猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

内含子中正筛选标记neo基因在转录中的剪切研究

目的:研究插入内含子中的正筛选标记基因neo在转录中的剪切情况。方法:克隆了猪血清白蛋白基因5'端调控序列,以猪基因组DNA为模板,P10/P11为引物,PCR扩增猪血清白蛋白基因翻译终止密码子后2.9kb的3'端调控序列;以pEGFP-1为模板,P400/P401为引物PCR扩增绿色荧光蛋白(EGFP)基因,插入猪血清白蛋白基因5'端调控区之后,在3'端调控序列的内含子中靠近N端的序列中插入正筛选标记基因neo,构建了表达EGFP的真核表达载体pEXp11。转染人肝癌细胞系HepG2,通过G418药物筛选获得稳定转染的抗药性细胞克隆。提取抗性细胞克隆基因组RNA并进行反转录,获得cDNA序列。结果:用引物D400/D401及分别位于neo基因和3'端调控序列上的一对引物D394/D357进行PCR检测,其中D394/D357并未扩增出目的条带。结论:插入内含子中的neo基因在转录过程中可随内含子一起被剪切。     

SV40PolyA片段串联拷贝数目影响GFP报告基因表达

Alu元件（约280bp）是人类基因组中的重要重复序列,串联Alu呈长度依赖性下调GFP报告基因表达,在Alu串联序列上游正向或反向插入SV40PolyA（简称PolyA,240bp）,解除Alu串联序列对GFP基因的抑制作用.PCR法扩增PolyA反序（PolyAas）不同位置的60bp片段,插入pAlu14质粒（14个Alu正向串联插入pEGFP-C1）的GFP基因和Alu14之间,瞬时转染HeLa细胞,通过荧光显微镜观察和Northern检测,1F1R（PolyAas5′端第1个60bp片段）和4F4R（自PolyAas5′端计算第4个60bp片段）不能活化基因;2F2R和3F3R（PolyAas中间的2段）可以解除Alu14对GFP基因的抑制作用.将2F2R和3F3R各自反复首尾串联,分别插入pAlu14质粒GFP基因和Alu串联序列之间,瞬时转染HeLa细胞,用插入4个2F2R的pAlu28,插入4个3F3R的pAlu18,插入4个3F3R的pAlu28作长度对照,以排除由于插入片段长度增加可能对结果造成的干扰,发现2～4个拷贝的2F2R和3F3R活化基因作用高于一个拷贝的同样片段,但是多于8拷贝以后,活化GFP基因作用减弱.本实验证明,SV40 PolyAas至少含有两段活化基因序列,活化基因序列（2F2R,3F3R）的最适活化基因条件需要合适的拷贝数.     

黑曲霉pepB基因缺失菌株的构建及其功能分析

以黑曲霉(Aspergillus niger)GICC2773基因组DNA为模板,用PCR方法分别扩增pepB基因中的上游约1．4kb和下游约1．3kb两段DNA序列,将此两段序列按同一方向分别插入质粒pMW1中潮霉素抗性基因(hph)表达单元的5′和3′端,构建成重组质粒pMW1-pepB,用于通过同源重组靶向破坏基因组中的pepB基因。同源重组则采用原生质体-PEG方法,将酶切pMW1-pepB得到的线性片段转化A．niger GICC2773菌株,通过潮霉素选择平板得到62个Hgy抗性转化子,然后采用PCR方法从这些抗性转化子中筛选到1个由于同源重组产生的pepB基因缺失突变菌株pepB29。功能分析显示该突变株的酸性蛋白酶活性有明显下降,外源蛋白漆酶的分泌表达有所提高。     

