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Whole‑genome transcriptome and DNA methylation dynamics of pre‑implantation embryos reveal progression of embryonic genome activation in buffaloes

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Journal of Animal Science and Biotechnology 2023年第6期14卷 2411-2427页

作者： Penghui Fu Du Zhang Chunyan Yang Xiang Yuan Xier Luo Haiying Zheng Yanfei Deng Qingyou Liu Kuiqing Cui Fei Gao deshun shi State Key Laboratory for Conservation and Utilization of Subtropical Agro‑Bioresources&Guangxi Key Laboratory of Animal Breeding and Disease Control Guangxi UniversityNanning 530004China. College of Animal Science and Technology Southwest UniversityChongqing 402460China. Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120China. Guangxi Key Laboratory of Buffalo Genetics Reproduction and BreedingGuangxi Buffalo Research InstituteChinese Academy of Agricultural ScienceNanning 530001China Guangxi Academy of Medical Sciences and the People’s Hospital of Guangxi Zhuang Autonomous Region Nanning 530016China. Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding School of Life Science and Engineering Foshan UniversityFoshan 528225China. Comparative Pediatrics and Nutrition Department of Veterinary and Animal SciencesFaculty of Health and Medical SciencesUniversity of CopenhagenDK 1870 C FrederiksbergDenmark.

Background During mammalian pre-implantation embryonic development(PED),the process of maternal-to-zygote transition(MZT)is well orchestrated by epigenetic modification and gene sequential expression,and it is related to the embryonic genome activation(EGA).During MZT,the embryos are sensitive to the environment and easy to arrest at this stage in vitro.However,the timing and regulation mechanism of EGA in buffaloes remain obscure.Results Buffalo pre-implantation embryos were subjected to trace cell based RNA-seq and whole-genome bisulfite sequencing(WGBS)to draw landscapes of transcription and DNA-methylation.Four typical developmental steps were classified during buffalo PED.Buffalo major EGA was identified at the 16-cell stage by the comprehensive analy-sis of gene expression and DNA methylation dynamics.By weighted gene co-expression network analysis,stage-spe-cific modules were identified during buffalo maternal-to-zygotic transition,and key signaling pathways and biological process events were further revealed.Programmed and continuous activation of these pathways was necessary for success of buffalo EGA.In addition,the hub gene,CDK1,was identified to play a critical role in buffalo EGA.Conclusions Our study provides a landscape of transcription and DNA methylation in buffalo PED and reveals deeply the molecular mechanism of the buffalo EGA and genetic programming during buffalo MZT.It will lay a foundation for improving the in vitro development of buffalo embryos.

关键词： Buffalo DNA methylome Embryonic genome activation Maternal-to-zygote transition Transcriptome

Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

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Journal of Animal Science and Biotechnology 2015年第4期6卷 395-401页

作者： Fanli Meng Hui Li Xiaoli Wang Guangsheng Qin Bj?rn Oback deshun shi Animal Reproduction Institute State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresourcesGuangxi University AgResearch Ltd. Ruakura Research CentreReproductive Technologies

Background： Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i） how much linear DNA to inject and ii） when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein （EGFP） plasmid into buffalo zygotes. Results： First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/pL DNA was significantly higher than from those injected with 5 pg/mL. However, 50 ng/pL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/pL DNA. Second, zygotes injected early （7-8 h post-insemination [hpi]） developed better than those injected at mid （12-13 hpi） or late （18-19 hpi） time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections （16.4 % vs 7.7 %）. With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions： Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

关键词： Buffalo Cytoplasmic injection Plasmid Transgenic Zygote

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猪DUXA基因克隆及过表达载体的构建

中国兽医学报 2024年第4期44卷 815-822,862页

作者：吕美云张云川齐赟佳佘柳石德顺罗婵广西大学动物科学技术学院、广西畜禽繁育与疾病防控重点实验室广西南宁530004

收集猪体外受精(IVF)和体细胞核移植(SCNT)的2-细胞、4-细胞、8-细胞和16-细胞时期的胚胎,对各细胞阶段DUXA基因的表达进行比较分析;提取猪卵巢的RNA后通过RT-PCR克隆得到DUXA目的基因,对DUXA基因进行生物信息学分析;将目的基因片段连接... 详细信息

