AS-PCR在牛羊异种核移植上的应用

利用PCR反应延伸过程中,3′端碱基不配对,PCR反应不能启动的等位基因特异性PCR分析法(AS-PCR)原理,采用生物软件DNASTAR分析牛、羊线粒体细胞色素b(Cytb)基因,以牛、羊碱基差异较大位点作为上游引物的3′端来设计引物,进行PCR扩增反应,分析牛羊异种克隆胚中线粒体的变化情况。结果表明,AS-PCR法是一种快速、有效鉴定异种重构胚中线粒体异质性的方法。     

马铃薯晚疫病菌对甲霜灵抗性的快速检测方法

为快速检测马铃薯晚疫病菌——致病疫霉Phytophthora infestans对甲霜灵的抗性,基于已知致病疫霉RPA190基因的T1145A变异引起的F382Y氨基酸点突变与甲霜灵抗性有关,通过设计4对特异性引物F382Y-F1/F382Y-R、F382Y-F2/F382Y-R、F382Y-F3/F382Y-R和F382Y-F4/F382Y-R建立等位基因特异性PCR(allele specific-polymerase chain reaction,AS-PCR)快速分子检测法,其中F382Y-R引物在4对引物里保持不变,并分析所建分子检测法的特异性和灵敏度。结果表明,正向特异性引物F382Y-F2、F382Y-F3和F382Y-F4的3''末端在致病疫霉抗甲霜灵菌株原有核苷酸突变位点1145A(对应引物为F382Y-F1)的基础上,再人工引入第1144位的核苷酸突变,将1144T分别突变成1144G、1144C和1144A,并优化退火温度和特异性引物与内参引物ITS1/ITS4比例,最终确定最适退火温度分别为54、60和58℃,特异性引物与内参引物最适浓度比例均为5∶1。以该3条引物对应的3对特异性引物和内参引物ITS1/ITS4组成的3组多重AS-PCR引物对甲霜灵敏感和抗性菌株具有良好的特异性,敏感菌株的扩增产物含1条879bp的内参片段,抗性菌株的扩增产物为1条879bp的内参片段和1条461bp的目的片段。3组AS-PCR检测体系均具有较高的灵敏度,其中引物F382Y-F4/F382Y-R对致病疫霉DNA的检测灵敏度达0.4pg/μL,引物F382Y-F2/F382Y-R和F382YF3/F382Y-R的检测灵敏度达4pg/μL。     

水稻氮高效、耐冷基因OsGRF4功能标记的开发及其利用

通过分子标记辅助选择(molecular marker-assisted selection,MAS)培育氮高效水稻品种是减少氮肥使用量、发展绿色农业和可持续农业的一个重要途经。水稻OsGRF4基因编码生长调节因子蛋白,编码区第487和488碱基由TC变异为AA,导致丝氨酸突变为赖氨酸,使得水稻具有氮高效利用、高产和耐冷的特性。为了提高OsGRF4基因在育种中的选择效率,本研究根据功能SNP(single nucleotide polymorphism)位点设计和筛选出等位基因特异PCR(polymerase chain reaction)功能标记PF+DMR+PR和PF+XMR+PR。利用此功能标记对不同品种(品系)和川大粒/巨穗稻的F2群体进行基因型检测,结合测序分析验证,能准确快速鉴定OsGRF4的纯合显性、纯合隐性和杂合基因型,且具有操作简单、成本低的特点。本研究开发的功能标记为通过MAS方法利用OsGRF4基因培育氮高效、高产和耐冷水稻新品种提供了技术支撑。     

小麦高代重组自交系中HMW-GS基因的分子鉴定

以JY970012群体内11个F8代重组自交系为材料，采用SDS—PAGE技术和AS—PCR技术对HMW—GS组成进行鉴定。结果表明：每个重组自交系在Glu-A1、Glu—B1、Glu—D1位点上HMW—GS组成稳定。F8代，AS—PCR技术在苗期鉴定表明：含有1亚基的株系具有约1500bp的特异带，而含有null亚基的株系没有此带；含有null亚基的株系在约920bp处出现特异带，而1亚基没有。SDS—PAGE和AS—PCR两种技术鉴定结果一致，同时，PCR技术简便、快速、准确、科学，可检测优质亚基基因，为小麦品质选育提供理论依据。     

The Establishment of AS-PCR Method to Distinguish Canine Distemper Asia-I Type Wild Strains and Vaccine Strains

