原生质体融合选育赖氨酸高产菌种的研究

北京棒杆菌1134衍生株与枯草芽孢杆菌151衍生株的原生质体融合得到了一株可以甜菜糖蜜为原料,在摇床与小罐发酵均能稳定产赖氨酸6 50％以上的高产菌株——Q4413。Q441 3菌落形态类似于11 34株,而菌体形态区别于双亲,呈较短的杆状,无鞭毛,但有较稀疏的纤毛。生理生化研究表明：Q4413耐盐性、蔗糖发酵,石蕊牛奶试验产碱状况酷似于151株,而与1134株略有差异;运动性、糊精、柠檬酸盐利用,MR试验、酪素水解、明胶水解、石蕊牛奶还原,又酷似于1134株而区别于151株;04413的最适生长温度、pH值居双亲之间;Qq4413为原养型加三重抗性[Rifr,AECr Nalr];其细胞DNA含量与GC比测定结果均居双亲之中,而且生物量测定结果也均与双亲有一定的差异。     

高产融合子Q4413的形态学与生理生化特征的研究

本文通过对枯草芽孢杆菌BR151衍生株与北京棒杆菌1134衍生株的赖氨酸高产融合子Q4413株的形态学、生理生化特性等方面的研究,揭示了融合子与双亲株在这些方面的差异,为Q4413株确系双亲株的重组子或新的融合子增加了佐证.     

高产融合子Q4413的遗传学研究

本文通过对枯草芽孢杆菌BR151衍生株与北京棒杆菌1134衍生株的赖氨酸高产融合子Q4413的遗传型研究、生物量(菌体湿重、菌重、菌体体积和菌体密度)测定、DNA含量测定和GC比的研究,揭示了融合子在遗传学方面与双亲株之间的差异,进一步证实了该株确系稳定的重组子或新的融合子.     

欧文氏菌和棒杆菌的属间隔合研究

研究了用原生质体融合技术获得欧文氏菌和棒杆菌的融合细胞,串联发酵Ｄ－葡萄糖产生２－酮基－Ｌ－古龙酸的第一步发酵菌株欧文氏菌ＳＣＢ２４７经０．８ｍｇ／ｍＬ溶菌酶酶解０．５ｈ后,原生质体的形成率和再生率分别为９９．８％和２７．８％。第二步发酵菌株棒杆菌ＳＣＢ３０５８经预处理后由１．３ｍｇ／ｍＬ溶菌酶酶解２ｈ,原生质体的形成率和再生率分别为９９．５％和５６．３％,用携带氨苄青霉素抗性标记的ＳＣＢ２４７和     

欧文氏菌和棒杆菌的属间融合研究*

研究了用原生质体融合技术获得欧文氏菌和棒杆菌的融合细胞。串联发酵Ｄ－葡萄糖产生２－酮基－Ｌ－古龙酸的第一步发酵菌株欧文氏菌ＳＣＢ２４７经０．８ｍｇ／ｍＬ溶菌酶酶解０．５ｈ后,原生质体的形成率和再生率分别为９９．８％和２７．８％。第二步发酵菌株棒杆菌ＳＣＢ３０５８经预处理后由１．３ｍｇ／ｍＬ溶菌酶酶解２ｈ,原生质体的形成率和再生率分别为９９．５％和５６．３％。用携带氨苄青霉素抗性标记的ＳＣＢ２４７和经热灭活的ＳＣＢ３０５８为亲本,在４０％ＰＥＧ６０００和０．２ｍｏｌ／Ｌ新生磷酸钙等适宜条件下融合,融合频率为３．６×１０~(－６)。在非选择和选择培养基上连续传代十几次后,对融合子的单菌形态、染色结果、菌落形态、色泽、总蛋白量、同工酶、发酵性能等方面与亲本进行了比较。结果表明,融合子确系棒杆菌和欧文氏菌的融合细胞。摇瓶发酵结果显示,所得的３８株稳定的融合子中约４０％能转化葡萄糖为维生素Ｃ前体２－酮基－Ｌ－古龙酸。     

提高枯草杆菌原生质体融合率的研究

利用改良的HM制备液,在适宜的条件下酶解制备亲本株的原生质体,在DNase存在的条件下,用PEG(MW6,000)做为聚合剂,经低温短时间处理,将枯草杆菌(B,subtilisK)Ki—2—132衍生株与BR151衍生株的原生质体融合,再在适宜的条件下使用改良的DM_3再生培养基,补加适量经处理的人血清白蛋白,同时加入适量适合于枯草杆菌细胞壁再生的引物进行再生。使再生率达到了55—58.15％左右,融合率高达1.82×10~(-2)。筛选出51种不同类型的融合子923株。经继代八代测得其融合子的稳定率达32 46％。从而在工业菌株的选育中使获得高产菌株成为可能。     