收集猪体外受精(IVF)和体细胞核移植(SCNT)的2-细胞、4-细胞、8-细胞和16-细胞时期的胚胎,对各细胞阶段DUXA基因的表达进行比较分析;提取猪卵巢的RNA后通过RT-PCR克隆得到DUXA目的基因,对DUXA基因进行生物信息学分析;将目的基因片段连接至pCDNA3.1-EGFP载体中,再将载体转染至293T细胞中。结果显示,与IVF胚胎相比,DUXA基因在SCNT胚胎中的表达滞后;克隆的DUXA基因编码区全长678bp,与预期相符;核苷酸序列比对分析显示,猪DUXA和马DUXA基因同源性最高;各物种间DUXA蛋白氨基酸保守性较高;进化树结果显示,猪DUXA基因与骆驼相应序列聚为一支,与山羊、水牛、长江江豚等哺乳动物的遗传距离相对较近;利用在线分析工具对猪DUXA编码蛋白的理化性质进行预测,结果显示猪DUXA的化学分子式C1082H1732N344O354S7,相对分子质量为25448.17,理论等电点为9.4;猪DUXA二级结构预测包含11个α-螺旋、3个β-折叠、19个T-转角和20个无规则卷曲,具有2个HOX结构域,预测其为细胞膜外蛋白,其三级结构和黄牛、山羊、人相似。构建的pCDNA3.1-EGF-DUXA质粒载体长度为6797bp,转染结果显示构建的过表达载体可以在293T细胞中表达。结果表明,本试验成功克隆了猪DUXA基因,并成功构建了过表达载体pCDNA3.1-EGFP-DUXA。

关键词：猪 DUXA基因基因克隆序列比对分析载体构建

金银花的生物学功能及其在动物生产中的应用研究进展

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中国畜牧杂志 2024年第2期60卷 7-11页

作者：莫红芳石宗承何东贤覃振斌黄稳妃熊飙石德顺熊晓妍广西农业职业技术大学广西南宁530007 广西大学动物科学技术学院广西南宁530004 中国农业科学院哈尔滨兽医研究所黑龙江哈尔滨150069

金银花(Honeysuckle)最早记载于东晋《肘后备急方》,在我国已有1 000多年的使用历史,是传统的药食同源植物,被广泛应用于医药、食品、化妆品、保健品及畜牧等领域。金银花富含黄酮类、有机酸类、挥发油类和环烯醚萜苷类等多种活性成分,... 详细信息

金银花(Honeysuckle)最早记载于东晋《肘后备急方》,在我国已有1 000多年的使用历史,是传统的药食同源植物,被广泛应用于医药、食品、化妆品、保健品及畜牧等领域。金银花富含黄酮类、有机酸类、挥发油类和环烯醚萜苷类等多种活性成分,具有抑菌抗炎、抗病毒、抗肿瘤、抗氧化、降低糖脂、调节免疫及保护肝脏等药理功能。金银花作为一种良好的新型饲料添加剂,其功能与饲用抗生素相似,可预防疾病、促进生长和保证肉品质,但目前关于金银花及其提取物在动物生产领域的应用研究相对较少。本文综述了金银花及其提取物的生物学功能以及其在动物生产中的应用,以期为金银花的后续研究及资源的合理开发利用提供参考依据。

关键词：金银花生物学功能动物生产应用

重组蛋白KDM4D对水牛成纤维细胞生长及组蛋白甲基化修饰的影响

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基因组学与应用生物学 2024年第4期43卷 627-635页

作者：俸云李思佳陈莫斯楠赵鑫陆灿强罗婵陆凤花石德顺亚热带农业生物资源保护与利用国家重点实验室南宁530004 广西畜禽繁育与疾病防控重点实验室南宁530004

本研究旨在探讨重组蛋白赖氨酸特异性去甲基化酶4D(lysine K-specific demethylase 4D,KDM4D)对水牛成纤维细胞(buffalo fetal fibroblasts,BFFs)生长及组蛋白甲基化修饰的影响,为提高水牛体细胞重编程效率提供理论基础。首先,使用不同... 详细信息

本研究旨在探讨重组蛋白赖氨酸特异性去甲基化酶4D(lysine K-specific demethylase 4D,KDM4D)对水牛成纤维细胞(buffalo fetal fibroblasts,BFFs)生长及组蛋白甲基化修饰的影响,为提高水牛体细胞重编程效率提供理论基础。首先,使用不同浓度的重组蛋白KDM4D处理BFFs,摸索出最适宜的处理浓度和处理时间。其次,采用实时定量PCR技术和EdU方法检测重组蛋白KDM4D对BFFs增殖凋亡的影响。最后,使用细胞免疫荧光和Western blot对组蛋白H3第9位赖氨酸三甲基化(histone 3 lysine 9 trimethylation,H3K9me3)修饰水平和异染色质蛋白1α(heterochromatin protein 1α,HP1α)基因的表达水平进行检测。结果发现,适宜浓度的重组蛋白KDM4D(0.10μg/mL)处理36 h对BFFs形态无明显影响,可以显著提高细胞活力(P<0.05)。实时定量PCR分析结果显示,与对照组相比,重组蛋白KDM4D可以显著提高细胞周期蛋白依赖性激酶4(cyclin dependent kinase 4,CDK4)和B淋巴细胞瘤2(B-cell lymphoma 2,BCL2)的mRNA相对表达水平,显著降低Bax(BCL2 associated X)的mRNA相对表达水平(P<0.05)。EdU细胞增殖检测结果表明,重组蛋白KDM4D处理后BFFs的增殖率显著高于对照组的(P<0.05)。细胞免疫荧光和Western blot结果显示,培养基中添加重组蛋白KDM4D后,BFFs的H3K9me3修饰水平和HP1α蛋白表达水平显著降低(P<0.05)。上述结果表明,适宜浓度的重组蛋白KDM4D(0.10μg/mL)处理36 h,可以促进BFFs的增殖,降低BFFs的H3K9me3修饰水平和HP1α蛋白的表达水平。本研究为进一步探讨重组蛋白KDM4D对水牛体细胞重编程的作用机制提供了理论依据。

