荧光发光杆菌TT01品系pirA2B2 基因的克隆表达与杀虫活性

【目的】Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50%和45%的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性。【方法】PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDS-PAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2蛋白。用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性。【结果】生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性;PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性。【结论】pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因。【意义】pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirA1B1的关系以及该基因的表达调控等打下了基础。     

嗜线虫致病杆菌血腔毒素Tp40的纯化和特性分析

[目的]嗜线虫致病杆菌是一种昆虫病原线虫共生菌,它能够产生多种杀虫毒素.本研究旨在从嗜线虫致病杆菌Xenorhabdus nematophila HB310菌株的细胞内纯化新的杀虫蛋白毒素,并对其进行基因克隆和序列分析.[方法]应用盐析和制备型非变性凝胶电泳等方法纯化蛋白,再通过对5龄大蜡螟幼虫血腔注射进行活性筛选.对获得的目的蛋白与已知蛋白进行同源分析,克隆出该目的蛋白的基因序列,从而进行相应的基因和氨基酸序列分析.[结果]本研究纯化的Tp40蛋白对大蜡螟LD50为68.54 ng/头,其SDS-PAGE电泳图谱只显示出一条分子量约为42 kDa的多肽.Western印迹分析表明Tp40与已知的Txp40为同源蛋白,并且仅存在于细胞内.编码该蛋白的基因开放读码框全长1107bp(GenBank登录号:EU095326),编码368个氨基酸残基,预测分子量为41.5 kDa,等电点为8.66,与GenBank中的其余13株昆虫病原线虫共生菌所包含的相似基因核苷酸序列及推导的氨基酸序列比较,同源性分别为85%～99%和70%～99%.[结论]Tp40蛋白具有很高的血腔杀虫活性,其基因序列具有较强的保守性,是昆虫病原线虫共生菌复合体杀虫过程中的一种关键因子.     

异小杆线虫XJZL1409(Heterorhabditis bacteriophora)共生菌的分离及致病性研究

本研究分离了来自新疆和田地区间作绿豆的4年枣0-40 cm根际土壤中的昆虫病原线虫XJZL1409Heterorhabditis bacteriophora的共生菌,并进行致病性测定。通过侵染期线虫和线虫致死的大蜡螟幼虫血腔直接分离,采用传统形态学观察结合分子生物学方法对菌株进行鉴定。同时大蜡螟Galleria mellonella 5龄幼虫接种菌株细胞发酵液测定致病性。菌株形态学特性结合16S r DNA序列系统发育分析,线虫XJZL1409的共生菌被鉴定为发光杆菌属Photorhabdus luminescens subsp. laumondii。室内菌液注射毒性测定结果显示,侵染致死的幼虫体色呈灰绿色;接种48 h后,每头幼虫接种菌液量为500 CFU时校正死亡率就可以达到100%; 48 h时致死中浓度LC50为10. 74 CFU/m L。同时菌液口服杀虫毒性结果表明,第5天时待测菌株菌液对大蜡螟幼虫的校正死亡率为21. 21%,体重抑制率为20. 02%。因此,昆虫病原线虫XJZL1409的共生菌为P. luminescens subsp. laumondii对大蜡螟幼虫具有很强的注射毒性,为研究线虫的致病机制及发掘新抗性基因奠定基础。     

发光杆菌碳端截短的Mcf毒素杀虫活性研究

发光杆菌能产生很多毒素杀死昆虫宿主.这些毒素中有一种叫Mcf致软因子.可以在大肠杆菌中表达并毒杀毛虫.通过同源性比对分析.在Mcf氨基酸序列中N端有一个BH3的区域,拥有BH3结构域的蛋白具有细胞凋亡的功能,推测保留N端BH3结构域的碳端截短Mcf毒素具有杀虫毒力.从发光杆菌属(Photorhabdus luminescens subsp.laumondii)中克隆了杀虫毒素基因mcf的5'端3 960 bp的核酸序列(包含BH3结构域).将该基因连接到E.coli表达载体pET28a上,并转入BL21(DE3).经IPTG诱导后,在SDS-PAGE上发现145 kD的目的蛋白条带.利用亲和层析纯化得到纯毒素,分别对甜菜夜蛾一龄幼虫喂食生测和对甜菜夜蛾四龄幼虫(Spodoptera exigua)注射生测,显示该毒素具有喂食毒力和血腔毒力,证明含有BH3区域的碳端截短Mcf毒素有杀虫的生物活性.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

