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。     

Isolation ofosRACD gene encoding a small GTP-binding protein from rice

Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N pan icle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA of osRACD gene that encodes a rice small GTP-binding protein. Asco mpared with maize RACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates that osRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice.     

差异展示法鉴定GA3诱导的水稻差异表达的mRNA

赤霉素是植物生长发育过程中一类重要的调节激素，在水稻不育系上喷施合适浓度的ＧＡ３（赤霉素的一种）可以克服其包颈现象。运用反转录和聚合酶链式反应建立了一套旨在分离差异表达ｃＤＮＡ的差异展示方法。     

一种优化的mRNA差异显示技术分析白刺盐碱胁迫表达相关基因研究

mRNA差别显示PCR技术是分离、检测差异基因表达的有力工具 我们研究了存在的可能影响DDRT-PCR研究结果的因素 通过改变DDRT-PCR方法中的引物、PCR反应中的退火温度等条件,得到了一种优化的DDRT-PCR技术,运用该项技术研究了白刺盐碱胁迫相关基因的差异表达 结果发现,运用这种方法得到的差异表达基因片段假阳性率较低,且差别条带较长,多为800bp以上,克服了DDRT-PCR技术中的主要问题,为更广泛地运用该项技术奠定了基础     

Molecular cloning of rice cDNAs related to pollen development using the subtraction hybridization technique

The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensitive mutant, Annong S-1 were used as the plant material. The mRNA has been extracted from the young panicle at the meiotic stage. By using the cDNA subtraction hybridization technique, three cDNA fragments, RP-1, RP-2 and RP-3 have been successfully cloned from Annong N. Northern blot analysis reveals that the mRNA of these three clones are expressed only in anthers, and not leaves. The mRNA levels of these clones are lower in anthers of Annong S-1 than in Annong N. Furthermore, the amount of mRNA extracted from anthers of Annong S-1 growing under high temperature (28℃) is lower than plants growing at lower temperature (25℃). Sequence analysis and homology search indicate that these three clones display no similarity to the current database. It is concluded that the three novel cDNA cloned are related to pollen development in rice.     

筛选差异表达基因的方法进展

植入前胚胎差异表达基因的筛选是分离、鉴定发育相关基因的关键 ,是克隆动物发育分子机理研究的基础 .哺乳动物植入前胚胎和克隆胚胎材料的获得十分不易 ,使得筛选差异表达基因的方法在该领域的应用均受到较大的限制 .以DDRT -PCR为基础的单胚mRNA差异表达技术的建立 ,为克隆植入前胚胎发育相关基因及哺乳动物克隆胚胎发育分子机理研究提供有力的工具 ,本文将在多年研究基础上对该技术及目前筛选差异表达基因的方法进行综述 .     

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.     

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.     

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.     

Differential display of carrot radicle genes in different developmental states

mRNA differential display was used in analyzing the expression of carrot somatic embryo radicle gene in different developmental stages brought about by controlled cultivation. As shown by experiment, marked differences in electrophoresis patterns of DNA exist between the normally developing carrot radicle and those with their development inhibited. 12 series of specific bands have been cloned, and structural analysis of them is under way.     

克隆胃粘膜细胞癌变相关基因的思路

采用ｍＲＮＡ和ＡＰ－ＰＣＲ等技术克隆胃粘膜细胞癌变相关基因在技术上是可行的。胃粘膜细胞癌变相关基因的获得，在阐明胃癌发生机制及胃癌的早期诊断和临床治疗方面，具有重要意义和广阔的应用前景。     

用cDNA-AFLP银染技术研究与花椰菜花色相关的基因

将smart cDNA PCR和AFLP银染技术结合，建立了一种新的mRNA差别显示技术，用这种方法对花椰菜黄花和白花的两个近等基因系进行了研究，找到一条差异带，经过Northern点杂交检测，发现此带为白花株系特有的谱带。     

棉铃虫滞育解除的相关基因鉴定

运用差异显示PCR(DD-PCR)方法分析棉铃虫滞育解除蛹差异表达的基因,结果得到了56个差异片段.通过RT-PCR和Real-time PCR的方法,鉴定了滞育解除过程中有3个基因表达量在下调,有4个基因表达量在上调.这7个差异表达基因中有3个和已知的基因有较高的同源性:FKBP12,esr16和NADH dehydrogenase 1 alpha subcomplex subunit 6.这些差异基因为今后研究滞育解除的分子机制提供了新的线索.     

Application of FLUPD-DD-PCR to the study of mRNA expression of glioma cells cultured under the condition of serum starvation

Fluorescently labeled universal primer directed differential display polymerase chain reaction technique (FLUPD-DD-PCR), an improved DD-PCR, is reported for the study of mRNA expression of glioma cells cultured with the serum starvation. The fluorescently labeled universal primer (FLUP) facilitates analysis of PCR products on an auto-sequencer. Compared with the traditional DD-PCR, FLUPD-DD-PCR has advantages of separation of target bands and measurement of mRNA expression quantitatively by adopting Cy5-labeled universal primer. And then the silver staining method has been used to recover the bands for use as template in re-pcr. In this study, 4 differential ESTs, of which 3 are novel ESTs, have been obtained. This work indicates some special novel genes, which can be induced to express under serum absent condition, are involved in coping with serum starvation stress on glioma cells growth.     

Application of FLUPD-DD-PCR to the study of mRNA expression of glioma cells cultured under the condition of serum starvation

Fluorescently labeled universal primer directed differential display polymerase chain reaction technique (FLUPD-DD-PCR), an improved DD-PCR, is reported for the study of mRNA expression of glioma cells cultured with the serum starvation. The fluorescently labeled universal primer (FLUP) facilitates analysis of PCR products on an auto-sequencer. Compared with the traditional DD-PCR, FLUPD-DD-PCR has advantages of separation of target bands and measurement of mRNA expression quantitatively by adopting Cy5-labeled universal primer. And then the silver staining method has been used to recover the bands for use as template in re-pcr. In this study, 4 differential ESTs, of which 3 are novel ESTs, have been obtained. This work indicates some special novel genes, which can be induced to express under serum absent condition, are involved in coping with serum starvation stress on glioma cells growth.     

In situ localization of proteinase inhibitor mRNA in rice plant challenged by brown planthopper

Proteinase inhibitor (PI) mRNA was localized by in situ hybridization in tissue sections of root, stem and leaf of the resistant rice (B5) plant fed by brown planthopper nymphs. In the rice material without BPH feeding, PI gene was expressed in the root, stem and leaf, while the abundance of PI mRNA was low. In the rice material fed by BPH,PI gene was expressed substantially in the parenchyma of rice stem and leaf, but weakly in the root. The results indicated that the PI gene was up-regulated in the rice plant challenged by brown planthopper. For the first time, we reported the expression changes of proteinase inhibitor gene in plant which was infested by a piercing/sucking insect.     

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.     

水稻生产受气象因素影响的动态评估方法——回归积分法

采用多要素回归积分的差分式方法可实现水稻生产受气象因素影响的动态评估。以金堂县水稻生产和气象因素资料为例,建立了差分式回归积分法,讨论了评价方法的可行性,介绍了评估方法的操作步骤及应用范围。