关键词：赖氨酸特异性去甲基化酶4D(KDM4D) 水牛成纤维细胞细胞增殖组蛋白甲基化修饰

An Insight on Small Molecule Induced Foot-Print Free Naive Pluripotent Stem Cells in Livestock

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Stem Cell Discovery 2015年第1期5卷 1-9页

作者： Mengmei Li Lanyu Li Jun Zhang Vinod Verma Qingyou Liu deshun shi Ben Huang School of Animal Science and Technology Guangxi University Nanning China State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangxi University Nanning China Centre of Biotechnology Nehru Science Centre University of Allahabad Allahabad India

Bona fide embryonic stem cell (ESC) lines from livestock species have been challenging to derive and maintain, contrasting mouse and human ESCs. However, induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells tender an option, as they display characteristic features of ESC. The comprehension that induced pluripotent stem cells (iPSC) could be created with in no time also holds the potential of allowing pluripotent cells to be derived from animal models vital in biomedical research. Endeavors to produce bona fide pluripotent stem cells (PSC) from livestock have been going on for more than two decades. But, attempts to derive bona fide livestock iPS cells have met with limited success. Recently it’s been reported that small molecules can augment reprogramming efficiency and may be used to substitute few or all transcription factors used for reprogramming. It is assumed that the reprogramming factors are conserved among species, and this small molecule reprogramming approach will probably apply to livestock species as well. So this review will focus mainly on the accomplishments of small molecules on accelerating cell reprogramming and obtaining naive pluripotency, and raise a new insight on, exogenous genes free, livestock naive iPSC generation with a new bullet, small molecule.

关键词： Small Molecule Foot-Print Free Naive Pluripotent Stem Cell Livestock

Understanding divergent domestication traits from the whole-genome sequencing of swamp-and river-buffalo populations

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National Science Review 2020年第3期7卷 686-701页

作者： Xier Luo Yu Zhou Bing Zhang Yi Zhang Xiaobo Wang Tong Feng Zhipeng Li Kuiqing Cui Zhiqiang Wang Chan Luo Hui Li Yanfei Deng Fenghua Lu Jianlin Han Yongwang Miao Huaming Mao Xiaoyan Yi Cheng Ai shigang Wu Alun Li Zhichao Wu Zijun Zhuo Do Da Giang Bikash Mitra Mohammad Farhad Vahidi Shahid Mansoor Sahar Ahmed Al-Bayatti Eka Meutia Sari Neena Amatya Gorkhali Sigit Prastowo Laiba Shafique Guoyou Ye Qian Qian Baoshan Chen deshun shi Jue Ruan Qingyou Liu Animal Biotechnology Department Agricultural Biotechnology Research Institute of Iran-North Region 11. National Institute for Biotechnology and Genetic Engineering 12. Animal Genetic Sources DepartmentDirectorate of Animal Resources Ministry of Agriculture 13. Department of Animal Science Faculty of Agriculture Syiah Kuala University 14. Animal Breeding DivisionNational Animal Science Research Institute Nepal Agriculture Research Council 15. Animal Science Department Universitas Sebelas Maret 16. International Rice Research Institute State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangxi University CAS Key Laboratory of Genome Sciences and Information Beijing Institute of Genomics Chinese Academy of Sciences National Engineering Laboratory for Animal Breeding Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural AffairsCollege of Animal Science and Technology China Agricultural University Guangdong Laboratory for Lingnan Modern Agriculture Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genomics Institute at Shenzhen Chinese Academy of Agricultural Sciences CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources Institute of Animal ScienceChinese Academy of Agricultural Sciences International Livestock Research Institute Faculty of Animal Science and Technology Yunnan Agricultural University Bacgiang Agriculture and Forestry University Cellular Immunology Lab Department of Zoology University of North Bengal

Domesticated buffaloes have been integral to rice-paddy agro-ecosystems for millennia, yet relatively little is known about the buffalo genomics. Here, we sequenced and assembled reference genomes for both swamp and river buffaloes and we re-sequenced 230 individuals(132 swamp buffaloes and 98 river buffaloes)sampled from across Asia and Europe. Beyond the many actionable insights that our study revealed about the domestication, basic physiology and breeding of buffalo, we made the striking discovery that the divergent domestication traits between swamp and river buffaloes can be explained with recent selections of genes on social behavior, digestion metabolism, strengths and milk production.

