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Dissecting PCNA function with a systematically designed mutant library in yeast

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Journal of Genetics and Genomics 2019年第6期46卷 301-313页

作者： Qingwen Jiang Weimin Zhang Chenghao Liu Yicong Lin qingyu wu Junbiao Dai Center for Synthetic and Systems Biology School of Life Sciences Tsinghua University Beijing 100084 China Shenzhen Key Laboratory of Synthetic Genomics and Center for Synthetic Genomics Institute of Synthetic Biology Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences Shenzhen 518055 China Department of Biology. Brandeis University. Massachusetts 02454 USA

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sen sitivity to DNA damaging reagents were identified using high-throughput phe no type screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

关键词： Mutagenesis High-throughput DNA damage tolerance Template switching Genetic interaction

Distinct roles of alternative oxidase pathway during the greening process of etiolated algae

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Science China(Life Sciences) 2021年第5期64卷 816-827页

作者： Ying Liu Hua Zhang Yan Cui Yanli Zheng Hui Chen Zhangli Hu qingyu wu College of Life Sciences and Oceanography Shenzhen UniversityShenzhen 518055China Shenzhen Academy of Environmental Science Shenzhen 518001China Gansu Innovation Center of Microalgae Technology Hexi UniversityZhangye 734000China Key Laboratory of Algal Biology Institute of HydrobiologyChinese Academy of SciencesWuhan 430072China University of the Chinese Academy of Sciences Beijing 100039China State Key Laboratory of Crop Stress Adaptation and Improvement School of Life SciencesHenan UniversityKaifeng 475004China

The vital function of mitochondrial alternative oxidase(AOX) pathway in optimizing photosynthesis during plant de-etiolation has been well recognized. However, whether and how AOX impacts the chloroplast biogenesis in algal cells remains unclear. In the present study, the role of AOX in regulating the reassembly of chloroplast in algal cells was investigated by treating Auxenochlorella protothecoides with salicylhydroxamic acid(SHAM), the specific inhibitor to AOX, in the heterotrophy to autotrophy transition process. Several lines of evidences including delayed chlorophyll accumulation, lagged reorganization of chloroplast structure, altered PSI/PSII stoichiometry, and declined photosynthetic activities in SHAM treated cells indicated that the impairment in AOX activity dramatically hindered the development of functioning chloroplast in algal cells. Besides, the cellular ROS levels and activities of antioxidant enzymes were increased by SHAM treatment, and the perturbation on the balance of NAD+/NADH and NADP+/NADPH ratios was also observed in A. protothecoides lacking AOX activity, indicating that AOX was essential in promoting ROS scavenging and keeping the redox homeostasis for algal chloroplast development during greening. Overall, our study revealed the essentiality of mitochondrial AOX pathway in sustaining algal photosynthetic performance and provided novel insights into the physiological roles of AOX on the biogenesis of photosynthetic organelle in algae.

关键词： alternative oxidase pathway chloroplast reconstruction greening microalgae reactive oxygen species redox

Effects of hydrolysis and bacterial degradation in the cells on hydrocarbons and biomarkers generated by pyrolysis of green alga Chlorella protothecoides

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Chinese Science Bulletin 1998年第9期43卷 749-753页

作者： qingyu wu Bin Zhang Yitao Song Guoying Sheng Jiamo Fu Tsing Hua Univ Dept Biol Beijing 100084 Peoples R China Inst Geol Sci Shengli Oilfield Dongying 257015 Peoples R China Chinese Acad Sci Inst Guangzhou Geochem Guangzhou 510640 Peoples R China

The relative contents of normal alkanes and isoprenoid alkanes from pyrolysed products of green alga Chlorella protothecoides increased when the algal cell subject to hydrolysis and bacterial degradation were used. Their value of C 23 -/C 24 + and the rate of Pr/C 17 increased greatly whereas the contents of C 20 alkenes were remarkably low compared with that from untreated sample (as control). These results are more analogous to the formation process of hydrocarbons derived from algae in natural geological environment.