琥珀蚕丝腺转录因子基因AaSGF-1的克隆、原核表达及组织表达分析

【目的】本研究旨在克隆琥珀蚕Antheraea assama丝腺转录因子基因AaSGF-1,分析其序列特征及表达模式并制备多克隆抗体,为探讨该基因的生理功能奠定基础。【方法】采用RT-PCR和RACE技术从琥珀蚕丝腺中克隆AaSGF-1的cDNA序列,并进行生物信息学分析;利用qPCR检测AaSGF-1在琥珀蚕5龄第4天幼虫不同组织(头、中肠、脂肪体、丝腺、血液、表皮)中的表达模式;构建原核表达质粒载体,在大肠杆菌Escherichia coli BL21中表达AaSGF-1,利用纯化的融合蛋白免疫新西兰兔子,获得高效的抗体。利用免疫荧光技术检测AaSGF-1在琥珀蚕蚁蚕丝腺和表皮及4龄幼虫丝腺中的表达情况。【结果】克隆了琥珀蚕AaSGF-1的cDNA序列(GenBank登录号： MK889510.1),开放阅读框(ORF)序列长1 050 bp,编码349个氨基酸残基,预测蛋白分子质量为38.8 kD,理论等电点(pI)为8.74。qPCR检测结果显示AaSGF-1在琥珀蚕5龄幼虫丝腺组织尤其是后部丝腺中高量表达,而在其他组织中几乎不表达。免疫荧光结果表明AaSGF-1在蚁蚕及4龄幼虫的丝腺中表达。【结论】本研究原核表达了琥珀蚕AaSGF-1,制备了多克隆抗体,证实了AaSGF-1在琥珀蚕幼虫的丝腺中高表达,为进一步研究该基因在琥珀蚕丝腺发育及丝蛋白合成中的作用奠定了基础。     

盐藻PDS基因的同源克隆及在大肠杆菌中的表达

【目的】八氢番茄红素脱氢酶PDS为真核膜结合蛋白,我们通过更换不同的表达策略,探索在大肠杆菌中表达真核膜结合蛋白的方式。【方法】利用RACE的方法克隆盐藻PDS的全长c DNA序列。利用原核表达载体p ET-28a构建p ET-28a-PDS表达载体;使用PLtac启动子替换T7启动子构建p ET-PLtacPDS表达载体;合成Mistic序列融合入p ET-28 a中构建了p ET-Mistic-PDS融合表达载体。分别转化入大肠杆菌BL21(DE3)中进行原核表达。【结果】获得了盐藻PDS基因的全长c DNA序列2237 bp,开放阅读框为1749 bp,共编码582个氨基酸(NCBI登录号为GQ923693.1)。利用p ET-28a-PDS和p ET-PLtacPDS表达的PDS蛋白表达量低,并以包涵体形式存在;利用p ET-Mistic-PDS载体表达的PDS蛋白表达量明显提高,且大部分以可溶蛋白形式存在,具有脱氢酶活性。【结论】实验结果表明Mistic作为促溶标签能促进膜蛋白的正确折叠,提高蛋白的可溶性。蛋白酶活测定结果证明了Mistic的融合可以保持蛋白的天然活性。     

