霉酚酸产生菌原生质体转基因方法的建立

摘要:【目的】建立霉酚酸产生菌短密青霉菌的遗传转化体系。【方法】以腐草霉素抗性为选择标记,利用聚乙二醇介导原生质体融合,进行外源基因转化。【结果】聚乙二醇介导的短密青霉菌原生质体转化效率为每微克DNA 2－3个转化子;转化子的PCR检测结果显示外源基因已经整合到短密青霉菌基因组中,转化子抗性稳定。【结论】霉酚酸产生菌短密青霉菌转基因体系的建立为该菌进行分子生物学研究以及基因工程育种奠定了基础。     

农杆菌介导转化黑曲霉分生孢子及产孢缺陷突变子T-DNA插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)T-DNA系统,建立转化黑曲霉(Aspergillus niger)分生孢子的方法,构建T-DNA插入突变子文库,为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105,诱导转化黑曲霉分生孢子,筛选具有潮霉素抗性的突变子。分析抗性稳定突变子菌株的表型,采用反向PCR方法分析T-DNA插入位点相邻位置的序列,并推测突变基因可能具有的功能。【结果】实验获得具有稳定潮霉素抗性转化子193株,转化率为5.6×102转化子/108分生孢子。部分转化子表型出现较为明显改变,其中一株不能产孢,对其T-DNA插入位点序列分析比对结果显示,突变基因属于超级转运家族(major facilitator superfamily,MFS)。【结论】本研究建立的农杆菌转化黑曲霉分生孢子平台,结合T-DNA插入突变位点分析,可以为黑曲霉基因组功能注释研究提供一种简便有效的途径。     

杨树腐烂病菌(Cytospora chrysosperma)原生质体遗传转化体系的构建

【目的】通过分析不同酶解条件对金黄壳囊孢菌[Cytospora chrysosperma(Pers.)Fr.]原生质体释放的影响,建立高效制备原生质体及其遗传转化体系的方法,为开展杨树腐烂病菌的致病分子机制研究奠定基础。【方法】以杨树腐烂病菌菌株CFCC 89981为受体,在细胞壁降解酶作用下产生用于转化所需的原生质体,通过PEG(Polyethylene glycol)介导将g GFP DNA导入杨树腐烂病菌的原生质体中获得转化子。经PCR扩增、Southern blot和荧光观察验证g GFP DNA插入到杨树腐烂病菌基因组中并表达出GFP(Green fluorescent protein)蛋白。【结果】以p H 5.5的1.2 mol/L KCl为稳渗剂,杨树腐烂病菌菌丝经Driselase和Lysing enzymes共同酶解4 h可获得1.2×108个/m L原生质体,再生率可达63.74%±9.73%,FDA(Fluorescein diacetate)溶液染色结果显示98%左右的原生质体具有较高的活力。利用PEG介导的遗传转化方法,转化效率可达76个/μg DNA。PCR检测和Southern blot均可在转化子基因组中检测到GFP基因片段,且荧光检测转化子的菌丝均呈绿色荧光,表明GFP基因在杨树腐烂病菌中表达。此外,GFP转化子在无潮霉素抗性的PDA培养基中多代转接后仍稳定遗传并表达GFP蛋白。【结论】通过筛选酶解条件,获得高质量、高活性的杨树腐烂病菌原生质体,并利用PEG介导的转化方法建立了高效稳定的原生质体遗传转化体系。该体系的建立为杨树腐烂病菌的后续研究奠定了技术基础。     

农杆菌介导的携带egfp基因载体转化寡雄腐霉

【目的】寡雄腐霉(Pythium oligandrum Drechsler)是一种对动、植物和环境无害,兼具杀菌和增产效果的生防真菌。通过研究建立农杆菌介导的寡雄腐霉遗传转化体系。【方法】选用EHA105、AGL-1、LBA4404三种农杆菌菌株对寡雄腐霉进行遗传转化研究,通过对影响遗传转化效果的条件参数试验优化,确立适宜寡雄腐霉遗传转化的农杆菌菌株及转化条件,建立农杆菌介导的寡雄腐霉遗传转化体系。【结果】经研究发现,所选3种农杆菌菌株中EHA105菌株对寡雄腐霉的遗传转化效果最好,其次是AGL-1菌株,LBA4404菌株转化效果不好。EHA105菌株经IM(含300μmol/L AS)诱导培养至OD_(600)=0.6时,与浓度为10~6–10~7个/m L的寡雄腐霉孢子悬浮液以1–10:1的比例混合,在25–26°C以液体振荡的方式避光共培养72 h(pH 5.0,含300μmol/L AS),寡雄腐霉菌体液体振荡恢复培养24 h,涂布抗性选择平板筛选寡雄腐霉转化子,即可得到寡雄腐霉基因工程菌株,其转化率可达到130个转化子/106个孢子。【结论】本研究首次构建了农杆菌介导的寡雄腐霉遗传转化体系,研究结果可为寡雄腐霉的生防机制及分子育种研究提供技术支撑。     