关键词： Chlorella protothecoides hydrolysis bacterial degradation alkane biomarker thermal simulation.

Hydrocarbons pyrolysed from nannoplanktonic algae:An experimental organism system for study on the origin of petroleum and natural gas

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Chinese Science Bulletin 1999年第8期44卷 767-770页

作者： qingyu wu Ruiyong Wang Junbiao Dai Yitao Song Yoshihiro Shiraiwa Tsing Hua Univ Dept Biol Sci & Biotechnol Beijing 100084 Peoples R China Inst Geosci Shengli Petro Adm Dongying 257015 Peoples R China Univ Tsukuba Inst Biol Sci Tsukuba Ibaraki 3058572 Japan

Enukuania huxleyi is marine nanoplanktonic alga with calcify scale called coccolith om its cell *** ubiquitous species with the Lafgest biomass in marice forms gigantic blooms covering thousands

关键词： Hydrocarbons pyrolysed from nannoplanktonic algae

Identification of long chain isoprenoid hydrocarbons from pyrolytic product of Dunaliella

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Chinese Science Bulletin 1999年第18期44卷 1700-1705页

作者： Ruiyong Wang Wen Zhou Junbiao Dai qingyu wu Guoying Sheng Jiamo Fu Nanjing Univ Dept Biol Nanjing 210093 Peoples R China Tsing Hua Univ Dept Biol Beijing 100084 Peoples R China Chinese Acad Sci Inst Guangzhou Geochem Guangzhou 510640 Peoples R China

In the pyrolytic research on Dunaliella salina cultured under natural conditions, abundant long chain regular isoprenoid hydrocarbons have been first detected and identified from algal pyrolytic product. Specially, the discovery of high abundant 2,6,10,14,18-pentamethyle-icosane indicated that regular iC25 probably originated from specific holophilic algae in hyper-

关键词： Dunaliella salina pyrolysis long chain isoprenoids 2 6 10.14 18-pentamethyleicosan.

Distribution of aromatic bio-markers in pyrolysates of co-ccolithophore

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Chinese Science Bulletin 2001年第3期46卷 246-252页

作者： ZHOU Wen wu qingyu WANG Ruiyong SONG Yitao ZHANG Linye LIU Zhili Department of Biological Science and Technology Nanjing UniversityNanjing 210093China Department of Biological Science and Technology Tsinghua UniversityBeijing 100084China Geological Science and Research Institute Shengli Petroleum Administration BureauDongying 257015China

Analyses of the original and pyrolytic products of coccolithophore at various temperatures suggest that the contribution of coccolithophore to the formation of large immature oil reservoirs should attract keen interest. Through biochemical processes algae can change inorganic sulphur into organic sulphur, which could be one of the most important precursors of organic sulphur compounds in oil and source rocks. When Methylphenanthrene Index (MPI) and Methylphenanthrene Ratio (MPR) indices were used to evaluate the evolution degree of source rocks and oil maturity, other maturity indices must be used together for correction. In low maturity, the relative abundances of diben-zothiophene (DBT), fluorine (F) and dibenzofuran (DBF) can be used to identify the oxidation-reduction environments.

关键词： coccolithophore pyrolysates aromatic biomarkers.

Molecular source of biomarkers by genetic engineering techniques

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Chinese Science Bulletin 2000年第11期45卷 1025-1030页

作者： DAI Junbiao wu qingyu WANG Ruiyong PENG Weimin SHEN Guoyin FU Jiamo Department of Biological Sciences and Biotechnology Tsinghua UniversityBeijing 100084China Institute of Guangzhou Geochemistry Chinese Academy SciencesGuangzhou 510640China

