抗逆相关基因GmAREB转基因小麦的获得与鉴定

从大豆中克隆一个抗逆相关的bZIP类转录因子基因GmAREB,功能分析表明:GmAREB基因的过表达可以显著提高转基因拟南芥和烟草的抗旱、耐盐和耐寒性。为了获得抗逆转基因小麦,本研究利用玉米的Ubiqutin启动子控制GmAREB基因表达,构建了用于小麦转化的载体pSK-GmAREB。采用基因枪共转化法转化小麦品种郑147和济麦22。通过PCR检测共获得T0代的阳性植株70株,转化率为1.37%。其中,郑147阳性植株共31株,转化率为2.14%;济麦22阳性植株39株,转化率为1.08%。目前,已经获得T1代转基因株系18个,其中以郑147为受体的株系4个,以济麦22为受体的株系14个。对部分株系进行Southern blotting分析,进一步证实GmAREB基因已经整合到小麦基因组中。在低温胁迫条件下,3个以济麦22为受体的转基因株系体内脯氨酸的积累与受体小麦相比有显著增加,初步证明在小麦中过表达GmAREB基因,可以促进渗透调节物质脯氨酸的积累,可能有助于转基因小麦抗逆性的提高。本研究为进一步筛选抗逆转基因小麦新材料奠定了基础。     

小麦多子房性状外显率影响因子的初步研究

为了提高杂交小麦的繁制种效益,选用具有不同山羊草细胞质的同质同核、同质异核、异质同核多子房小麦为试验材料,于成熟期调查多子房外显率,并用DPS7．05专业数据处理软件对调查结果进行方差分析,研究了多子房外显率表达的影响因素。结果表明,具有相同细胞核和细胞质背景的多子房小麦,各小花内第一、二、三粒子粒多子房外显率表达效应无显著差异;异质同核背景下的多子房外显率的表达效应也无显著差异;相同细胞质不同细胞核背景下的多子房外显率的表达效应达到显著水平。因此,欲提高小麦多子房性状表达的外显率,选择合适核型至关重要。     

λDNA导入引起小麦染色体变异的研究

小麦种子从萌动至２叶期,用λＤＮＡ进行浸滴处理,Ｄ１代出现花粉母细胞染色体变异的植株占观察株的７３．３％,染色体变异的细胞占观察细胞数的２８．３％。主要有染色体落后、单价体、染色体桥、多价体、染色体多极分离,染色体螺旋化不同步,异常二分体和卤分体及微核等类型,随世代增进,染色体行为趋于稳定,但不同个体间存在的较大差异。这种复杂的染色体变异可能是外源ＤＮＡ在受体整合过程中的细胞学反应。     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

小麦与羊草体细胞杂种的细胞核和细胞质基因组分析

通过原生质体融合与培养获得小麦与羊草的体细胞杂种,其核基因组成以羊草(供体)为主,用线粒体特异探针atp6与叶绿体特异探针rbcL进行的RFLP分析结果表明,胞质基因组成偏向羊草并发生了重组.讨论了受体核基因组消减对杂种再生及对受体胞质基因组消减的影响.     

小麦新型细胞质雄性不育类型的研究

从普通小麦中国春细胞核置换到小麦属和山羊草属不同细胞质的同核异质稳定系中,筛选出１０个全不育的细胞质雄性不育类型,对它们一些主要特性的研究结果表明：（１）各不育类型在小孢子败育埋藏和特点上无显著差异;但雄蕊心皮化频率因细胞质不同差异显著;（２）各不育类型的生长特性、抗病性、光合特性主要农艺性状等因细胞质不同具有不同的表现;（３）在供试的各不育类型中,拟斯卑尔脱山羊草、阿拉拉特小麦,野生二粒小麦和菇     

类固醇激素受体复合物的装配与核内转运

类固醇激素受体 (ＳＲ)包括糖皮质激素受体 (ＧＲ)、孕激素受体 (ＰＲ)、雌激素受体(ＥＲ)、雄激素受体 (ＡＲ)等 ,其中以前两者的研究较多。ＳＲ主要存在于类固醇激素的靶细胞胞质和胞核中 ,当细胞外液中类固醇激素通过细胞膜进入胞质后 ,它能与胞质中ＳＲ结合 ,通过胞质和胞核中ＳＲ的穿梭 ,从而调节核基因组相关产物的转录、翻译及分泌一些生物活性物质 ,以发挥类固醇激素的作用。ＳＲ在细胞内有游离形式和复合物形式 ,而且存在几种不同的复合物形式 ,它们是怎样形成以及形成后如何转运到核内的 ?本文将对此作一综述。1 .ＳＲ复合物的组…     