苏云金芽孢杆菌vip3A基因的鉴定、克隆及活性分析

【目的】鉴定我国自行分离的苏云金芽孢杆菌（Bacillus thuringiensis, Bt）菌株中vip3A基因的分布和基因型,从其中对鳞翅目幼虫表现高毒力的Bt菌株中克隆vip3Aa基因。【方法】利用PCR-RFLP方法确定vip3A基因的分布和鉴定基因型,利用PCR方法克隆vip3A全长基因。【结果】171株野生型Bt菌株中共鉴定出63株含有vip3A基因,均与vip3Aa1类基因相似。从TF9和Bt16菌株中克隆得到2个vip3Aa基因,分别构建了携带vip3Aa基因的表达载体p30vip-26和p30vip-27,SDS-PAGE和Western Blot分析表明,IPTG诱导后均可表达88 kDa左右的Vip3A蛋白,蛋白可溶性分析表明约10%可溶。这两种基因序列已被国际Bt基因命名委员会分别正式命名为vip3Aa26和vip3Aa27。生物测定结果显示,Vip3Aa27蛋白对粉纹夜蛾（Trichoplusia ni）、甜菜夜蛾（Spodoptera exigua）和棉铃虫（Helicoverpa armigera）3种重要鳞翅目害虫初孵幼虫的毒力较高,LC50值分别为0.125 μg/mL,0.238 μg/mL和9.238 μg/mL。而Vip3Aa26蛋白仅对粉纹夜蛾有活性,LC50值为4.423 μg/mL。【结论】本研究中的Vip3Aa27蛋白对粉纹夜蛾、甜菜夜蛾和棉铃虫幼虫均能表现高杀虫活性,而Vip3Aa26蛋白仅对粉纹夜蛾幼虫有活性,实验结果表明Vip3A蛋白个别氨基酸的变化对其杀虫活性影响很大。     

嗜线虫致病杆菌血腔毒素Tp40对大蜡螟幼虫体内酶活和中肠组织的影响

嗜线虫致病杆菌Xenorhabdus nematophila在侵入到寄主昆虫血腔后能够成功地逃避或抑制寄主昆虫的免疫反应并快速杀死昆虫。为深入了解嗜线虫致病杆菌的杀虫机理,明确关键的致病因子,作者应用盐析和制备型非变性凝胶电泳等方法,从嗜线虫致病杆菌HB310菌株的细胞内分离纯化了一种新的杀虫蛋白——Tp40,该蛋白对大蜡螟Galleria mellonella具有高血腔注射活性,对大蜡螟5龄幼虫的LD₅₀为68.54 ng/头。本文检测了该毒素对大蜡螟幼虫的致病特性,注射Tp40毒素后,大蜡螟幼虫表现出兴奋和痉挛等症状,当以不低于（70±0.02）ng/头的剂量注射Tp40,大蜡螟幼虫均在20 min内死亡,但试虫的体色 、血淋巴的颜色以及血细胞的形态没有发生明显的变化。对大蜡螟体内酶活性的测定结果显示,在注射LD₅₀剂量的Tp40蛋白后,试虫体内羧酸酯酶和乙酰胆碱酯酶活力都明显的高于对照（P<0.05）,而酚氧化酶活力显著低于对照（P<0.05）。对大蜡螟幼虫中肠的组织病理学研究显示：这种42 kDa蛋白能够破坏试虫的中肠组织,导致其肠壁细胞出现排列紊乱、脱落和围食膜消失。据此推测,Tp40与嗜线虫致病杆菌对寄主昆虫的免疫抑制有关,寄主中肠组织可能是其作用靶标之一。     

管氏肿腿蜂毒液丝氨酸蛋白酶同源物SgSPH对寄主血淋巴酚氧化酶活性的影响

【目的】本研究旨在通过克隆表达管氏肿腿蜂Scleroderma guani毒液丝氨酸蛋白酶同源物(serine protease homologue, SPH)基因SgSPH,探索其编码的毒液蛋白对寄主血淋巴酚氧化酶活性的影响。【方法】利用RT PCR技术克隆管氏肿腿蜂毒液SgSPH基因的开放阅读框(ORF),采用生物信息学软件分析其基因序列结构特征;采用qPCR技术检测该基因在不同发育阶段(卵、低龄幼虫、高龄幼虫、老熟幼虫、吐丝幼虫、黄茧蛹、黑茧蛹和羽化后1-5 d成虫)和雌成虫不同组织(头部、胸部、去除毒液器官的腹部和毒液器官)中的相对表达量;利用载体pSUMO Mut对该基因进行原核表达,用镍亲和层析柱纯化表达的重组蛋白,采用SDS-PAGE和Western blot对获得的重组蛋白进行鉴定;用酶活性测定方法,分析SgSPH重组蛋白对寄主黄粉虫Tenebrio molitor蛹血淋巴酚氧化酶活性的抑制作用。【结果】克隆获得管氏肿腿蜂毒液SgSPH基因(GenBank登录号: MT920663)的ORF,长798 bp,编码265个氨基酸,其中第1-20位氨基酸为信号肽,理论分子量为30.53 kD,等电点为9.59。多序列比对分析表明,SgSPH与其他寄生蜂毒液丝氨酸蛋白酶和SPH具有较低的氨基酸序列一致性(9%~17%),且缺乏保守的催化三联体。qPCR分析表明,SgSPH基因在管氏肿腿蜂成虫阶段和毒液器官中高表达。SDS-PAGE电泳和Western-blot检测发现,成功表达SgSPH重组蛋白,并纯化得到了高纯度SgSPH重组蛋白。酶活性检测结果显示,SgSPH能抑制寄主黄粉虫蛹血淋巴的酚氧化酶活性。【结论】结果提示管氏肿腿蜂毒液SgSPH具有干扰寄主酚氧化酶级联反应的功能。     