The mutant lacking ORF469 fragment in Synechocystis sp. PCC 6803 (cyanobacterium) was created by means of DNA recombination. In its genome, ORF469, the key DNA fragment controlling the light-independent pathway of chlorophyll biosynthesis was deleted and replaced by erythromycin resistance cassette. The operation resulted in the fact that the content of chlorophyll in mutant cells was fully controlled by illumination and two kinds of cells were harvested, one is high chlorophyll with concentration of 9.427 μg · mg-1 and the other is low chlorophyll with concentration of 0.695 μg ·mg-1. They were subjected to thermal simulation respectively at 300℃ for 100 h. The alkanes biomarkers from pyrolysates were analyzed by GC-MS and main difference between high and low chlorophyll cells was found at their contents of isoprenoid hydrocarbons. Pr/nC17 and Ph/nC18 from pyrolysate of low chlorophyll cells were 0.192 and 0.216 respectively, which were about 1/3 and 1/7 of that from high chlorophyll cells. The

关键词： Synechocystis sp. PCC 6803 DNA recombination biomarkers thermal degradation isoprenoid hydrocarbons.

NIGT1 represses plant growth and mitigates phosphate starvation signaling to balance the growth response tradeoff in rice

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Journal of Integrative Plant Biology 2023年第8期65卷 1874-1889页

作者： Yuxin Zhang Qianqian Zhang Meina Guo Xueqing Wang Tianjie Li qingyu wu Lihui Li Keke Yi Wenyuan Ruan National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences Beijing 100081China State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China/Key Laboratory of Plant Nutrition and Fertilizer Ministry of AgricultureInstitute of Agricultural Resources and Regional PlanningBeijing 100081China National Engineering Research Center of Tree Breeding and Ecological Restoration College of Biological Sciences and TechnologyBeijing 100083China

Inorganic phosphate(Pi)availability is an important factor which affects the growth and yield of crops,thus an appropriate and effective response to Pi fluctuation is ***,how crops orchestrate Pi signaling and growth under Pi starvation conditions to optimize the growth defense tradeoff remains *** we show that a Pi starvationinduced transcription factor NIGT1(NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1)controls plant growth and prevents a hyper-response to Pi starvation by directly repressing the expression of growth-related and Pisignaling genes to achieve a balance between growth and response under a varying Pi ***1 directly binds to the promoters of Pi starvation signaling marker genes,like IPS1,mi R827,and SPX2,under Pi-deficient conditions to mitigate the Pi-starvation responsive(PSR).It also directly represses the expression of vacuolar Pi efflux transporter genes VPE1/2 to regulate plant Pi *** further demonstrate that NIGT1 constrains shoot growth by repressing the expression of growth-related regulatory genes,including brassinolide signal transduction master regulator BZR1,cell division regulator CYCB1;1,and DNA replication regulator *** findings reveal the function of NIGT1 in orchestrating plant growth and Pi starvation signaling,and also provide evidence that NIGT1 acts as a safeguard to avoid hyper-response during Pi starvation stress in rice.

关键词： NIGT1 phosphate(Pi) PHRs Pi-starvation responsive(PSR) Pi starvation Pi signaling

Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor

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The Crop Journal 2020年第3期8卷 465-479页

作者： Shengcai Huang Shichao Xin Guoqiang Xie Jiao Han Zhonglai Liu Bing Wang Shuqing Zhang qingyu wu Xianguo Cheng Laboratory of Plant Nutrition and Biology Institute of Agricultural Resources and Regional PlanningChinese Academy of Agricultural SciencesBeijing 100081China Key Laboratory of Rubber Biology and Genetic Resources of the Ministry of Agriculture Rubber Research InstituteChinese Academy of Tropical Agricultural SciencesHaikou 571011HainanChina Jiujiang Academy of Agricultural Sciences Jiujiang 332101JiangxiChina Guangling No.5 Middle School Datong 037500ShanxiChina

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9(CRISPR-associated protein9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na^+ /K^+ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na^+ efflux but also of K^+ and Ca^(2+) influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.

关键词： MPT ATP CRISPR Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor