DDRT-PCR

摘要： Yunjie is closely related to important vegetables an d oil seed crops,and can be considered a genetic resource for all Brassiceae crops.Self compatibility is an important variant traits to Yunjie.This study aimed to probe the expression organ of self-compatible gene and isolate cDNA fragments related to self-compatibility of Yunjie. Leaf,anther and stigma from self-incompatible (SI)and self-compatible (SC)lines of Yunjie were examined by DDRT-PCR.The results showed that the amplification pattern of leaves and anthers were the same in SI and SC lines.However,different bands were obtained in stigmas of SI and SC lines before and after flowering.SC gene of E. sativa was not showing constitutive expression but showed tissue-specific expression in stigma.Only two fragments were amplified in SC lines before flowering,the one was less than 100 bp,the other was 750 -1 000 bp.A 300 bp fragment was found unique to self-compatible line after flowering.These three cDNA fragments might be closely re-lated to the self-compatibility in Yunjie.In addition,two cDNA fragments were generated in SI line before flowering, one was 500 bp,the other was 750 bp.One band of 500 -600 bp was identified in SI line after flowering.These cDNA fragments could efficiently distinguish self-compatible and self-incompatible lines,and could also be useful for cloning self-compatible gene of Yunjie.%芸芥与芸薹属植物具有亲缘关系，也是芸薹属植物的重要育种资源。自交亲和性是芸芥的一种重要变异性状。为了探明芸芥自交亲和基因的表达器官，并分离与自交亲和性有关的 cDNA 片段，以芸芥的一对近等基因系，自交亲和系（SC）与自交不亲和系（SI）为试材，利用差异显示 RT-PCR 技术分别对开花前和开花后的叶片、花药和柱头进行鉴定。结果表明，不论开花前还是开花后，芸芥自交亲和系与自交不亲和系的叶片或花药间的扩增带型是一样的，但在近等基因系的柱头间得到了差异表达条带。这证明芸芥自交亲和基因不是组成型表达，而是组织特异性表达，柱头是其唯一的表达器官。在开花前的自交亲和系中共得到了2条 cDNA 片段，一条小于100 bp，另一条为750～1000 bp，在开花后的自交亲和系中得到了1条300 bp 的 cDNA 片段。据分析这3条 cDNA 片段可能与芸芥的自交亲和性密切相关。在开花前的自交不亲和系中共得到了2条 cDNA 片段，一条为500 bp，另一条为750 bp，同时在开花后的自交不亲和系中得到了1条500～600 bp 的 cDNA 片段。这些 cDNA 片段可以用来区分芸芥的自交亲和系与自交不亲和系，还可用于克隆自交亲和基因。

摘要： 为深入了解玉米抗灰斑病的分子机理,本试验以抗/感玉米灰斑病自交系78599-1、OH43HtN/掖478、K12为试验材料,利用DDRT-PCR方法筛选玉米6叶期时接种玉米灰斑病菌粗毒素0、5、7和9d时玉米叶片中差异表达的mRNA并通过反式Northern进行验证.结果发现在抗/感材料78599-1、OH43HtN/掖478、K12中分别分离到6、5、5和3条差异表达片段;对19条差异表达片段进行测序和blast分析,得到6个候选基因片段,它们与玉米中MTN3、CONSTANS-CO5家族的基因、脱落酸受体基因和酰基辅酶A结合蛋白基因同源性分别为97％、99％、97％和100％;另外获得2个基因片段与未知功能基因同源性均为99％.研究结果将为深入研究抗灰斑病分子机理提供理论依据.

摘要： 为分离和筛选芸芥自交亲和相关基因,以芸芥自交亲和系(SC)和白交不亲和系(SI)开花前及开花后的花药和柱头为试材,利用mRNA差异显示技术筛选芸芥自交亲和相关差异表达基因,结合实时荧光定量PCR技术验证差异表达基因在SI和SC开花前后不同时期的表达水平差异.结果共筛选得到了11条差异片段.差异表达基因包含木葡聚糖半乳糖基转移酶、DnaJ伴侣蛋白、果胶酸裂解酶家族蛋白、40S核糖体蛋白s19-1和假设蛋白等.木葡聚糖半乳糖基转移酶基因在SC开花前柱头中表达量最大,其表达量是SI开花前柱头中的4.41倍;SC8假设蛋白基因在芸芥SC开花后花药中表达量最大,是SI开花后花药中的23.98倍.初步推测这些差异基因可能在芸芥SI和SC性状调控过程中发挥重要作用.

