梨自交不亲和基因cDNA芯片杂交条件优化及应用北大核心

[目的]优化梨自交不亲和基因(S-RNase或S基因)c DNA芯片杂交条件,利用芯片检测梨品种S基因型。[方法]提取梨品种雌蕊RNA,Cy3标记引物RT-PCR获得S基因荧光标记特异c DNA序列。设置不同杂交条件,用已知S基因型品种荧光标记的PCR产物在不同条件下分别与芯片杂交,杂交信号分析芯片杂交效果。用芯片优化杂交体系鉴定梨品种未知S基因型,DNA测序验证芯片鉴定结果。[结果]芯片杂交最佳条件:杂交温度42℃,杂交时间8~9 h,PCR纯化产物终浓度为200 ng·μl-1。优化杂交条件下芯片鉴定晚咸丰、秀水、丽江马占梨1、湘菊、木通梨、甘甜、弥渡小红梨、丽江大中古、金晶和弥渡火把等梨品种S基因型分别为:Pp S15Pp S52、Pp S4Pp S5、Pb S22Pp S37、Pp S1Pp S2、Pp S1Pp S3、Pp S13Pp S15、Pp S12Pb S42、Pb S21Pb S22、Pp S3Pp S60和Pp S5Pp S5。DNA测序验证各品种所含S基因与芯片鉴定结果一致。[结论]梨自交不亲和基因c DNA芯片优化杂交条件后可准确鉴定梨品种所含已鉴定的S基因资源。     

中国梨品种S基因型鉴定的初步研究

采用PCR技术和聚丙烯酰胺凝胶电泳法对6个中国梨品种的s基因型进行了鉴定研究,并与已知S基因型的日本梨品种进行了比较。研究结果表明,供试的6个中国梨品种S基因型均不相同,‘西子绿’、‘金花’和‘金水酥’各包含了S1～S7以外的新的S基因,为这些品种田间授粉品种的选配提供了参考。     

白梨和砂梨S基因型确定及S34新基因的分离

梨是配子体型自交不亲和植物, 确定不同品种的S基因型是科学杂交授粉及提高梨产量和品质的基础。本文根据砂梨S1-9等位基因一级结构特征, 设计特异引物PF和PR, 以白梨(Pyrus bretschneideri)品种鹅梨(Pyrus bretschneideri ‘El i’) 和砂梨(Pyrus pyrifolia)品种博多青(Pyrus pyri folia ‘Hakataao’) 的叶片基因组DNA为模板, 通过 PCR-RFLP系统检测、克隆测序以及生物信息学分析, 分离鉴定了它们的片段大小相似的2条S等位基因, 从中获得1条新的S基因, 命名为S34-RNase基因, 并确定了这2个梨品种的S基因型, 分别为鹅梨S13S34和博多青S22S34。     

白梨和砂梨S基因型确定及S34新基因的分离

梨是配子体型自交不亲和植物,确定不同品种的S基因型是科学杂交授粉及提高梨产量和品质的基础。本文根据砂梨S1-9等位基因一级结构特征,设计特异引物PF和PR,以白梨（Pyrus bretschneideri）种鹅梨（Pyrus bretschneideri‘Eli’）和砂梨（Pyrus pyrifolia）品种博多青（Pyrus pyrifolia‘Hakataao’）的叶片基因组DNA为模板,通过PCR·RFLP系统检测、克隆测序以及生物信息学分析,分离鉴定了它们的片段大小相似的2条S等位基因,从中获得1条新的S基因,命名为S34-RNase基因,并确定了这2个梨品种的S基因型,分别为鹅梨S13S34和博多青S22S34。     

甜樱桃(Prunus avium L.)品种S基因型鉴定

根据蔷薇科S-RNase基因(S基因)高度保守区C2和RC4区设计一对特异引物PruC2和PruC4R,对甜樱桃品种的基因组DNA进行S基因特异PCR扩增。克隆S基因的扩增片段,核酸序列在GenBank上搜索,确定了4种S基因的核酸序列和大小。结果表明,在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因。4种S基因扩增片段的大小分别是：S1为677bp,S3为762bp,S4为945bp,S6为456bp。参试的自交不亲和品种的S基因型分别是：红灯、红艳、早红宝石和先锋相同,为S1S3;抉择、红丰和那翁相同,为S3S4;大紫为S1S6;长把红为S1S4;养老为S2S6;自交亲和品种外引7号和斯太拉为S3S4。     

