利用聚鸟氨酸提高大豆原生质体外源基因的转化效率(简报)

有不少利用PEG法把外源基因导入到大豆原生质体,获得了转基因植株的报道,目前PEG的转化效率有所提高,但还是不能满足转化的需要,如何提高原生质体的转化效率是基因转化工作中的关键问题。本实验为了解决这个难题,以大豆幼子叶原生质体为材料,利用聚鸟氨酸(PLO:Poly-L-Ornithine,MW:114900,SIGMA)对Bt基因的转化进行了探讨。     

真菌免疫调节蛋白基因LZ-8过表达载体的构建及转化灵芝原生质体的研究

灵芝Ganoderma lingzhi是我国著名的药用真菌。但是,作为一种营养和保健价值都非常高的大型担子菌,灵芝还缺乏完善的转基因方法和安全转基因体系。本研究建立了免疫调节蛋白基因的过表达系统,并利用真菌特异性启动子GPD、终止子NOS和目的基因LZ-8构建真菌特异性双T-DNA表达载体p SB130NG-LZ8;利用溶壁酶提取灵芝原生质体,并用FDA染色法检测灵芝原生质体活性,原生质体成活率约为80%。通过PEG转化法对灵芝原生质体成功进行了转化,转化得到的原生质体在带有潮霉素抗性平板上长出,转化率为3–4/μgpSB130NG-LZ8+107个原生质体。转化子通过PCR检测和荧光定量PCR检测,获得LZ-8在灵芝中的过表达。     

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。     

一种有效的嗜热真菌杜邦嗜热菌靶向基因替代系统

以嗜热真菌杜邦嗜热菌Thermomyces dupontii NRRL 2155为研究材料,利用同源重组原理和真菌原生质体转化方法,以潮霉素抗性基因替换嗜热真菌目标基因,获得抗潮霉素的靶向基因敲除突变菌株。优化的遗传转化体系为：用15mg/mL裂解酶,在28℃下酶解2g杜邦嗜热菌菌丝5.5h以获得原生质体,经STC缓冲液洗涤重悬后,利用PEG（polyethylene glycol）介导的遗传转化方式,将10μg线性敲除全长片段转化至杜邦嗜热菌原生质体中,通过潮霉素筛选及PCR验证得到基因替换突变菌株,同源重组率达到20%。本研究首次将原生质体转化方法应用在杜邦嗜热菌,并成功建立稳定高效的基因替换体系,为快速构建杜邦嗜热菌的遗传转化体系和研究该嗜热真菌的基因功能提供有效方法。     

红曲菌9903A转化体系影响因素的研究

采用PEG介导的原生质体转化方法,研究了将含有潮霉素抗性基因的质粒pMP-Hygro转入红曲菌9903A的影响因素。实验结果表明,原生质体转化最佳条件为:1.0mol/L的山梨醇为渗透压稳定剂;以含有50mmol/L的Ca2 和最终质量分数为40%的PEG4000为转化介质;最佳原生质体浓度和载体DNA用量分别为1×108个/mL、1μg/100μL原生质体;原生质体再生培养基采用不含无机盐的培养基,再生方式采用原生质体液涂布单层再生培基平板法。得到的平均DNA转化率可达160个/μgDNA。本文所研究的PEG介导的原生质体转化方法可以较好的向红曲菌细胞导入外源DNA,并使外源DNA在红曲菌细胞内大量表达。     

禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究

目的:旨在敲除禾谷镰刀菌Fusarium graminearum Fg PDE1基因,确定其缺失突变体表型,从而分析该基因的生物学功能。方法:应用Split-marker技术构建含有潮霉素基因敲除盒,通过PEG介导原生质体转化,PCR筛查抗潮霉素转化子以获得缺失突变体ΔFg PDE1,根据突变体表型变化及致病性的检测对Fg PDE1基因的功能进行分析。结果:采用Split-marker技术,成功构建了Fg PDE1基因敲除盒;PEG介导转化禾谷镰刀菌原生质体后成功获得转化子。经PCR筛查,得到3个PCR确认的敲除突变体;表型观察发现,ΔFg PDE1菌落的外型及菌落生长速度与野生型没有明显差异。孢子侵染西红柿果实实验证明:以西红柿为侵染宿主,相对于野生型,突变体致病性没有明显减弱;但突变体分生孢子产量显著下降。结论:Fg PDE1基因可能与禾谷镰刀菌分生孢子的形成有关。     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

PEG介导的外源基因对绿豆叶肉原生质体的转化和瞬间表达研究(简报)

作者曾利用Krens等(1982)的PEG法将NPT-Ⅱ丛因直接导入绿豆原生质体并获得稳定表达,但该法的转化率极低。为了建立绿豆原生质体有效的转化系统,我们将PEG法在大豆原生质体转化的基础上进一步改进,利用GUS基因为报告基因,对三个不同品种的绿豆叶肉原生质体进行直接转化。并对影响PEG转化的有关因素进行了研究。     

糖多孢红霉菌A226 的原生质体转化和染色体同源整合

糖多孢红霉菌的原生质体转化和染色体同源整合,是红霉素生物合成基因改造的重要途径。本研究对糖多孢红霉菌A226原生质体制备和转化条件进行了优化,结果表明以对数生长后期和稳定期菌丝体制备的原生质体转化效率较高;质粒、原生质体和PEG－T缓冲液体积比例为15：40：200（μl)时转化效果较好;比重小原生本的转化效率虽高,但在转化子中有效整合的比例较低;PEG1000和PEG3350对转化效率没有显差异;而Yamamoto转化系统优于Weber转化系统。PCR鉴定、抑菌活性鉴定和质谱分析均表明,转化质粒已整合到染色体红霉素合成基因位点。     

香菇B交配型位点的分子遗传学结构研究II.一个香菇单核体的B位点结构的分子解析

在先前的工作中,曾经运用简并PCR和染色体步行的方法从香菇中获得了1个信息素受体编码基因和1个信息素前体编码基因。根据香菇135菌株的原生质体单核体的全基因组测序信息,设计了4对引物,用于扩增香菇苏香菌株的原生质体单核体SUP2中的信息素受体编码基因STE-3的同源物及其侧翼保守基因。实验结果共获得了33,655bp的DNA序列,运用BlastX搜索对所获得的序列进行同源性分析后,发现了7个推定基因,其中有3个为信息素受体编码基因。再根据信息素前体所具有的保守基序特征,在2个信息素受体编码基因附近发现了4个信息素前体编码基因。首次对香菇的B交配型位点的分子遗传学结构有了比较全面的了解。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

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     

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.