摘要： In the process of breeding research by ways of self-incompatible lines in E . Sativa Mill .,there is often a phenomenon that the seed setting percentage from the cross of two parents is high in bud stage ,but the number of seeds is low in late stage owing to the natural segregation .By using the method of complete diallel crossing ,the purity of S al-leles in 10 self-incompatible lines of E . Sativa Mill .was studied ,and the expression tissue of self-incompatible genes was analyzed with DDRT -PCR technique .The results showed that the self-compatible index of Yunjie 1 ,Yunjie 3 , Yunjie 5 ,Yunjie 6 ,Yunjie 8 ,Yunjie 9 and Yunjie 13 was below 1 after selfing or outcrossing among these 7 self-incom-patible lines ,that is to say ,their self-incompatibility was stable .However ,the self-incompatibility of Yunjie 2 ,Yunjie 4 and Yunjie 10 was not stable .DDRT-PCR analysis showed that the self-incompatible genes of E .Sativa Mill .didn’ t belong to constitutive expression ,but to specific tissue expression .Therefore ,when creating F1 hybrids by ways of self-incompatible lines ,it is necessary for us to determinate the purity of S alleles and their genotypes besides agronomic traits and self-incompatible stability .%在利用芸芥自交不亲和系途径开展育种研究的过程中，为了避免在人工试配组合时，双亲蕾期杂交结籽率较好，但在自然隔离条件下配制杂交种时出现双亲花期交配不亲和的现象，本试验对10份芸芥进行了系内株间和系间的完全双列杂交，同时对自交不亲和基因的表达器官进行了DDRT-PCR扩增研究。结果表明，芸芥1、芸芥3、芸芥5、芸芥6、芸芥8、芸芥9和芸芥13这7份材料系内株间异交和套袋自交的亲和指数均小于1，即表现为不亲和性，说明这些材料属于S等位基因纯合的不亲和系；而芸芥2、芸芥4和芸芥10这三份材料系内株间异交和套袋自交时，呈现出不亲和性不稳定的现象，即一部分杂交表现为亲和，另一部分杂交表现为不亲和，说明这3份材料的自交不亲和性尚未稳定。DDRT-PCR扩增结果表明，芸芥自交不亲和S基因只在柱头组织中表达，其属于组织特异性表达。因此，在利用自交不亲和系途径培育一代杂种时，在农艺学性状和自交不亲和性基本稳定时，有必要进一步测定自交不亲和系S等位基因的纯合性和基因型，这一工作可以有效地指导育种实践。

摘要： [目的]利用mRNA差异显示(mRNA differential display PCR,DDRT-PCR)技术,寻找、筛选并确定与库尔勒香梨萼片脱落和宿存相关基因的差异表达时期以及相关的差异基因.[方法]以新疆库尔勒香梨盛花前期、盛花期及其盛花后期三个时期同一花序的第2和第4朵花为试材,通过DDRT-PCR技术分离和克隆库尔勒香梨萼片脱落和宿存相关基因片段.[结果]分离得到42条差异片段,其中双引物片段有7条,在GenBank中同源性比较发现有2条与控制开花以及激素调节有密切关系的SPL和MYB类转录因子有很高的同源性,分别是78％和86.83％.[结论]实验为获得香梨萼片脱落与宿存差异表达基因建立的体系.为相关基因全长的克隆奠定基础,并为进一步验证其功能提供依据.%[ Objective ] The expression of differential gene was studied in calyx falling - off Korla Fragrant Pears and their persistent related genes through the method of DDRT - PCR in order to seek, screen and define the genes and blooming. [Method] Korla Fragrant Pear flower (the second flower and the fourth flower) of the same inflorescence of three anthesis stages (early full bloom, full bloom, the end of bloom) in the same tree were taken as material. Improved DDRT - PCR method was used to investigate the differential display of genes relate to calyx leaving and persistent related genes of Korla Fragrant Pears. [Result] The result showed that the differential display exist in early full bloom stage, 42 different expressed cDNA fragments were obtained and only 7 double - primer bands were amplified. The alignments of nucleotide and amino acid sequences indicated that there were 2 fragments which have high homology that might be closely related to SPL and MYB, the two translation factors important to regulation on plant phytohormone and flowering. The homology was 78% and 86.83% , respectively. [Conclusion] This study had established the system of DDRT - PCR to obtain genes which relate to calyx of leaving and persistent. It laid on a foundation for cloning full length of relative gene,which also makes insurance to the further studying its physiological and biochemical characteristics and functions of thd gene expression product.

摘要： 概述了mRNA差异显示技术(DDRT-PCR)的基本原理.对近年来DDRT-PCR技术在中药作用机制、中药复方作用机制、中药植物生物学方面的研究现状做了综述.展望了该技术在中药研究方面的应用前景.

摘要： 采用DDRT-PCR技术,对低温(4°C)胁迫处理不同时间(0、8、12、24和48 h)后茶树[Camellia sinensis(Linn.)O.Ktze.]抗寒品种‘紫阳圆叶’(‘Ziyangyuanye’)叶片中差异表达的基因进行分离和测序,并采用半定量RT-PCR对差异表达基因的表达特性进行了比较.结果表明:有12个引物对扩增出有明显差异的cDNA片段,其中3个片段是与抗寒性相关的差异片段,分别被命名为Csgsf、Cscaf1和Cscaf2,碱基数分别为217、316和232 bp.比对结果显示:Csgsf与茶树品种‘安吉白茶’(‘Anjibaicha’)和‘龙井43’(‘Longjing 43’)的谷氨酰胺合成酶基因的同源性分别为96％和91％,与菜豆(Phaseolus vulgaris Linn.)、蒺藜苜蓿(Medicago truncatula Gaertn.)、油棕(Elaeis guineensis Jacq.)、西洋参(Panax quinquefolius Linn.)和水稻(Oryza sativa Linn.)等植物的谷氨酰胺合成酶的基因序列同源性均达到90％以上,因此,Csgsf应为茶树谷氨酰胺合成酶基因片段;Cscaf2与干旱胁迫条件下茶树表达的cDNA的同源性为100％,为茶树应答干旱和低温胁迫的基因片段;Cscaf1未检索出同源序列,推测其为与冷胁迫相关的未知基因片段.半定量RT-PCR分析结果表明:Csgsf和Cscaf1在低温胁迫的初始期即开始表达且表达量随低温胁迫时间延长逐渐下调;而Cscaf2的表达量随低温胁迫时间延长逐渐上调并在胁迫48 h后达到最大,3个片段的表达特性均与差异扩增结果相符.

