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Strategy for efficient cloning of biosynthetic gene clusters from fungi

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Science China(Life Sciences) 2019年第8期62卷 1087-1095页

作者： Ruixin Li ZiXin Li Ke Ma Gang Wang Wei Li Hong-Wei Liu Wen-Bing Yin Peng Zhang Xing-Zhong Liu School of Life Sciences University of Science and Technology of ChinaHefei230027China State Key Laboratory of Mycology Institute of MicrobiologyChinese Academy of SciencesBeijing100101China

Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do ***, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.

关键词： biosynthetic gene clusters Saccharomyces cerevisiae homologous recombination DNA assembly

Investigations of Retention Rule in Reversed-Phase High Performance Liquid Chromatography——Theoretical Consideration of Retention Convergence and Experimental Verification

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Science China Chemistry 1993年第1期36卷 1-9页

作者：焦庆才陈耀祖师治贤 State Key Laboratory of Applied Organic Chemistry Lanzhou University Lanzhou 730000 PRC Northwest Plateau Institute of Biology Academia Sinica Xining 810000 PRC

This paper studied the effect of different chromatographic parameters in RP-HPLC (for example,composition of mobile phase, temperature, number of carbon atoms within the solute molecules, solvent molecules and alkyl ligand on bonded phase surface) on the retention convergence based on the displacement adsorption multi-interaction model,and derived a few retention convergent equations by using thermodynamic method,which have been verified by a great deal of experimental data of homologous ***,we have developed a general method, which can calculate the coordinate values of various retention convergent points by computer directly from experimental data,and the results predicted are in good agreement with that obtained by using plotting method.

关键词： RPLC retention model retention convergence homologous series.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway

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Chinese Journal of Integrative Medicine 2019年第8期25卷 604-612页

作者： CHEN Ming-tai HUANG Ruo-lan OU Li-jun CHEN Ying-nan MEN Ling CHANG Xiao WANG Ling YANG Yu-zhu ZHANG Zhong Cardiovascular Department Shenzhen Traditional Chinese Medicine Hospital Guangzhou University of Traditional Chinese Medicine

Objective: To test the hypothesis that the inhibition of endoplasmic reticulum(ER) stress-induced apoptosis in oxidized low-density lipoproteins(ox-LDL)-induced human aortic-vascular smooth muscle cells(HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase(PERK)-eukaryotic translation initiation factor 2α(e IF2α)-activating transcription factor 4(ATF4)-CCAAT/enhancer binding protein homologous protein(CHOP) signaling pathway by Pollen Typhae total flavone(PTF). Methods: Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group(70 μg/m L high ox-LDL), an HPTF group(70 μg/m L high ox-LDL+500 μg/m L PTF), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), and a LPTF group(70 μg/m L high ox-LDL+100 μg/m L PTF) in the first part;and a normal control group, an ox-LDL group(70 μg/mL high ox-LDL), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), a sh RNA group(transducted with PERK shRNA lentiviral particles), a scramble shRNA group(transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group(250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group(70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their m RNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was applied to test cel viability, and the level of apoptosis was monitored by flow cytometry. Results: The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and sh RNA groups. Moreover, the ox-LDL group had increased protein and m RNA levels of binding immunoglobulin protein and ER-associated

关键词： Pollen Typhae total flavone endoplasmic reticulum stress protein kinase RNA-like endoplasmic reticulum kinase-eukaryotic translation initiation factor 2a -activating transcription factor 4-CCAAT/enhancer binding protein homologous protein pathway apoptosis vulnerable atherosclerotic plaque

GPER agonist G1 suppresses neuronal apoptosis mediated by endoplasmic reticulum stress after cerebral ischemia/reperfusion injury

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Neural Regeneration Research 2019年第7期14卷 1221-1229页

作者： Zi-Wei Han Yue-Chen Chang Ying Zhou Hang Zhang Long Chen Yang Zhang Jun-Qiang Si Li Li Department of Physiology Medical College of Shihezi University Key Laboratory of Xinjiang Endemic and Ethnic Disease Shihezi University School of Medicine Affiliated Teng Zhou Central People's Hospital Jining Medical University Department of Physiology Jiaxing College of Medicine

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion(I/R) *** this study,three key proteins in the endoplasmic reticulum stress pathway(glucose-regulated protein 78,caspase-12,and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen *** Sprague-Dawley rats received ovariectomy(OVX),and then cerebral I/R rat models(OVX+ I/R) were established by middle cerebral artery *** after I/R,rat models were injected with 100 μg/kg E2(OVX + I/R +E2),or 100 μg/kg G protein-coupled estrogen receptor agonist G1(OVX + I/R + G1) in the lateral *** scoring was used to detect neurobehavioral changes in each *** volumes were measured by 2,3,5-triphenyltetrazolium chloride *** changes in neurons were observed by Nissl *** dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group,neurological function was remarkably improved,infarct volume was reduced,number of normal Nissl bodies was dramatically increased,and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 *** detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum,caspase-12 distribution and expression were detected by immunofluorescence,and mRNA and protein levels of glucose-regulated protein 78,caspase-12,and C/EBP homologous protein were determined by polymerase chain reaction and western blot *** results showed that compared with the OVX+ I/R group,E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78,C/EBP homologous protein,and ***,the G protein-coupled estrogen receptor antagonist G15(OVX + I/R + E2 + G15) could eli