运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法

【背景】萎锈灵抗性基因作为筛选标记在植物和真菌中得到广泛应用。【目的】构建可以使用萎锈灵作为筛选标记的香菇遗传转化技术。【方法】利用溶壁酶消化培养4 d的香菇菌株411-4的菌丝体获得原生质体,加入适量pL-cbx质粒和聚乙二醇溶液,混合物涂布于再生筛选培养基上,培养后挑选菌落进行验证试验。【结果】在原生质体数目108个和添加4μg质粒DNA的情况下,得到了40个抗性转化子。利用PCR实验和转代实验对转化子进行验证,结果显示38个转化子的抗性可稳定遗传,表明萎锈灵抗性基因整合进入了供试菌株的基因组中。【结论】利用香菇411-4菌株建立了一套运用萎锈灵抗性基因作为分子标记的遗传转化技术体系。     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF.gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化,利用氯嘧磺隆抗性标记筛选转化子,对转化子PCR验证及荧光显微观察;采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选,并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库,转化效率为150 400个转化子/106孢子,从3 721个转化子中筛选得到致病性缺陷转化子25个;随机选取20个转化子进行遗传稳定性测定,在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性,且表型稳定,表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化,构建橡胶树胶孢炭疽菌T-DNA插入突变体库,筛选致病缺陷突变菌,为进一步研究该菌致病相关基因提供材料。     

PEG 介导的苹果腐烂病菌原生质体转化

摘要: 【目的】建立PEG 介导的苹果腐烂病菌原生质体遗传转化体系。【方法】本文利用带有hph 基因的质粒,以苹果腐烂病菌(Valsa mali var.mali) 03-8 为受体菌株,通过PEG 融合法对其原生体进行转化。【结果】于YEPD 内培养48 h 的菌丝,在酶解液浓度为50 mg /mL Driselase + 10 mg /mL Lysing Enzymes 情况下,按10 mL酶液/0. 5 g湿菌体比例,酶解2 h时可以释放出4 × 107 个/mL 原生质体,其转化效率为44 个/μg DNA。对转化子的PCR 检测和Southern 杂交分析表明,hph 基因已经整合进苹果树腐烂病菌的基因组中。转化子在PDA 培养基中继代5 次后,87. 5% 的转化子仍能正常生长,表明外源基因hph 能在苹果树腐烂病菌中稳定遗传。【结论】该转化体系的建立为苹果树腐烂病菌致病相关基因的深入研究奠定了基础。     

农杆菌介导香菇菌丝遗传转化体系的研究

香菇Lentinula edodes是世界第二大食药用真菌,随着其全基因组测序的完成,功能基因组学研究也逐渐展开,建立稳定的香菇转化体系是目前的研究热点。本文将探索以农杆菌为介导的遗传转化体系在香菇中进行随机插入突变的转化效率以及稳定性。构建的质粒p YN6982以潮霉素抗性基因hyg作为筛选标记基因,以增强型荧光蛋白基因egfp作为报告基因,以农杆菌EHA105和LBA4404为介导,同时转化了孢子单核体、原生质体单核体和双核体菌株。结果表明采用小米粒培养基进行菌丝培养和转化,经潮霉素抗性筛选,以及5代传代后,对转化子中的hyg和egfp基因进行PCR扩增和测序,验证了转化子的遗传稳定性。经过荧光显微观察表明,EGFP在转化子中可稳定表达。本研究探索出一种采用小米粒培养基培养菌丝并进行转化的新方法,建立了稳定的农杆菌介导的香菇遗传转化体系,为进一步开展香菇的基因功能研究奠定了良好的基础。     

潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化

为了建立适合米根霉的遗传转化体系,应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体p BS-hygro-ldh A;分别采用PEG/Ca Cl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体p BS-hygro-ldh A转化入米根霉AS 3.819菌株中,并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响;通过荧光定量PCR(q PCR)对米根霉转化子基因组中质粒整合拷贝数进行了检测,并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体p BS-hygro-ldh A的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高,原生质体电转化最佳电场强度为13 k V/cm,孢子萌发2.5 h转化率最高,萌发孢子电转化最佳电场强度为14 k V/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明,在一定范围内,高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系,为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.