水分胁迫下不同进化型小麦抗氧化能力比较

以6种不同基因型小麦为试验材料,研究了水分胁迫下不同生长期小麦体内超氧化物歧化酶（SOD）活性以及超氧自由基（O2）含量变化,并分析了两者之间的相关关系。结果表明,水分胁迫下6种基因型小麦SOD活性及超氧自由基含量在拔节期和灌浆期均有不同程度的增加;栽培型品种SOD活性增幅高于野生型品种,超氧自由基增幅较低;同样,二粒小麦与一粒小麦相比,二粒小麦SOD活性增幅高于一粒小麦,超氧自由基增幅较低;但现代栽培小麦种表现不明显。结果说明,栽培型与野生型小麦相比,二粒小麦与一粒小麦相比,具有较强的抗氧化能力。     

PELP1/MNAR:一种新的核受体辅助调节因子

脯氨酸-谷氨酸-亮氨酸富集蛋白1/雌激素受体非基因组活性辅助调节因子(proline-,glutamic acid-,leucine-rich protein 1/modulator of nongenomic activity of estrogen receptor,PELP1/MNAR)是一种新近发现的核受体辅助活化因子,具有较为复杂的分子结构,在多种组织中广泛表达。与先前发现的核受体辅助调节因子不同的是:作为一种支架蛋白,PELP1/MNAR既参与核受体调控靶基因转录的基因组作用,又参与了核受体激活激酶信号系统的非基因组作用,并且可能在核受体信号与生长因子信号串话(cross talk)中发挥重要作用。近年的研究表明,PELP1/MNAR在乳腺癌、卵巢癌、子宫内膜癌、前列腺癌等激素依赖性肿瘤中均有异常表达和分布,在激素依赖性肿瘤的发生、发展、转移、耐药形成过程中可能具有重要意义,可望成为内分泌依赖性肿瘤治疗的一个新的靶点。     

小麦密植

一.为什么要实行小麦密植在一定的耕地面积上播种一定数量的种子,使每个植株都有足够的营养面积,充分利用地力和日光能,是获得单位面积上高额产量的重要农业技术措施之一。“密植”二字具有相对的意义,所谓合理密植,实际上是在一定的自然条件和农业技术水平下的播种方法和播种量的问题,决不能误解为愈密愈好。目前苏联播种小麦的行距多为15厘米(4.5寸),每公顷的播种量由400万粒到530万粒不等(合我国每亩26万7千粒到35万4千粒),很多先进工作者在创造高额丰产中,每公顷播种量有达到700万粒或700万粒以上者.近年来又采用了窄行条播行距缩小到7.5厘米(2.25     

Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton (Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-delta-cadinene synthase cDNA. Southern analysis of leaf DNA digested with HindIII, Pst or KpnI restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation-regeneration process.     

导入外源DNA对小麦基因表达的影响

用（ＳＤＳ）ＰＡＧＥ对外源豌豆ＤＮＡ导入小麦体的不良变异后代种子蛋白质和酯酶同工酶（ＥＳＴ）、超氧化物歧化酶（ＳＯＤ）、过氧化同工酶（ＰＯＤ）进行分析,发现变异小麦的种子蛋白质消减４个组分带,ＥＳＴ和ＰＯＤ消减２条酶谱带,ＳＯＤ的活性明显降低,并且变异小麦植株的发育生长表型呈现出脆弱,早衰迹象,表明导入外源ＤＮＡ抑制了某些基因的表达,同时分析导致这种负作用的原因。     

Expression of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> gene for Δ6 desaturase results in the accumulation of high levels of γ-linolenic acid in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ6,9,12) is a nutritionally important fatty acid (FA) playing a vital role in biological structures and cellular functions, which is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a FA that could be converted into GLA by the enzyme Δ6 desaturase, if it is present. As a first step to produce GLA in oil seed crops, we isolated a cDNA encoding the Δ6-FA desaturase from filamentous fungus Mucor circinelloides M29. Expression of this gene in transgenic tobacco resulted in the accumulation of GLA to the levels of 23.1% of the total FA. The results suggested that it is feasible to introduce the M. circinelloides Δ6 desaturase gene into conventional oil crop to produce a large amount of GLA for functional foods and pharmaceutical products. This text was submitted by the authors in English. Y.L. Hao, X.H. Mei, and Y.B. Luo contributed equally to this work.     

Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome.

The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane filter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pAD1 to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI- and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicin-resistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids.     

外源DNA导入小麦引起遗传变异的验证

对由波兰小麦ＤＮＡ注射到普通小麦鄂恩１号子房获得的Ｄ５代稳定遗传变异２个转化株系的种子醇溶蛋白,进行单向和双向聚丙烯酰胺凝胶电泳分析,结果表明由外源ＤＮＡ导入获得转化株系的变异,与其种子醇溶蛋白电泳图谱出现供体的某些组分和缺少受体的某些组分相印证。     

