correlated with the contents of palmitic acid and linolenic acid as well as seed vigor. These results provide essential information for further exploring the role(s) of the specific GhFATs in determining oil biosynthesis and cottonseed compositions.     

废用条件下大鼠和达乌尔黄鼠骨骼肌氧化应激和抗氧化防御能力与肌萎缩的比较研究

有鳞类(蛇和蜥蜴)具有较发达的嗅器和犁鼻器,对其不同种类嗅觉结构的认识有助于阐明爬行动物化学感觉的进化。本文采用组织学方法比较了草原沙蜥(Phrynocephalus frontalis)、荒漠沙蜥(P. przewalskii)、密点麻蜥(Eremias multiocellata)和秦岭滑蜥(Scincella tsinlingensis)的嗅器及犁鼻器。结果发现,草原沙蜥的鼻腔较为狭长,秦岭滑蜥呈梨形,其他两种蜥蜴的鼻腔略成圆形。秦岭滑蜥的嗅上皮最厚,其次是密点麻蜥和草原沙蜥,荒漠沙蜥最薄。犁鼻器主要由犁鼻腔、犁鼻感觉上皮、犁鼻神经及蘑菇体等组成,没有腺体。草原沙蜥和荒漠沙蜥的犁鼻腔较为宽阔,密点麻蜥和秦岭滑蜥的较窄。4种蜥蜴的犁鼻感觉上皮均较嗅上皮厚,蘑菇体向后逐渐缩小至消失,犁鼻感觉上皮成闭环状,包围犁鼻腔。密点麻蜥和秦岭滑蜥的犁鼻感觉上皮位于犁鼻器的背侧,蘑菇体位于腹侧;与此不同,两种沙蜥的犁鼻感觉上皮偏向于犁鼻器的腹内侧,蘑菇体位于背外侧。密点麻蜥的犁鼻感觉上皮最厚,其次为秦岭滑蜥,两种沙蜥最薄;秦岭滑蜥犁鼻感觉上皮的感觉细胞密度最高,其次是密点麻蜥,两种沙蜥最低。这些结果提示,密点麻蜥和秦岭滑蜥对嗅觉信号的依赖和投入较两种沙蜥多;4种蜥蜴犁鼻器的结构差异间接地佐证了有鳞类犁鼻器系统发生的特异性。