Canine distemper (CD) is a contagious disease, which can damage the immune system, respiratory system, digestive system, and even nervous system, leading to systemic pathological changes, and has a huge threat to pet dogs, fur animals, etc. At present, the commonly used colloidal gold test cannot effectively distinguish vaccine immunity from animal natural infection. To establish an efficient and accurate detection method for identifying wild strains and vaccine strains of canine distemper virus(CDV), the whole genome of six canine distemper wild strains isolated from dogs and three widely used CDV vaccine strains collected from Wuhan area were sequenced. After comparing and analyzing the amino acid and the base sequences, the H gene was determined as the target gene for AS-PCR primer design. By genotyping the H gene, it was found that the prevalent CDVs in Wuhan were all Asia-Ⅰ, while the vaccine strain Y2 was America-I, and the vaccine strains Y1 and Y3 were both America-Ⅱ. Comparing the CDV-H gene sequences of 179 Asia-Ⅰtypes (6 wild-type strain samples + 173 GenBank Asia-Ⅰtype stains) and 3 vaccine strains, using AS-PCR technology (3'mismatch) to design a pair of primers. It can effectively distinguish CDV Asia-I wild strain and vaccine strain. The upstream primer sequence is 5'-TTAATATAATAATGACAGTG-3', and the downstream primer sequence is 5'-CCTCAAGGGGCACA-3'. The results showed that the primer had a strong specificity and the wild strains could amplify an 894 bp fragment, while the vaccine strains could not. There are 9 more regular bases (amino acids) variation sites in H gene of wild strain, and the 277th amino acids of all vaccine strains are Asparagine. These mutations may lead to an increase in N-glycosylation sites, which will have an impact on the virulence of canine distemper virus vaccine strains. The AS-PCR method established in this study can effectively distinguish the canine distemper vaccine and Asia-Ⅰwild strains.     

Marker assisted selection to improve HMW-glutenins in wheat

The present work presents the application of new markers based on thePCR technology to amplify the complete coding sequence of the specificalleles of the high molecular weight (HMW) glutenin genes. A set ofAS-PCR molecular markers of the Glu-A1 and Glu-D1 genes wasdesigned, making use of the minor differences between the sequences of thex1, xNull and x2 of Glu-A1, x2, x5, y10 and y12 of Glu-D1. These markers were applied to assist selection inbackcross progenies to improve the glutenin quality in Spanish wheat. Theselection was also assisted using other polymorphic systems (AFLPs) inrecovering the genetic background of the recurrent parent.     

簇毛麦HMW-GS基因的位点特异PCR(AS-PCR)标记及其多态性

簇毛麦含有丰富的HMW-GS基因变异.本研究旨在建立簇毛麦HMW-GS基因的位点特异PCR(AS-PCR)标记,并对18个居群簇毛麦HMW-GS基因遗传多样性进行系统评价,为开发和利用簇毛麦HMW-GS改良普通小麦品质奠定基础.根据先前克隆的3个簇毛麦HMW-GS基因序列(1Va、1Vb和1Vc)和已发表的普通小麦HM...     

中国原产果梅自交亲和变异品种花粉决定基因SFB的插入突变

 果梅是配子体自交不亲和树种,但在长期进化过程中也出现了自交亲和品种。以果梅典型的自交不亲和品种‘南高’和自交亲和品种‘甲州小梅’为对照,采用田间自交授粉试验对原产于中国的‘四川白梅’和‘长农17’两个果梅品种进行了自交亲和性鉴定,结果表明：其自花授粉坐果率分别为30.73%和16.27%,属于自交亲和品种。进一步通过AS-PCR分析发现花粉SFB基因存在一段插入序列,造成了分子水平的基因突变。推测该基因的突变使‘四川白梅’和‘长农17’SFB功能发生改变,从而自交亲和。     

甜樱桃自交不亲和基因型AS-PCR鉴定体系的建立与应用

采用改良CTAB法从甜樱桃幼嫩叶片中提取基因组DNA,通过对影响PCR反应的因素和反应程序进行研究,建立了优化的AS-PCR体系,并利用此体系扩增出6个自交不亲和基因(S基因)片段,通过对扩增结果分析,确定了这6个基因分别为S1、S3、S4、S5、S6和S9,并确定了部分甜樱桃品种S基因型。     

Molecular characterization of a novel HMW-GS 1Dx5′ associated with good bread making quality (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and the study of its unique inheritance

A novel 1Dx type high molecular weight glutenin subunit (HMW-GS) associated with good bread-making quality was identified in the bread wheat line W958. This glutenin subunit was designated as 1Dx5′ here for the same electrophoretic mobility as the traditional one 1Dx5 in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In this work, the 5′ flanking region, N-terminal as well as partial central repetitive domain of this allele was cloned and sequenced. Comparison of the amino acid sequence of 1Dx5′ with that of 1Dx5 and 1Dx2 showed the main difference being the substitution of one cysteine residue located in the central repetitive domain of 1Dx5 with serine residue in 1Dx5′ or an additional proline being inserted at the N-terminal of 1Dx5′ compared with 1Dx2. Allelic specific-polymerase chain reaction (AS-PCR) molecular markers discriminating the alleles of Glu-D1 locus were designed and applied to two F₂ segregation populations. Meanwhile, the dough properties of the F₂ population were measured and analyzed, showing that the 1Dx5′ subunit is as good as 1Dx5 and superior than 1Dx2 in terms of dough property. The result illustrated that the inter-chain hydrogen bonds formed between the subunit repetitive domains rather than the cysteine may play the main role in bread making quality. Notably, the novel 1Dx5′ have a great portion in the F₂ segregation population, suggesting the phenomenon of segregation distortion which could facilitate the introduction of 1Dx5’ into the wheat breeding program.