利用mRNA差异显示技术筛选绵羊毛性状相关基因

利用mRNA差异显示技术（DD-PCR）,比较绵羊肩、腹、腿三部位皮肤中mRNA的差异表达,共获得差异表达的cDNA片段16条,其中肩部、腹部与腿部之间的差异较大;对所有16条差异片段进行测序并在GENBANK中进行检索,未发现有同源序列.提示16条cDNA片段可能为未发现的基因片段,也可能存在假阳性片段.     

以mRNA差异显示法分离牙鲆白化相关基因的初步研究

以正常和白化牙鲆表皮组织总RNA为模板,用Oligo d(T)13M(M分别为A、C、G)3种锚定引物和26种差异显示随机引物组合进行差异显示,PAGE凝胶电泳后,用新型高灵敏度核酸荧光染料SYBR GREEN I显示差异DNA条带,得到区分度较好的差异表达图谱,共分离49条牙鲆白化相关DNA片段,片段长度260～800bp左右,经克隆测序后将可作为新的ESTs进行同源序列比较.     

用差异显示反转录PCR银染技术研究小鼠前胃癌相关基因

目的:以N-亚硝基-肌氨酸乙酯(NSEE)作为诱变剂建立NIH小鼠前胃癌动物模型,研究小鼠前胃癌差异表达基因,探索细胞癌变的分子机理.方法:采用mRNA差异显示(DDRT-PCR)、反向Northern点杂交、克隆、测序和生物信息学分析等方法,对其癌变组织中差异表达的基因进行系统的研究分析.结果:得到5条在小鼠前胃正常对照组织、癌变组织之间差异表达的cDNA片段.筛选后,对其中3个差异条带进行DNA序列测定,有1个与已知基因Tpt1高度同源,2个与同1基因片段AK085193.1高度同源.提示这2个基因与小鼠前胃癌的形成有关.     

用差异显示技术分离空心莲子草抗旱相关基因

采用mRNA差异显示技术,分离空心莲子草在干旱胁迫1 d后差异表达的基因,获得139个基因片段.经过Reverse Northern检测,初步证实有两个片段受干旱诱导表达上调.对其中一个片断克隆测序并进行核甘酸序列比对,显示与其他基因同源性都很低,表明可能是新的基因.半定量PCR技术证实该基因受干旱诱导表达上调.     

mRNA差异显示技术研究低温条件下深黄被孢霉基因的表达差异

mRNA差异显示技术已经广泛应用于研究植物、动物和微生物在各种环境条件下基因的差异表达研究.研究以15℃和30℃培养的深黄被孢霉M6-22菌体为材料,利用mRNA差异显示技术研究两种培养条件下基因的表达差异.经实时荧光定量PCR验证,共获得7条差异片段,相似性搜索结果表明它们是6-磷酸葡萄糖异构酶、单糖核苷酸转运蛋白、Ras1鸟苷酸转移因子、依赖于NAD的苹果酸脱氢酶、Δ12-脂肪酸脱氢酶、CLK4关联的丝氨酸/精氨酸丰富蛋白和假定蛋白,涉及糖酵解、蛋白质修饰、信号传导、脂肪酸合成和mRNA加工等生命过程,表明深黄被孢霉M6-22低温适应性是多种途径协同调控的结果.     

mRNA差异显示法筛选和克隆东亚飞蝗抗药性相关基因

筛选和克隆东亚飞蝗抗药性相关基因.采用mRNA差异显示法(mRNA diffFerential display PCR,DD-PCR),对比了东亚飞蝗在受到氯氰菊酯杀虫剂的诱导前后,基因表达发生的变化.对差异片段进行了回收,克隆、测序,Northern点杂交验证以及序列分析和同源性比较.东亚飞蝗在受到氯氰菊酯杀虫剂的诱导前后,存在明显的基因表达差异,发现差异表达条带9条,对9条差异表达条带进行克隆、测序,经序列分析和同源性比较和RNA点杂交验证,表明确认其中1条带与东亚飞蝗的抗药性有关.东亚飞蝗的mRNA差异显示证明,该EST序列可能在抗药的发生发展中具有一定的作用.     

mRNA差异显示PCR技术在水稻研究中的应用

mRNA差异显示PCR技术是在基因转录水平上研究基因差异表达和性状差异的有效方法之一,该方法在生物的发育、分化和对各种生物、理化因子作用时应答过程基因表达的研究中应用十分广泛。就mRNA差异显示PCR技术的基本原理、优点与不足、改进与完善等作了简要归纳,综述了该方法在水稻的发育分化、激素调控、抗逆性和抗病性等方面所取得的研究成果,并对该方法在水稻方面的应用前景作了简单分析。     