樱桃品种S基因型及自交不亲和性分子机制研究进展

系统介绍了樱桃S基因型鉴定的主要方法,全面列出上百个已知甜樱桃品种的S基因型,其中共涉及16个S基因;着重讨论了樱桃S基因座内S-RNase和SFB基因的研究概况、结构特点及位置关系,并提出该领域善待解决的问题。     

cDNA芯片阳性对照的制备及在芯片敏感性分析中的应用

cDNA芯片是一种高通量基因表达谱分析技术,在生理病理条件下细胞基因表达谱分析,新基因发现和功能研究等方面具有广阔应用前景。CDNA芯片阳性对照的选取以及CDNA芯片检测敏感性是芯片成功应用的关键问题之一。以在系统发育上与人类基因同源性小的荧火虫荧光素酶基因材料,制备了用于人类和其他动物基因表达谱CDNA芯片的通用型阳性对照探针和相应的mRNA参照物,经反转录对mRNA参照物进行Cy3荧光标记并与DNA芯片杂交后发现,mRNA参照物能特异性地与荧光酶基因cDNA片断杂交,而与人β-肌动蛋白基因,人G3PDH基因以及λDNA/HINDⅢ无杂交反应。把mRNA参照物以不同比例加入HepG2总RNA中,以反转录荧光标记后与CDNA芯片杂交,结果发现当总RNA中的MRNA含量为1/10^4稀释（即mRNA分子个数约为10^8个）时,CDNA芯片基本检测不出mRNA标记产物的杂交信号。而且,cDNA芯片检测的信号强度与芯片上固定的探针浓度密切相关,当探针浓度为2g/L时,杂交信号最强,随着探针浓度下降芯片的杂交信号趋于减弱。CDNA芯片通用型阳性参照物的制备以及应用于CDNA芯片检测敏感性研究为CDNA芯片应用于人和其他动物基因表达谱高通量分析和新基因功能研究提供了技术基础和理论依据。     

应用cDNA芯片分析79个新基因的人胚组织表达谱

大规模cDNA测序和生物信息学技术相结合,得到来自于商品化的人胚肾cDNA文库79个代表新基因的表达序列标签(EST).随后,采用高速度机械手制备这些cDNA的基因芯片,用于鉴定79个新基因的ESTs在20周、26周两个胚胎时期6种组织中的基因表达状况,以研究这些EST片段代表的新基因功能提供线索.通过芯片杂交及结果分析,得到同一个组织两个不同时相8个差异表达的基因,随后的RNA印迹分析的结果与芯片杂交的结果相一致.     

cDNA芯片差异表达基因检测的非转换方法

cDNA微阵列数据中包含许多变异因素,用于检测差异表达基因和其它统计分析前,必须将这些“噪音”剔除。对数比法（背景校正、对数比转换和数据标准化）已经被广泛应用于cDNA微阵列数据分析中,然而这种方法却存在着一些亟待解决的缺陷。对此,该文提出一种非转换方法,它可免去对数比的转化过程,直接在背景校正后进行数据标准化,可以有效剔除实验“噪音”。研究结果表明：在检测差异表达基因的效率方面,非转换方法比常规的对数比法具有更好的稳健性和更高的检测功效,基因检出率和准确性大大提高。     

梨自交不亲和及其亲和突变品种花柱内S4（S4^SM）基因的表达与作用的比较

提取梨 (PyrusserotinaRehd .)自交不亲和品种“二十世纪”(基因型为S2 S4 )、自交亲和的突变品种“奥嗄二十世纪”(S2 SSM4 ,SM =Stylar_partmutant;花柱部分突变 )及其亲和后代花柱的可溶性蛋白。经等电聚焦电泳 (IEF_PAGE)分析表明 ,“奥嗄二十世纪”及其后代花柱仍存在SSM4 蛋白 ,但其含量逐代减少 ,同时发现“奥嗄二十世纪”的SSM4 基因仅在柱头表达 ,而“二十世纪”的S4 基因表达的部位除了柱头外 ,还包括花柱上部及花柱下部 ,且表达量呈现从柱头到花柱下部下降的趋势。S蛋白经等电聚焦电泳的凝胶板进行RNase活性染色处理 ,也得到相同的结果。从花柱 (包括柱头 )中纯化出的S蛋白经SDS_PAGE电泳后进行RNase活性染色的结果表明 ,S4 与SSM4 蛋白的分子量相近 (约 30kD) ,并且均具有RNase活性。进一步以酵母RNA为基质测定的比活性也基本相等 ,约为 2 75U·min-1·mg-1蛋白。在离体条件下 ,上述两种S蛋白 (S_RNase)也以相同的程度抑制S4 或SSM4 花粉发芽及花粉管伸长。研究证明 ,自交亲和突变品种“奥嗄二十世纪”的SSM4 基因也具有原始自交不亲和品种“二十世纪”S4 基因的功能。因此 ,其自交亲和的原因可归结为SSM4 基因的表达量较少及SSM4 基因仅在柱头中表达的缘故。     