原生质体融合构建高效产脂肽工程菌

芽孢杆菌因其可产生多种生理活性物质,在环境污染修复、生物防治、微生物采油等领域具有广阔的应用前景。莫哈韦芽孢杆菌Bacillus mojavensis JF-2和解淀粉芽孢杆菌B. amyloliquefaciens BQ-6是从油田筛选出的产脂肽类表面活性剂菌株, 但在微生物采油实际应用中受到氧气浓度、盐度及pH的限制。原生质体融合是改变微生物代谢功能的一种简便有效的方法,以上述两株芽孢杆菌为对象,利用4因素3水平正交试验来探索菌龄、溶菌酶浓度、酶解温度和酶解时间对原生体制备、再生的影响。此外,对两菌株进行了双亲灭活原生质体融合,通过筛选得到了一株工程菌HY-4,并对其进行了初步的评价。结果表明:菌龄、溶菌酶浓度和酶解时间显著影响芽孢杆菌原生质体制备率及再生率(P<0.05),且在溶菌酶处理前用生理盐水多次洗涤菌体细胞,可提高制备率。两种芽孢杆菌原生质体制备及再生的最优条件均为:菌龄7 h、溶菌酶浓度2.5 mg/ml、酶解时间30 min、酶解温度42 ℃。融合子HY-4的最高耐盐度为15%,可耐50 ℃高温,代谢产脂肽的pH范围为4.0~9.5,且在好氧及厌氧条件下均能够代谢产脂肽,在厌氧条件下生长迅猛(细胞干重>1.6 g/L)。综上所述,融合子HY-4具有较大的应用潜力,该研究为芽孢杆菌的遗传育种打下了方法学基础,并对驱油微生物菌种的选育具有指导意义。     

蛋白酶产生菌种间原生质体融合的研究

Baci llus属中的一些种是生产蛋白酶的重要菌株。 采用2500u／ml溶菌酶制备的两种芽孢杆菌原生质体在30％聚乙二醇(Mw6000)的作用下,成功地获得了枯草芽孢杆菌(Bacillusfubtilis)和地衣状芽孢杆菌(Bacillus licheniformis)的种间融合重组子。融合频率为7．47 x10—2．28×10-5经过四代的移接验证,融合子的稳定率达到12—20％。结果表明,两种产蛋白酶的芽孢杆菌融台后,营养缺陷和抗药特性方面可以互补;菌落形态发生某些变化;生产性能有较大的改进。     

适于罗布麻生物脱胶的果胶酶产生菌分离筛选与表征

在青海柴达木盆地沤制的罗布麻表皮中分离得到两株适于罗布麻脱胶的高产果胶酶菌株,经形态、生理生化指标鉴证和16SrDNA菌种鉴定,确定一株为新的果胶酶产生菌琼氏不动杆菌（Acinetobacter junii）,一株为枯草芽孢杆菌（Bacillus subtilis）。水解圈实验得出前者H／C值（H／C为水解圈直径H与菌落直径C之比）为5,后者为3;经酶活力测定,在37℃下,琼氏不动杆菌（Acinetobacter junii）在11h达到产酶高峰（酶活力为103．2IU／mL）,枯草芽孢杆菌（Bacillus subtilis）培养至9h达到产酶高峰（酶活力为91．6IU／mL）,但前者最高酶活力比后者高12．5％;经脱胶实验得出两菌残胶率分别为18．47％和17．31％,对罗布麻生物脱胶有较好的适用性。     

棒杆菌(Corynebacterium pekinense)1134菌株原生质体化及其再生条件的研究

Corynebacterium属中的某些菌种是生产氨基酸的重要菌株。但它对蛋清溶菌酶很不敏感,较难制备其原生质体和再生。本文对供试菌株在培养时,加入氨基苄青霉素(ampicillin)、丝氨酸(serine)或甘氨酸(glycine);在酶解前采用不同的预处理,探讨了棒杆菌1134株原生质体化及其再生的条件。经过多次试验,最后选用补加适量的氨基苄青霉素和丝氨酸的培养液培养菌体,用果胶酶(Pectinase)和巯基乙醇(mercaptoethanol)进行酶解前的予处理,使其原生质化的培养液时缩间短至2.5小时,再生率提高至21.1％。为实现该菌株的原生质体融合及工业微生物育种奠定了基础。     

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.