盐胁迫对小麦耐盐突变体苗期超氧化物歧化酶活性的影响

对小麦盐突变体H8706－34，H8706－44，RH－48，RH8706－49室内水培的苗期（2叶小心），进行了0．7％的NaCl盐胁迫试验，结果表明，非盐胁迫时耐盐性强的突变体的超氧化物歧化酶（SOD）的活性极显著高于耐盐性差的突变体；在盐胁迫时，不同的突变体的SOD活性发生不同的变化，耐盐性强的突变体的SOD活性变化幅度较大，变化周期较长，表现出较好的适应性和对超氧自由基的反应能力；而耐盐性差的突变体的SOD活性的变化幅度小，且变化周期短，表现出较差的适应性和对超氧自由基的较的反应能力。     

mRNA差异显示技术及其在植物基因工程中的应用

mRNA差别显示技术是将mRNA反转录技术与PCR技术二者相互结合发展起来的一种RNA指纹图谱技术,目前已广泛应用于分离鉴定组织特异性表达的基因.本文介绍了mRNA差别显示的基本原理、技术路线以及反应条件的改进方法,最后指出了此技术在植物基因工程中的应用.     

银染mRNA差异显示法的建立及其应用

以银染mRNA差异显示方法对原发及转移灶胃癌标本总RNA为研究对象进行银染差示分析和回收差异条带,从中获得系列差异表达片段。随机选取5个从原发性胃癌样品回收的表达条带,以取自体外培养胃癌细胞株的RNA进行点杂交验证,均被证实为真实带。对2个差异表达片段进行分析、测序和GenBank同源比较结果表明：MGD1片段与肿瘤转移相关基因MTA1完全同源,属胃癌转移相关基因;PGD2与胃癌转移抑制相关,并与细胞周期抑制蛋白p27／Kip1具99％的同源性。结果表明,银染差异显示法具有快速、直观、假阳性率低等优点,同时也为进一步研究胃癌转移的相关基因提供了新线索。     

Isolation of a vernalization-related cDNA clone (VRC) using mRNA differential display in winter wheat

Vernalization is an essential factor which affects the flowering development in cold-requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization, we examined mRNA populations in differently-treated plumules of winter wheat (Triticum aestivum L. cv Yanda 1817) using mRNA differential display. One vernalizationrelated cDNA clone (VRC), VRC54, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that VRC54 was a novel vernalization-related clone found in higher plant which not only might play an important role in the floral induction in vernalization-requiring plants but also was different from the cold-acclimatized genes.     

Novel gene expressed during early embryogenesis of zebrafish identified by mRNA differential display

Following the revelation of the molecular mechanism of morphogenesis in fruitfly, research on the molecular mechanism of morphogenesis in vertebrate becomes the focus of developmental biology. The isolation of genes controlling the embryogenesis of zebrafish, a vertebrate model animal, is considered as an initial step toward investigating this issue. There are several approaches that can be used to isolate developmental genes, each of which is suited to a particular situation. In this note, mRNA differential display was utilized to demonstrate the mRNA differences among zebrafish embryos at 4, 5 and 6 h post fertilization (28.5℃, corresponding to oblong, dome and shield stages, respectively, called blastula, gastrula and neurula in this note). One cDNA tag that was specific to embryos at neurula stage was cloned and sequenced. After sequence comparison in Genbank, we found that this cDNA tag represents a novel gene. The expression of this gene in the developing zebrafish embryos was examined by whole mount in situ hybridization. The hybridization results confirmed that this gene was specifically expressed in zebrafish neurula embryos.     

Cloning and sequencing of cDNA fragment of gene of wheat (Triticum aestivum. L) induced by dehydration

cDNA fragment of the gene (dehydration induced,di1) of wheat (Triticum aestivum. L) induced by 30% PEG-6000 (−1.13 MPa) treatment was isolated with mRNA differential display technique. Northern blot analysis showed that the expression ofdi1 gene improved at 10 h reached the highest at 48 h under 30% PEG-6000 treatment. cDNA fragment ofdi1 gene has been cloned and sequenced (211 bp). DNA sequence analysis shows that there is no homologue in GenBank todi1 cDNA.     

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display （DDRT-PCR） technique was adopted to find out the genes related to settlement metamorphosis development process of Ruditapes philippinarum larvae. In this study, we have obtained three hundred and forty-six amplification bands in total from pediveliger larvae, veliger larvae, eye spot larvae and post-larvae. Sixty-five out of three hundred and forty-six bands are distinctly differential display from band pattern, which can be put into four groups, standing for different expression characters. Sixteen differential display bands were cloned, sequenced and analyzed and nine different sequences are obtained in the study. Three sequences have higher similarity to the cDNAs deposited in database and three are very similar to the rDNA of other species, considered as the rDNA of Ruditapes philippi narum. The rest three sequences are found to be novel sequences after analyzed. Their accession numbers are AY916799, AY916798, and AY916797 respectively. We thought the novel sequences are possibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide some fundamental understandings that are helpful for the improvement of scallop seed raising industry.