Identification of Self-Incompatibility Genotypes of Apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) by S-Allele-Specific PCR Analysis

A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S₁–S₉, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.     

确定梨自交不亲和基因型研究的技术进展

综述了运用杂交授粉试验和分子生物学方法等技术确定梨品种自交不亲和基因型研究的技术进展,分析了这些技术在确定梨品种自交不亲和基因型方面的优点和不足之处,并初步探讨了研究前景。因为HV区氨基酸的不同,不同S基因型也有所差异。因此,除了在分子生物学的水平上进行研究外,其他方法如mRNA、蛋白质和杂交授粉等水平上的研究在确定S基因型上也同样重要。     

Determination of self-locompatibility genotypes of Korean apple cultivars based on S-RNase PCR

To prevent self-fertilization, apple has a gametophytic self-incompatibility mechanism, part of a widespread intraspecific system, that is controlled by a multi-allelic locus. This attribute has been exploited in breeding programs for new cultivars. Likewise, many apple orchards depend on artificial pollination. Therefore, molecular analysis and early identification of the self-incompatibility (S) genotype could greatly improve breeding schemes and pollen donors selection. Here, we PCR-amplified the S-RNase PCR fragments from a total of 14 cultivars and parents, using new primers (ASPF3+ASPR3) common to 23 S-alleles in apple. The S-genotypes were determined for the following: ‘Hongro’ (S₁S₃), ‘Gamhong’ (S₁S₉), ‘Saenara’ (S₁S₃), ‘Chukwang’ (S₃S₉), ‘Hwahong’ (S₃S₉), ‘Seokwang’ (S₃S₃), ‘Hwarang’ (S₁S₉), ‘Sunhong’ (S₃S₉), ‘S.E.B.’ (S₁S₁₉), ‘S.G.D.’ (S₂S₃), and ‘Mollie’s Delicious’ (S₃S₇). We also confirmed the characteristics of the S-genotypes for eight Korean apple cultivars by PCR-Southern blot analysis, using seven S-RNases as probes.     

cDNA微阵列制作的优化

为了优化筛检cDNA微阵列中靶基因的最适长度、浓度及点样溶液的种类,设计持家基因betaactin和GAPDHRT PCR3对引物,产物长度在189～1078bp之间,以乙肝病毒DNA片段为阴性对照,扩增纯化后分别溶于3×SSC、50%DMSO及0.5mol/L碳酸盐缓冲液(pH=9.0)中,调整浓度分别为0.5μg/μL、1.0μg/μL和1.5μg/μL,比较上述不同条件的杂交结果。结果表明,杂交具有较好的特异性,阴性对照(乙肝病毒)和空白对照(点样溶液)均未见杂交信号;3种长度的同一靶基因杂交信号强度无明显差别(betaactinP=0.378;GAPDHP=0.866);3种点样溶液中以50%DMSO杂交信号最好,较强且均匀一致(P=0.0001),其余2种差异不显著(P=0.142);3种浓度靶基因杂交信号差异不显著(P=0.648),浓度高者信号略强。短片段靶基因(200bp左右)可获得与长片段靶基因(1000bp以上)一样较好的杂交信号,点样溶液以50%DMSO效果最好,靶基因浓度为0.5μg/μL时即可得到较好的杂交结果。     

25-O-乙酰升麻醇-3-0-β-D-木糖苷对HepG2细胞基因表达的影响

研究环菠萝蜜烷三萜化合物25-0-乙酰升麻醇-3-0-β-D-木糖苷对肿瘤细胞HepG2基因表达的影响,探讨其细胞毒作用机制。利用人低密度cDNA microarray研究细胞基因表达的改变,发现9个表达改变基因,6个下调,3个上调。结论表明25—O-乙酰升麻醇-3—0-β-D-木糖苷对HepG2细胞的细胞毒性与MAPK等信号传导途径相关。     

Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

多效生长因子相关基因的生物信息学分析

目的:用生物信息学方法分析多效生长因子(PTN)潜在的分子功能。方法:利用由美国亚利桑那癌中心提供的生物信息学数据库,对前期用小鼠全基因组表达谱芯片检测到的Ptn相关基因进行生物信息学分析,通过GO Terms分析这些基因所属的功能群体,用Pathway Miner分析这些基因参与调控的信号通路。结果:370个由芯片检测得到的Ptn相关基因中,在GO Terms数据库中找到231个基因,其中参与细胞成分构成的基因占31.83%,具有分子功能的基因占35.34%,而参与生物学过程的基因占32.83%;在Pathway Miner数据库中找到105个基因。这些基因相关的信号通路有230条,分别属于细胞和调控过程通路以及代谢通路。结论:PTN是一个重要的细胞因子,可能参与机体的免疫与防御反应、炎症反应,以及细胞的增殖、凋亡调控等。     

Programs of Gene Expression During the Laying Down of Memory Formation as Revealed by DNA Microarrays

Many experiments in the past have demonstrated the requirement of de novo gene expression during memory formation. In contrast to the initial reductionistic view that genes relevant to learning and memory would be easily found and would provide a simple key to understand this brain function, it is becoming apparent that the genetic contribution to memory is complex. Previous approaches have been focused on individual genes or genetic pathways and failed to address the massively parallel nature of genome activities and collective behavior of the genes that ultimately control the molecular mechanisms underlying brain function. In view of the broad variety of genes and the cross talk of genetic pathways involved in this regulation, only gene expression profiles may reflect the complete behavior of regulatory pathways. In this review we illustrate how DNA microarray-based gene expression profiling may help to dissect and analyze the complex mechanisms involved in gene regulation during the acquisition and storage of memory in the mammalian brain.     

Identification of aberrant cell cycle regulation in Epstein-Barr virus-associated nasopharyngeal carcinoma by cDNA microarray and gene set enrichment analysis

Previous studies have revealed that Epstein-Barr virus （EBV） was closely associated with nasopharyngeal carcinoma （NPC）. This study aimed to characterize the global pathways affected in the EBV-associated NPC. Combined with microdissection, gene expression profries in 22 NPCs and 10 non-tumor nasopharyngeal epithelial （NPE） tissue samples were analyzed. All NPC specimens served in the microarray analysis were positive for EBV, as judged by identification of the expression of EBV nuclear antigen 1 （EBNA1）. Through gene set enrichment analysis （GSEA）, we found that cell cycle pathway was the most disregulated pathway in NPC （P = 0.000, false discovery rate q-value = 0.007）, which included some aberrant expressed components. We first found that overexpression of CDK4, cyclin D1, and Rb proteins, and loss of expression of proteins p16, p27, and p19 were statistically significant in NPC tissues compared with non-cancerous NPE （P〈 0.05） by real-time RT- PCR and tissue microarray. EBV-encoded small RNA-1 （EBER-1） hybridization signals in the NPC showed significant associations with the overexpression of Rb （P = 0.000）, cyclin D1 （P = 0.000）, CDK4 （P = 0.000）, and the loss of expression of p16 proteins （P = 0.039）. In the final logistic regression analysis model, EBER-1 and abnormal expression of p16, Rb, cyclin D1, and E2F6 were inde- pendent contributions to nasopharyngeal carcinogenesis. Through survival analysis, only cyclin D1 could predict the prognosis of NPC patients. These results suggested that cell cycle pathway was the most disregulated pathway in the EBV-associated NPC, and EBER-1 was closely associated with p16, CDK4, cyclin D1, and Rb. cyclin D1 could be the prognosis biomarker for NPC.     

分析基因表达图式的新方法

随着基因组研究的深入进行,基因的分子生物学除了要寻找在生物学上重要的个别基因并研究其结构与功能外,更重要的应是了解整个基因组的功能活动,即细胞全部基因的表达图式.要解决如此复杂的问题就必须在研究方法上有所创新,基因表达系列分析法、cDNA微阵列分析法、DNA微芯片分析法等正是近几年发展起来的分析基因表达图式的新方法.