关键词： nerve regeneration cerebral ischemia/reperfusion injury estrogen G protein-coupled estrogen receptor G1 G15 endoplasmic reticulum stress glucose-regulated protein 78 caspase-12 C/EBP homologous protein neuronal apoptosis neural regeneration

Promoter variations in DBR2-like affect artemisinin production in different chemotypes of Artemisia annua

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Horticulture Research 2023年第9期10卷 220-232页

作者： Xingwen Wang Lan Wu Li Xiang Ranran Gao Qinggang Yin Mengyue Wang Zhaoyu Liu Liang Leng Yanyan Su Huihua Wan Tingyu Ma Shilin Chen Yuhua Shi Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine Artemisinin Research CenterInstitute of Chinese Materia MedicaChina Academy of Chinese Medical SciencesBeijing 100700China Institute of Herbgenomics Chengdu University of Traditional Chinese MedicineChengdu 611137China Amway(China)Botanical R&D Center Wuxi 214115China Key Lab of Chinese Medicine Resources Conservation State Administration of Traditional Chinese Medicine of the People’s Republic of ChinaInstitute of Medicinal Plant DevelopmentChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100193China

Artemisia annua is the only known plant source of the potent antimalarial artemisinin,which occurs as the low-and high-artemisinin producing(LAP and HAP)***,the different mechanisms of artemisinin producing between these two chemotypes were still not fully ***,we performed a comprehensive analysis of genome resequencing,metabolome,and transcriptome data to systematically compare the difference in the LAP chemotype JL and HAP chemotype *** analysis revealed that 72.18%of sesquiterpenes was highly accumulated in HAN compared to *** omics analysis found a DBR2-Like(DBR2L)gene may be involved in artemisinin ***2L was highly homologous with DBR2,belonged to ORR3 family,and had the DBR2 activity of catalyzing artemisinic aldehyde to dihydroartemisinic *** resequencing and promoter cloning revealed that complicated variations existed in DBR2L promoters among different varieties of *** and were clustered into three variation *** promoter activity of diverse variant types showed obvious ***,the core region(-625 to 0)of the DBR2L promoter was identified and candidate transcription factors involved in DBR2L regulation were ***,the result indicates that DBR2L is another key enzyme involved in artemisinin *** promoter variation in DBR2L affects its expression level,and thereby may result in the different yield of artemisinin in varieties of *** provides a novel insight into the mechanism of artemisinin-producing difference in LAP and HAP chemotypes of ***,and will assist in a high yield of artemisinin in ***.

关键词： thereby insight homologous

ALTERNATE PREDOMINANCE IN COMPETITION FOR CONJUGATION IN NITROGEN BRIDGE COMPOUND

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Science China Chemistry 1990年第4期33卷 409-420页

作者：蒋明谦虞忠衡戴萃辰 Institute of Chemistry Academia Sinica Beijing 100021 PRC

It is demonstrated quantitatively that the serial number N is not m + n + 4 in the nitrogen bridge conjugated system (Ⅸ), so the lone pair of electrons on the nitrogen cannot conjugate with acenyl branch and formyl polyenic branch simultaneously. The character of UV spectra and substituent equivalents (△Ns) show that acenyl branch competes for the lone pair of electrons with formyl polyenic branch. If the length m of acenyl branch is lengthened while n keeps constant, a point where the acenyl branch is predominant over formyl polyenic branch in competition, will eventually be reached. On the other hand, the effect of the latter will become greater than the former. The effect of the two branches predominates each other alternately with the alternate increase in m and n values.

关键词： rolo of homologous linearity nitrogen bridge conjugated system alternative competition for conjugation conjugated stem conjugated polarization.

Loss of BRCA1 expression leads to worse survival in patients with gastric carcinoma

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World Journal of Gastroenterology 2013年第12期19卷 1968-1974页

作者： Zi-Zhen Zhang Yuan Jie Charles Liu Xiao-Lu Yin Ping Zhan Yi Gu Xing-Zhi Ni Department of General Surgery Renji Hospital School of Medicine Shanghai Jiaotong University Astra-Zeneca Innovation Center China

AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall ***: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of

关键词： homologous recombination deficiency Gastric cancer Breast cancer type 1 susceptibility protein Mediator of DNA damage checkpoint protein 1 Ataxia telangiectasia mutated Ataxia telangiectasia mutated-Rad3-related

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

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Science China(Life Sciences) 1999年第5期42卷 554-560页

作者：于嘉林晏立英苏宁侯占军李大伟韩成贵杨莉莉蔡祝南刘仪 State Key Laboratory of Agrobiotechnology China Agricultural University Beijing China Department of Plant Pathology China Agricultural University Beijing China

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7 629 nucleotides encoding a polyprotein with 2 407 amino acids, from which seven putative proteins may be produced by an autolytic cleavage processing besides the viral coat protein. The RNA2 is 3 639 nucleotides and codes for a polyprotein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75% respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus .

关键词： wheat yellow mosaic virus nucleotide sequence homologous analysis.

Role of ubiquitination in meiotic recombination repair

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Science China(Life Sciences) 2010年第4期53卷 447-454页

作者： CUI Li & LI Wei State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China State Key Laboratory of Reproductive Biology Institute of Zoology Chinese Academy of Sciences Beijing China

Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associated with ubiquitination with regard to homologous recombination (HR)-dependent DSB repair.

关键词： ubiquitin meiosis homologous recombination DNA double-strand breaks