黑暗链霉菌中tbmA基因的功能研究

PCR获得tbmA基因内部863 bp片段,构建基因阻断穿梭载体pSPU112-1,经接合转移导入Strepto-myces tenebrariusH6,筛选单交换阻断变株,并用Southern blot验证阻断变株的tbmA已经被破坏。经发酵产物分析,阻断变株不再合成氨甲酰妥布霉素,只合成安普霉素。首次从分子水平证明了tbmA只参与氨甲酰妥布霉素生物合成,而不参与安普霉素的生物合成。     

一株妥布拉霉素高产菌株特性的研究

用高温和NTG处理暗黑链霉菌410-II(Streptomyces tenebrarius410-II)的孢子,得到六株无色突变株。所产抗生素只有两个组分,经分析为氨甲酰妥布拉霉素(carb…yltobramycin)和阿普拉霉素(Apramycin),不再含有出发菌株产生的氨甲酰卡那霉素(Carbamoylkanamycin)。在适宜的发酵条件下,所含两个组分的比例约为I：1。突变株基本不产生可溶性紫色素,发酵液离心所得上清液虽加入Fe冲,也不变成紫色,有利于提取工艺。六株突变株中W1028一M,所产抗生紊总效价比原株410-Il高20％以上,发酵液中氨甲酰妥布拉霉素含量比4100l提高约45—50％。由f组分减少,提取分离效率提高,在实验室用10升发酵液进行提取实验,妥布拉霉素单组分的收率达到25％o突变株w1028一M5是一株产量增高、收率提高、适用于生产的菌株。     

产生妥布拉霉素的ER—16突变株的选育

以妥布拉霉素产生菌暗黑链霉菌AS 4.1098(410-Ⅱ)为出发菌株,经高温和亚硝基胍处理,获得6株无色突变株,对其中的W1028-M5用亚硝基胍及甲基磺酸乙酯继续处理,得到突变株E228,再经抗自身代谢终产物妥布拉霉素抗性株的选育,得到ER-16和ER-21等高产菌株。所产抗生素仅含两个组份,即氨甲酰妥布拉霉素和阿普拉霉素。不再含氨甲酰卡那霉素。测定的几项主要理化性质与出发菌株以及文献报道的数据完全一致。     

产生妥布拉霉素的ER-16突变株的选育

以妥布拉霉素产生菌暗黑链霉菌AS 4.1098(410-Ⅱ)为出发菌株,经高温和亚硝基胍处理,获得6株无色突变株,对其中的W1028-M5用亚硝基胍及甲基磺酸乙酯继续处理,得到突变株E228,再经抗自身代谢终产物妥布拉霉素抗性株的选育,得到ER-16和ER-21等高产菌株。所产抗生素仅含两个组份,即氨甲酰妥布拉霉素和阿普拉霉素。不再含氨甲酰卡那霉素。测定的几项主要理化性质与出发菌株以及文献报道的数据完全一致。     

南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究*

通过链霉素对南昌霉素 (Nanchangmycin)产生菌NS 41 80菌株孢子的致死浓度测定基础上 ,采用诱变剂甲基磺酸乙酯 (EMS)的不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含链霉素 ( 1 0 μg/mL)致死浓度的高氏平板上 ,获得了大量的链霉素抗性基因 (str)突变株。然后从 3,0 0 0株链霉素抗性基因 (str)突变株中通过初筛获得比诱变出发菌株产素能力提高 2 0 %以上的菌株 2 0 2株。再进一步通过摇瓶复筛 ,获得比出发菌株产素能力分别提高 1     

阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

以野生型阿维链霉茵NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643.将pilL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷.通过Southern杂交分析,证明whiEa基因簇被置换.通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象.     

环境放线菌中的达托霉素抗性机制

[目的]研究环境放线菌中的达托霉素抗性机制,为临床耐药机制的出现提供预警.[方法]通过测定土壤放线菌(49株)和药用植物内生放线菌(10株)的达托霉素耐受谱,筛选达托霉素抗性菌株;通过达托霉素灭活实验,确定抗性菌株的灭活能力;通过形态观察和16S rRNA序列分析分类鉴定达托霉素降解菌.通过PCR扩增检测达托霉素去酰基化酶基因在降解菌株中的分布情况.[结果]本研究中所有的环境放线菌均耐受达托霉素.在土壤放线菌中和药物植物内生放线菌中,分别有24株(49.0％)和4株(40％)能够灭活达托霉素,25 (51.0％)株和6株(60％)通过其他机制耐受达托霉素.序列测定表明,链霉菌属(Streptomyces)、小单孢菌属(Mcromonospora)和诺卡氏菌属(Nocardia)的部分菌株有灭活达托霉素的能力.PCR扩增表明,5株(17.9％)放线菌含有编码达托霉素去酰基酶的基因.[结论]环境放线菌具有超高的达托霉素抗性频率,灭活达托霉素是主要抗性机制之一.     

梅岭霉素高产菌株链霉素抗性基因突变株筛选

通过链霉素对梅岭霉素 (Meilingmycin)产生菌南昌链霉菌NS 41 80菌株孢子致死浓度的测定 ,采用诱变剂EMS 4种不同剂量对菌株孢子进行诱变处理 ,然后涂布在含链霉素致死浓度的高氏平板上 ,获得了大量的链霉素抗性基因 (str)突变株。并进一步筛选到梅岭霉素高产菌株 80 5 1 1 2 2 1 ,在摇瓶条件下 ,只产梅岭霉素不产南昌霉素 ,梅岭霉素活性单位达 1 ,52 1 μg/mL,比NS 41 80的摇瓶发酵单位 855μg/mL提高了 77 9% ,该菌株连续传 6代进行摇瓶发酵     

圈卷产色链霉菌san7324和san7324L基因阻断对形态分化和尼可霉素产生的影响

【目的】圈卷产色链霉菌全局性调控基因wblA阻断突变后,尼可霉素不再产生。RNA-seq和转录分析表明san7324基因在野生型菌株中可以正常转录,而在wblA阻断突变株(ΔwblA)中不能转录,为此本文旨在揭示san7324与尼可霉素产生的关系。【方法】利用同源双交换策略对san7324进行基因阻断,而后通过基因遗传回补及对尼可霉素生物合成相关基因的转录分析等方法研究san7324的功能。【结果】在相同培养条件下,阻断突变株Δsan7324与野生型菌株相比失去了合成尼可霉素的能力。我们通过同源比对发现圈卷产色链霉菌中还存在一个与san7324同源的基因san7324L,该基因的阻断导致尼可霉素产量降低。当san7324和san7324L两个基因同时被阻断后,得到的突变株Δsan7324-san7324L生长稀疏而且不能正常发育分化形成灰色表型的孢子或孢子链,只能形成白色表型的气生菌丝,同时也丧失了合成尼可霉素的能力。当这两个基因(san7324-san7324L)回补双突变株后,则恢复了野生型的表型(能形成孢子链并恢复尼可霉素的产生)。进一步的研究初步表明san7324和san7324L的阻断主要影响了尼可霉素生物合成基因簇中途径特异性调控基因sanG的转录水平,从而影响圈卷产色链霉菌的发育分化和尼可霉素的产生。【结论】该结果为链霉菌形态分化与生理代谢关系的研究提供了更多的证据,同时为多效调控基因wblA作用机制的阐明奠定了基础。     

通过获得链霉素抗性基因突变株筛选小诺霉素高产菌株

通过链霉素对小诺霉素产生菌 (Micromonospora purpura) 49 1 2 #菌株孢子致死浓度的测定 ,采用诱变剂EMS 3种不同诱变剂量对菌株的孢子进行诱变处理 ,诱变处理的孢子涂布在含链霉素致死浓度的改良高氏平板上 ,获得大量的链霉素抗性基因突变株 ,然后从链霉素抗性基因突变株进一步筛选小诺霉素高产菌株 ,获得小诺霉素菌株 49 1 2 3菌株。在摇瓶条件下 ,其产小诺霉素生物活性单位比出发菌株 49 1 2 #的摇瓶发酵单位提高了 40 %以上。小诺霉素的组分比由出发菌株的C2b∶C1a的 5∶5提高到 8∶2。C2b有效组分提高了 30 %;链霉素抗性基因突变与小诺霉素发酵单位突变之间 ,小诺霉素正突变率达到 40 %,负突变率达 2 6%,正突变大于负突变     

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid β-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD⁺ levels following changes in NAD⁺-dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD⁺) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.     

MYOZ2通过负调控TCAP的表达促进C3H10T1/2细胞成脂分化

本研究旨在明确钙调磷酸酶肌小节结合蛋白2（myozenin2,MYOZ2）的组织表达特性,阐明其对C3H10T1/2细胞成脂分化的影响及可能的作用机制。采集180日龄马身猪、60日龄ICR小鼠、35日龄罗斯肉鸡和12月龄小尾寒羊背最长肌、皮下脂肪和肝组织,检测MYOZ2基因mRNA表达。结果显示,MYOZ2基因在所检测的物种中表现出相似的组织表达规律,在背最长肌表达量最高,皮下脂肪与肝组织低水平表达。在C3H10T1/2细胞中沉默MYOZ2基因后,qRT-PCR结果显示,与对照组相比,成脂关键基因PPARγ、FABP4表达极显著下调（P<0.01）;Western 印迹结果表明,PPARγ蛋白含量显著降低（P<0.05）;油红O染色发现,沉默MYOZ2基因后脂滴数明显减少,甘油三酯含量显著降低（P<0.05）。利用qRT-PCR技术检测沉默MYOZ2基因后,脂肪代谢相关基因SCD、FASN、SREBP1、NR1H3、DGAT1、PNPLA2、HSL、CES1、CPT1等的表达,结果显示,SCD、FASN、SREBP1、PNPLA2、HSL极显著下调（P<0.01）,NR1H3显著下调（P<0.05）,DGAT1表达下调但差异不显著,CES1、CPT1显著上调（P<0.05）。利用STRING数据库构建MYOZ2相关蛋白质互作网络图,发现MYOZ2可能通过肌联蛋白帽（Titin-cap,TCAP）与PPARγ相互作用进而影响成脂分化。沉默TCAP基因,qRT-PCR结果显示,与对照组相比,成脂关键基因PPARγ、FABP4表达极显著上调（P<0.01）;Western 印迹结果表明,与对照组相比,PPARγ蛋白含量显著升高（P<0.05）;油红O染色发现,沉默TCAP基因后脂滴数明显增多,甘油三酯含量显著升高（P<0.05）。利用qRT-PCR技术检测沉默MYOZ2后TCAP基因的表达结果显示,沉默MYOZ2后TCAP的表达极显著升高（P<0.01）。综上所述,MYOZ2基因在背最长肌中表达量最高,在皮下脂肪和肝组织中也有少量表达。此外,MYOZ2可能通过MYOZ2-TCAP-PPARγ途径影响成脂关键基因PPARγ和FABP4的表达,进而调控脂肪酸代谢相关基因SCD、FASN、SREBP1、NR1H3、DGAT1、PNPLA2、HSL、CES1、CPT1,在成脂分化过程中发挥重要作用。     

Cover Caption

Previous studies have confirmed HaCad (cadherin), HaABCC2 and HaABCC3 are functional receptors of Bt toxin Cry1Ac in cotton bollworm, Helicoverpa armigera. Aminopeptidase N1 (APN1) has been suggested as a putative receptor in several lepidopteran insects including H. armigera through evidence from RNAi‐based gene silencing approaches. In the current study, we tested the role of APNs in the mode of action of Bt toxins using CRISPR/Cas9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible SCD strain of H. armigera to establish three homozygous knockout strains. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera (see pages 440–448). Photo by Yi‐Dong Wu.     

Multiple origins of allopolyploid wheatgrass Elymus caninus revealed by RPB2, PepC and TrnD/T genes

We examined evolutionary mechanisms in the tetraploid Elymus caninus by comparing the phylogenetic relationships of 21 accessions suggested by sequence data from two single copy nuclear genes, the largest subunit of RNA polymerase II (RPB2) and phosphoenolpyruvate carboxylase (pepC), and one non-coding chloroplast region, TrnD/T. Elymus caninus is known combining two different genomes, an St genome and an H genome. Data from two single copy nuclear genes showed that there are two versions of the St genome in the species, St1 and St2. Most accessions combined one of these versions with an H genome version but two accessions had both versions of the St sequence for RPB2. This suggests that the RPB2gene may have been duplicated without chromosome doubling, possibly induced by transposable element. Our data also indicate that the H genome sequences in E. caninus have multiple origins, and a close phylogenetic relationship between Hordeum bogdanii and H sequences in some accessions of E. caninus. Thus, it is more likely that H. bogdanii is one of the major donors of the H copy in E. caninus. The maternal origin of E. caninus is the St genome species. There was no correlation between the geographic origin of the accessions and their sequence divergence.