强直性肌营养不良症DMPK基因CTG重复序列与Alu±1kb单倍型研究

强直性肌营养不良(myotonic dystrophy,DM)是由于DMPK基因3′非翻译区CTG重复序列异常扩展所致的、主要累及神经肌肉系统的常染色体显性遗传病。在该基因的第8内含子中还存在一个Alu重复序列的1kb插入/缺失多态性,即Alu±1kb多态性。为了帮助阐明汉族人群中DM突变的起源,并为解释DM在不同群体中发病率的差异提供更多依据,本文从300例已知CTG拷贝数的正常汉族群体中随机挑选60例,首先通过PCR扩增确定其Alu±1kb多态性,然后对Alu±1kb和CTG双杂合的标本,采用长PCR方法先行扩增含Alu±1kb和CTG重复序列的DNA片段,再分别对含Alu(＋)和Alu(－)的DNA片段中的CTG拷贝数进行常规PCR分析,以确定二位点的单倍型。结果表明60例正常人中二位点间呈连锁不平衡。其单倍型为:（CTG）5均与Alu(＋)连锁;多数(CTG)11～14与Alu(－)连锁;在两个(CTG)≥19的等位基因中一个与Alu(＋)连锁,另一个与Alu(－)连锁。各民族相关资料的比较提示,汉族人群中(CTG)11～14与非洲黑人的起源可能不同;(CTG)19～30/Alu－1kb在汉族人群中的频率远比欧洲人群的高;(CTG)19～30/Alu－1kb与(CTG)19～30/Alu＋1kb在汉族人群中是以一定比例共存的;(CTG)19～30在不同民族间的起源不尽相同;如果从(CTG)5到(CTG)19～30的假设成立的话,则很可能是一个较为复杂的过程。 Abstract：Myotonic dystrophy(DM),an autosomal dominant disease of the neuromascular system,is caused by expansion of the CTG repeats in the 3＇untranslated region of the DMPK gene.In the eighth intron of this gene,there is another polymorphism－the insertion/deletion of 1kb Alu repeat(Alu±1kb).In order to help elucidate the origin of DM mutation in Chinese Han patients,and explain the difference of incidence of DM in different populations,60 normal Han individuals were randomly chosen from 300 Chinese,whose CTG copy number had been previously ascertained.The polymorphism of Alu±1kb of the 60 cases were firstly analyzed by in vitro amplification;then the 22 cases in which both sites were heterozygous were analyzed as following:the fragments containing both Alu±1kb and CTG repeat sequence were first amplified by long PCR method;and then the CTG copy numbers were analyzed in the Alu(＋)and Alu(－)alleles.In the 60 cases studied,a remarkable linkage disequilibrium between CTG triplet repeats and Alu±1kb were observed.All the(CTG)5 alleles were linked with Alu(＋),and most of the(CTG）11－14 alleles were linked with Alu(－);one of the two alleles of(CTG）≥19 was linked with Alu(＋),the other was linked with Alu(－).This suggests that the origin of(CTG)11－14 in Chinese Han may be different from that of African Blacks;the frequency of (CTG)19－30 /Alu－1kb in Chinese Han might be much higher than that in Europeans Caucasians.The(CTG)19－30/Alu－1kb and(CTG)19－30/Alu＋1kb coexist in Chinese Han in some proportion;the origin of (CTG)19－30 in different population may be different;if the hypothesis of(CTG)5 to(CTG)19－30 is true,then the progress should have been a relatively complicated process.     

水葫芦EcGS1b基因启动子的克隆及表达分析

利用染色体步移技术从水葫芦基因组DNA中扩增得到谷氨酰胺合成酶EcGS1b基因5'端上游约1.7 kb的一段序列,经PlantCARE序列分析表明,该序列中除了包括TATA-box和CAAT-box等基本元件外,还含有TATC-box、HSE、LTR和多种光反应相关的元件,初步推测其为EcGS1b基因启动子,命名为pEcGS1b。进一步通过PCR扩增了4个5'缺失的pEcGS1b片段,命名为p1200、p900、p600、p400。以pBI121为基础,构建了4个以GUS为报告基因的植物表达载体,命名为p1200::GUS、p900::GUS、p600::GUS、p400::GUS。利用农杆菌介导的叶盘法将4个植物表达载体转化烟草叶片,进行瞬时表达。结果表明4个缺失片段均具有启动功能,但弱于组成型表达的花椰菜花叶病毒(CaMV) 35S启动子,其中p1200的启动活性最强。     