精子介导转基因动物的制备

采用直接注射的方法,将脂质体包裹的含人乳铁蛋白基因的重组质粒pLNCXHLF注入兔睾丸组织和羊输精管中,一个月后分别与正常雌兔及正常的母羊交配。所产仔兔均为活体,经PCR和Southern检测,转基因仔兔阳性率平均为35%(11/31);羔羊经引物F1/R1系统PCR检测阳性率为33.3％（4/12）,F2/R2系统PCR检测阳性率为25%（3/12）。将流产羔羊组织解剖后提取舌、肺、肝脏、肌肉、皮肤、脑、生殖腺、脾脏、小肠、心脏、肾脏共11种组织的总DNA进行PCR检测,发现：F1/R1和F2/R2系统PCR检测出阳性信号存在于不同组织;F2/R2 PCR系统检测阳性信号较F1/R1 PCR系统少;而且各种组织的外源DNA存在的拷贝数不等,造成阳性信号的强弱程度不同。结果表明：1）脂质体包裹外源基因转染精子的方法,可将外源基因导入受精卵,并得到了较高的转基因阳性率;2）精子携带外源DNA的整合过程是随机的,在受精过程和胚胎早期分化过程中可能发生了片段丢失、不完全整合或游离于基因组存在而产生嵌合体。本文的研究结果证明了该方法是一种简捷有效的新途径,为进一步深入探讨精子介导转基因动物制备可行性奠定了基础,给精子载体法制备转基因哺乳动物研究提供了有价值的参考。 Abstract:The recombinant plasmids pLNCXHLF fused with human lactoferritin gene were directly injected into male rabbit's testis and male goat's spermaductus.The transfected males were fertilized with females one month later.Using F1/R1 PCR system and southern blotting,the transgenic positive rate of rabbit offsprings genomic DNA was 35%(11/31).From the PCR results of F1/R1 and F2/R2 system,the transgenic positive rate of genomic DNA of goat offsprings were 33.3% (4/12)and 25%(3/12) respectively.We prepared genomic DNA from 11 kinds of tissues of goat offsprings,which were tongue,lung,liver,muscle,skin,brain,gonad,spleen,intestines,heart,and kidney.The result of F1/R1 PCR system indicated that the abilities to uptake exogenous DNA were various in different tissues;the positive signals of F2/R2 PCR system were feebler than the ones of F1/R1 PCR system,and the density of positive signals attributed to the amount of copies of exogenous DNA in the tissues.In this experiment,spermatozoa-mediated gene transfer can produce the transgenic animals after exogenous DNA being entrapped by liposome.But during the course of fertilization and the early process of embryo proliferation,the exogenous DNA had lost segments,partly integrated,or existed outside of genomic DNA.So the rate of chimera was relatively high.According to this result,this method is not only a simple and effective way to produce transgenic animals but also make references to other researchers to prepare transgenic mammals by this means.     

Insertional inactivation of streptolysin S expression in Streptococcus pyogenes

The inactivation of a genetic determinant critical for streptolysin S production was accomplished by transfer and insertion of the transposon Tn916 into the DNA of a group A streptococcal strain. The group D strain CG110 was able to efficiently transfer Tn916 into the group A strain CS91 when donor and recipient cells were concentrated and incubated together on membrane filters. Among tetracycline-resistant transconjugants, nonhemolytic mutants that no longer produced streptolysin S and retained the capacity to produce streptolysin O were discovered. Hemolytic revertants from these mutants regained tetracycline sensitivity; other revertants still retained a tetracycline resistance phenotype. Hybridization studies employing Tn916 DNA located Tn916 sequences in EcoRI and HindIII fragments of DNA from mutants devoid of streptolysin S; one carried a single copy of Tn916, and the other two carried multiple copies of the transposon.     

Isolation of Stable Hemolysin and Catalase Variants of Staphylococcus aureus S6C Includes One with an Exoprotein-Deficient Phenotype

Previous studies suggested that the exoprotein-deficient phenotype of a Δ1058::Tn551 insertion/deletion mutant of Staphylococcus aureus S6C was not owing to the insertion/deletion event, but instead was owing to the inherent instability of the agrC gene during transduction of the Δ1058::Tn551 region into S6C. The purpose of the following study was to examine S6C as a potential source of exoprotein-deficient mutants that would account for their appearance after transposition and transduction. Four stable variants of S6C were isolated that differed in their hemolysin and catalase activities. Surprisingly, the agr regulatory molecule, RNAIII, was undetectable in one of these variants, which most likely accounted for the exoprotein-deficient phenotype of this variant. When the original Δ1058::Tn551 mutation was transduced into the hemolytic, catalase-positive variant of S6C, none of the transductants exhibited an exoprotein-deficient phenotype. These data suggest that, while the exoprotein-deficient phenotype of the S6C variant is most likely due to mutations in the agr regulatory system, these mutations are not caused by the transduction of the Δ1058::Tn551 region into S6C, but instead already exist in an exoprotein-deficient variant of S6C. Received: 6 November 2000 / Accepted: 16 January 2001     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Introduction of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis

The conjugative Streptococcus faecalis transposon Tn916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tetr) were detected at a frequency of approximately 7.0 X 10(-7) per recipient cell during filter matings, whereas transfer of Tn916 was not observed in broth matings. The Tetr phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn916 was demonstrated when Tetr B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tetr isolates contained Tn916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.