摘要： [Objective] This study aimed to investigate the variation rule of total RNA concentration during formation of microtubers of Pinellia ternala induced by sucrose. [Method] Plantlets of P. ternata were used as experimental materials. Total RNA concentration was determined with spectrophotometer,and the variation of total RNA was analyzed before and after formation of P. ternata microtubers treated with different concentrations of sucrose using DDRT-PCR technology. [Result] Total RNA concentration of P. ternata treated with different concentrations of sucrose varied greatly,which showed an upward trend in the first 15 d,declined from 15th d to 25th d,and slightly raised from 25th d to 30th d. Microtubers formed under various sucrose treatments showed an increase in RNA concentration after the induction, and it was assumed that the treatment of high concentrations of sucrose might have activated the expression of development-related genes in P. ternata microtubers. [Conclusion] This study provides the basis for revealing the related gene expression information and molecular mechanism during formation of P. ternata microtubers induced by sucrose.%[目的]探讨蔗糖诱导半夏试管块茎形成过程中RNA的变化规律.[方法]以半夏试管苗为材料,采用5种蔗糖浓度处理,通过分光光度计测定总RNA含量,并结合差异反转录PCR技术,分析各浓度蔗糖处理半夏试管小块茎前后其总RNA的变化规律.[结果]各蔗糖浓度处理的半夏总RNA含量变化幅度较大,但总趋势表现为前15 d处于上升状态,从15 d到25 d开始下降,从25 d到30 d又开始小幅度上升.试管块茎的形成伴随着总RNA含量的上升,高浓度蔗糖处理可能启动了半夏试管块茎发育相关基因的表达.[结论]该研究结果为揭示蔗糖调控半夏试管块茎形成过程中相关基因的表达信息和分子机理提供了依据.

摘要： 选取病拟南芥转基因NahG(抗病)、生态型WS-2(抗病)和Shakh-dara(感病)为材料,利用mRNA差异显示技术(DDRT-PCR)对抗、感病材料接种葱链格孢后抗性相关基因的差异表达情况进行了分析,共获得135个与拟南芥抗葱链格孢侵染相关的差异表达片段.反式Northern杂交结果表明,38个差异表达片段为阳性片段,阳性率为28.1％.同源性分析结果表明,拟南芥抗感生态型感染葱链格孢后,诱导了呼吸过程、参与细胞壁构建和蛋白质水解等相关基因的表达.其中,接种3h诱导表达的WS-2-47序列编码含有SH3保守域的Sla1蛋白,表明拟南芥受葱链格孢侵染后可能是通过寄主细胞壁的改变来提高寄主的抗病性.

摘要： 利用DDRT-PCR技术初步分析了小麦条锈病抗性品种‘89-27'和耐性品种‘咸农四号’在感染后叶片中的基因差异表达,共筛选到差异显示片段47个,包括诱导表达8个、增强表达21个和抑制表达18个.其中,7个稳定表现的差异片段DD1～DD7测序和序列相似性比对结果表明:DD1与far1基因相似性较高,可能与Phy A蛋白调控的特异性生理活性有关；DD2与rRNA基因完全同源,可能与感染后应激蛋白大量快速合成有关；DD7与光合系统Ⅱ中的叶绿素结合蛋白a/b基因相似性较高,可能与光捕捉效率和CO2固定能力的补偿作用有关；DD3～DD6功能不详,可能为新EST序列.%The gene expressions of the leaves infected with P. striiformis were compared by the DDRT-PCR technology between the cultivar ' 89-27' resistant to stripe rust and the cultivar ' Xian-nong 4' tolerant to stripe rust,and it was found that there were 47 differential display fragments,including 8 induced expressions, 21 enhanced expressions and 18 inhibited expressions. The sequencing and sequence alignment of the 7 stable-expression fragments DD1 - DD7 showed that the DD1 was quite similar to the gene far1 and might be related to some specific physiological activities regulated by Phy A;The DD2 and the rRNA gene were completely homologous and might be related to large and fast synthesis of stress protein after infection;The DD7 was very similar to chlorophyll a/b-binding protein of Photosystem II and might be related to the compensation action of light-receiving efficiency and CO2 fixation capacity;The DD3 - DD6 were not clear in functions and might be new EST sequences.