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.     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.     

Composition and diversity of bird communities in Swedish farmland–forest mosaic landscapes

CapsuleThe amount of forest (at local and landscape scales) and occurrence of residual habitats at the local scale are shown to be the major factors influencing bird community composition in farmland–forest landscapes in central Sweden.

Aims To investigate the importance of local habitat and landscape structure for breeding birds in farmland–forest landscapes in central Sweden.

Methods Breeding birds were censused at 292 points. A detailed habitat mapping was made within 300 m of the points. Within a 300–600 m radius only two major habitats (forests and arable fields) were identified.

Results Cluster analyses of bird communities identified three site types that also differed in habitat composition: (i) partially forested sites in forested landscapes; (ii) heterogeneous sites with residual habitats in mosaic landscapes; and (iii) field-dominated farmland sites in open landscapes. A total of 19 of 25 farmland bird species (restricted to farmland or using both farmland and forest) had the highest abundance in farmland sites with mosaics of forest and farmland, while only six farmland species had the highest abundance in field-dominated sites. The bird community changed from being dominated by farmland species to being dominated by forest species (common in forest landscapes without farmland) at small proportions (10–20%) of forest at the local scale. A major difference in habitat composition between heterogeneous and field-dominated sites was the occurrence of different residual habitats (e.g. shrubby areas and seminatural grasslands). These habitats seemed to influence bird community composition more than land-use, despite covering <10% of the area. Seminatural grasslands were important for bird community composition and species-richness, but grazing seemed to be less important. Among different land-use types, cereal crops were the least preferred fields. Set-asides with tall vegetation and short rotation coppices were positively associated with species-richness of farmland birds.

Conclusion In general, the composition of the landscape was important for bird community composition, although amount and distribution of forests, occurrence of residual habitats and land-use of fields at the local scale had the strongest influence on bird community composition. The possible implications of these patterns for managing farmland–forest landscapes are discussed.     

Chemical constituents from the fruits of Rhus typhina L. and their chemotaxonomic significance

This work describes the isolation and characterization of twenty-nine compounds from the fruits of Rhus typhina L., including eleven flavonoids (1–11), eleven phenols (12–22), two pentacyclic triterpenes (23–24), two organic acids (25–26), one lumichrome (27), one courmarin (28) and one pyrimidine (29) on the basis of their spectroscopic data. Compounds apigenin (1), daidzein (4), orobol (5), 3′, 5, 5′, 7-tetrahydroxyflavanone (6), naringenin (7), butein (8), (-)-catechin (9), quercetin-3-O-α-L-(3″-O-galloyl)-rhamnoside (11), 2-hydroxybenzoic acid (13), 4-hydroxybenzaldehyde (14), vanillin (15), methyl 3,4-dihydroxybenzoate (16), 3,5-dihydroxybenzamide (18), tyrosol (19), caffeic acid (20), 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (21), phlorizin (22), friedelin (23), oleanolic acid (24), 4,4-dimethyl-heptanedioic acid (25), anthranilic acid (26), lumichrome (27), scoparone (28) and uracil (29) have not been recorded before in this plant. This is the first report on the occurrence of compounds 4–7, 9, 11, 13–14, 16, 18–21, 25–29 from the genus Rhus. Moreover, the chemotaxonomic significance of these isolated compounds was also summarized.