关键词： Bubalus bubalis genome buffalo domestication re-sequencing traits

Histone hyperacetylation of porcine fetal fibroblasts,not affecting cellular characteristics,improves the preimplantation developmental competence,quality and epigenetic status of cloned embryos

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Histone hyperacetylation of porcine fetal fibroblasts,not af...

308 nm准分子激光联合CO_(2)点阵激光及PRP用于稳定期白癜风治疗的临床效果分析

第十五届中国实验动物科学年会

作者： JunMing Sun KuiQing Cui ZhiPeng Li BangJun Gao JianRong Jiang QingYou Liu Ben Huang deshun shi Laboratory Animal Center Guangxi Medical University State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources Guangxi University

It is investigated how histone hyperacetylation of porcine donor cells improves the preimplantation developmental competence and epigenetic status of cloned embryos.The results showed the enhanced level of Ac H3 K14 was in a dose and time dependent manner.Along with the SAHA concentration increasing except for extreme concentration of 6 μM for 48 to 96 h,the outcome of histone hyperacetylation had not significant effect on cell morphology,cell viability and cell cycle.Histone hyperacetylation of PFFs did not affect the levels of Bax transcripts,but a significant decrease in HDAC1 mRNA occurred at 48-96 h in the hyperacetylation of donor cells.The SCNT embryos with histone hyperacetylation of donor cells showed significantly higher blastocyst formation rates and the total nuclei number compared to the control embryos.In addition,the histone Ac H3 K14 level of SCNT-SAHA embryos were significantly higher than those of IVF embryos at the 2-cells and 4-cells stages,but they were not different at the blastocyst stage.The expression levels of Cdx2 in SCNT-SAHA blastocysts were comparative with that of IVF blastocysts.The expression levels of OCT4 in SCNT-SAHA blastocysts were significantly higher than that of SCNT-Control embryos,but lower than that of the IVF embryos.In conclusion,histone hyperacetylation of donor cells did not affect cellular characteristics,moreover,improved quality of porcine SCNT embryos and partly corrected the abnormal histone acetylation modification.

关键词： SCNT donor HDACi reprogramming HDAC

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中外医疗 2022年第2期41卷 22-25,29页

作者：石德顺广东省佛山市第一人民医院皮肤科广东佛山528000

目的探讨308 nm准分子激光联合CO2点阵激光及PRP用于稳定期白癜风治疗的临床效果。方法简单随机选取2020年6月—2021年6月佛山市第一人民医院皮肤科60例稳定期非节段型白癜风患者,数字随机抽取分成3组,每组20例。1组采用二氧化碳点阵激... 详细信息

目的探讨308 nm准分子激光联合CO2点阵激光及PRP用于稳定期白癜风治疗的临床效果。方法简单随机选取2020年6月—2021年6月佛山市第一人民医院皮肤科60例稳定期非节段型白癜风患者,数字随机抽取分成3组,每组20例。1组采用二氧化碳点阵激光+308 nm准分子激光治疗,2组采用富血小板血浆(PRP)+308 nm准分子激光治疗,3组采用二氧化碳点阵激光+富血小板血浆(PRP)+308 nm准分子激光治疗。比较3组各部位PASI评分、VASI、色素积分、不良反应及白斑消退时间。结果3组面颈部治疗后PASI评分(1.69±0.35)分、VASI(20.69±1.95)分低于1组(2.05±0.31)分、(22.82±1.92)分、2组(2.06±0.32)分、(22.76±1.94)分;色素积分(2.05±0.25)分高于1组(1.74±0.20)分、2组(1.64±0.21)分,差异有统计学意义(P0.05)。3组95%、50%、30%白斑消退时间均短于1组、2组,差异有统计学意义(P<0.001)。结论308 nm准分子激光联合CO_(2)点阵激光及PRP用于稳定期白癜风治疗具有明显效果,不良反应少,可促进白斑消退,应用价值高。

关键词： 308 nm准分子激光 CO_(2)点阵激光稳定期白癜风不良反应 PRP