强直性肌营养不良症DMPK基因CTG重复序列与Alu±1kb单倍型研究

强直性肌营养不良(myotonic dystrophy,DM)是由于DMPK基因3′非翻译区CTG重复序列异常扩展所致的、主要累及神经肌肉系统的常染色体显性遗传病。在该基因的第8内含子中还存在一个Alu重复序列的1kb插入/缺失多态性,即Alu±1kb多态性。为了帮助阐明汉族人群中DM突变的起源,并为解释DM在不同群体中发病率的差异提供更多依据,本文从300例已知CTG拷贝数的正常汉族群体中随机挑选60例,首先通过PCR扩增确定其Alu±1kb多态性,然后对Alu±1kb和CTG双杂合的标本,采用长PCR方法先行扩增含Alu±1kb和CTG重复序列的DNA片段,再分别对含Alu(＋)和Alu(－)的DNA片段中的CTG拷贝数进行常规PCR分析,以确定二位点的单倍型。结果表明60例正常人中二位点间呈连锁不平衡。其单倍型为:（CTG）5均与Alu(＋)连锁;多数(CTG)11～14与Alu(－)连锁;在两个(CTG)≥19的等位基因中一个与Alu(＋)连锁,另一个与Alu(－)连锁。各民族相关资料的比较提示,汉族人群中(CTG)11～14与非洲黑人的起源可能不同;(CTG)19～30/Alu－1kb在汉族人群中的频率远比欧洲人群的高;(CTG)19～30/Alu－1kb与(CTG)19～30/Alu＋1kb在汉族人群中是以一定比例共存的;(CTG)19～30在不同民族间的起源不尽相同;如果从(CTG)5到(CTG)19～30的假设成立的话,则很可能是一个较为复杂的过程。 Abstract：Myotonic dystrophy(DM),an autosomal dominant disease of the neuromascular system,is caused by expansion of the CTG repeats in the 3＇untranslated region of the DMPK gene.In the eighth intron of this gene,there is another polymorphism－the insertion/deletion of 1kb Alu repeat(Alu±1kb).In order to help elucidate the origin of DM mutation in Chinese Han patients,and explain the difference of incidence of DM in different populations,60 normal Han individuals were randomly chosen from 300 Chinese,whose CTG copy number had been previously ascertained.The polymorphism of Alu±1kb of the 60 cases were firstly analyzed by in vitro amplification;then the 22 cases in which both sites were heterozygous were analyzed as following:the fragments containing both Alu±1kb and CTG repeat sequence were first amplified by long PCR method;and then the CTG copy numbers were analyzed in the Alu(＋)and Alu(－)alleles.In the 60 cases studied,a remarkable linkage disequilibrium between CTG triplet repeats and Alu±1kb were observed.All the(CTG)5 alleles were linked with Alu(＋),and most of the(CTG）11－14 alleles were linked with Alu(－);one of the two alleles of(CTG）≥19 was linked with Alu(＋),the other was linked with Alu(－).This suggests that the origin of(CTG)11－14 in Chinese Han may be different from that of African Blacks;the frequency of (CTG)19－30 /Alu－1kb in Chinese Han might be much higher than that in Europeans Caucasians.The(CTG)19－30/Alu－1kb and(CTG)19－30/Alu＋1kb coexist in Chinese Han in some proportion;the origin of (CTG)19－30 in different population may be different;if the hypothesis of(CTG)5 to(CTG)19－30 is true,then the progress should have been a relatively complicated process.     

侧耳木霉T2-1植酸酶基因的序列分析及蛋白结构预测

利用GenBank发表的植酸酶A编码序列设计的引物,通过PCR的方法对侧耳木霉(Trichoderma pleuroticola)T2-1基因组DNA进行扩增,获得了一条长约1.7 kb的特异性DNA片段.序列测定结果表明,该DNA片段含有植酸酶编码基因的完整序列和3段内舍子序列,其中植酸酶基因全长1 443 bp,编码480个氨基酸,5'端有一段编码23个氨基酸的信号肤序列,其余的457个氨基酸残基为成熟植酸酶的氨基酸序列.对该基因编码的氨基酸序列进行三级结构预测,发现它为磷酸单酯酶.已将侧耳木霉T2-1植酸酶基因序列在GenBank中注册(登录号:GQ325590).这是目前中国在GenBank注册的第一个完整的木霉植酸酶编码基因.     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

苦瓜中与AGAMOUS相似基因启动子的克隆和表达载体的构建

McAG2基因是苦瓜中分离得到的与AGAMOUS相似基因,在花器官和果实中特异表达,参与花的第三轮和第四轮器官的形成.文章采用染色体DNA步移技术克隆得到长为1 417 bp的McAG2基因5'上游片段.序列分析显示此片段含有典型的TATA-box、CAAT-box、丰富的激素应答调控元件.为了研究这些调控元件,还构建了McAG2基因5'侧翼缺失和内含子缺失表达载体.     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

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.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细