五指山小型猪近交系白细胞抗原Ⅰ类3 基因的研究

 【目的】为探讨五指山小型猪（WZSP）近交系群体中SLAⅠ3 (SLA-3) 位点等位基因分布结构及其特性。【方法】利用4对引物，采用RT-PCR扩增了32头WZSP SLA-3基因部分外显子1、外显子2和大部分外显子3序列。根据家系和扩增结果，选择8头个体的PCR产物克隆测序。【结果】获得9个不同的核苷酸序列。与GenBank中所用SLA-3基因的核苷酸序列及其编码的氨基酸序列进行比较分析，确定这9个序列均为SLA-3位点上的新等位基因，但这些等位基因间核苷酸变异很少；氨基酸序列比较发现猪和人之间有很高的保守性。同时，构建了23个SLA-3的等位基因和1个HLA-A等位基因核苷酸序列的系统树。【结论】WZSP在SLA-3位点和其它猪品种有明显的差异，拥有其独特的遗传资源。从分子水平为WZSP SLA-3基因的分子分型及特异单倍体猪的培育和异种器官移植实验用动物的研究提供理论依据。     

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.     

藏绵羊DQA1基因多态性分析

 【目的】研究藏绵羊DQA1基因多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点及各等位基因间的遗传关系,同时分析其进化意义。【方法】采用PCR-SSCP方法检测了900只藏绵羊DQA1基因第2外显子多态性;克隆、测序群体内变异产生的各等位基因序列,并分析序列数据。【结果】发现了17个DQA1的等位基因,包括缺失的1种基因,其中5个为发现的新等位基因。16个单倍型序列中发现56个核苷酸多态位点,27个氨基酸多态位点。【结论】藏绵羊DQA1基因第2外显子具有丰富的多态性,群体中可能蕴藏着更多的遗传资源;藏绵羊DQA1基因最初可能是由2个等位基因突变分化成两大类等位基因的;藏绵羊DQA1基因第2外显子序列与牛的DQA1基因第2外显子序列具有较高的同源性,预示绵羊和牛的DQA1基因最早可能来源于它们分歧以前的共同祖先原始序列;DQA1基因在与其相关的特定抗原刺激下发生的免疫应答反应在绵羊和牛上具有相似性;新等位基因C的139位发现了1个新的核苷酸突变位点（A/G）,属同义突变;5个新发现的DQA1等位基因遗传关系较近,可能由同一等位基因突变产生。     

五指山猪近交系群体中DRA和DRB基因的SSCP检测

为确定五指山猪(WZSP)近交系群体SLA!ⅡDRA、DRB基因的等位基因数及其特性,利用PCR扩增SLA!ⅡDRA、DRB基因的第2外显子序列,经单链构象多态性即SSCP检测其等位基因数,选择纯合子个体的PCR产物直接测序。对所获得的序列与Genbank中所有相应的序列进行比对和聚类分析,特别是SLA!DRB!C、SLA!DRB!D、HLA!DRB*09012、HLA!DRB*1201、HLA!DRA*0101等位基因。结果表明:在WZSP近交系群体中,DRA、DRB各有2个等位基因且发现1个DRB新等位基因。DRA第2外显子有很强的保守性,而DRB基因呈现出高度的多态性,在核苷酸水平同源性为85%以上,氨基酸水平同源性为70%以上。该试验成功地检测了WZSP近交系SLA!DRA、DRB基因的等位基因及其特性,为建立WZSPSLA!DR基因抗原的分子分型及特异单倍型猪的培育研究奠定基础。     

伏牛白山羊6个微卫星标记的遗传多样性分析

选取位于绵羊第6号染色体上与牛多胎基因FecB紧密连锁的2个微卫星基因座OarAE1 01、BM1329,牛第3号染色体上的微卫星基因座BMS1248,绵羊第4号染色体上的2个微卫星基因座MAF70、OarHH35及牛第8号染色体上的微卫星基因座BM1227,对伏牛白山羊遗传多样性进行检测。结果表明,6个微卫星基因座在伏牛白山羊中共检测到50个等位基因,其中平均多态信息含量(PIC)为0.789,且每个微卫星基因座的PIC>0.5,平均有效等位基因数(Ne)、平均杂合度(H)分别为5.165、0.903。由此表明,6个微卫星标记均具有高度多态性,可用于伏牛白山羊遗传多样性的分析。     

Genome-wide Identification and Analysis of DNA Methyltransferases in Grape

DNA methylation is an important epigenetic regulation mechanism, which is catalyzed by DNA methyltransferases. In this study, eight DNA methyltransferase genes were identified in grape genome to analyze the selective pressure, gene expression and codon usage bias. The results showed grape DNA methyltransferase MET subfamily underwent relatively strong purifying selection during evolution, while chromomethylase CMT subfamily underwent positive selection during evolution. Under different abiotic(heat, drought or cold) stresses, the expression level of many grape DNA methyltransferase genes changed significantly. The expression level of these genes might be related with cis-regulatory elements of their promoters. The results of codon usage bias analysis showed that synonymous codon bias existed in grape DNA methyltransferase gene family, which might be affected by mutation pressure. These results laid a solid foundation for in-depth study of DNA methyltransferases in grape.     

轮枝镰孢SSR标记开发及在玉米分离群体遗传多样性分析中的应用

【目的】开发轮枝镰孢（Fusarium verticillioides）的SSR标记,为轮枝镰孢遗传多样性研究提供技术支持。【方法】利用Fastpcr在轮枝镰孢基因组中查找符合条件的SSR位点,采用Primer5.0软件设计引物,利用NTSYS及Popgene分析来自玉米的轮枝镰孢单孢分离物群体的PCR扩增结果。【结果】共设计有效扩增SSR引物158对,经筛选,109对（69.0%）可扩增出2条及以上的条带,其中55对引物（34.8%）多态性较好,可扩增出3条及以上的条带。从11条已组装的轮枝镰孢染色体上各选出2对多态性引物,计22对引物,对66株轮枝镰孢玉米分离物进行扩增,共获得125个等位变异,变异范围为2-11个,平均为5.68个。轮枝镰孢玉米分离物之间Nei’s基因多样性指数为0.1139-0.8687,平均为0.6199,表现出较高的遗传多样性。【结论】基于轮枝镰孢基因组序列开发的SSR标记具有很好的多态性,可用于轮枝镰孢的遗传多样性分析。用22对SSR引物扩增,以遗传相似系数0.3进行划分,可将66株轮枝镰孢玉米分离物分为3群;中国轮枝镰孢玉米分离物的遗传多样性与地理分布无相关性。     

甜樱桃DFR基因内含子2和内含子3的多态性

【目的】研究70个甜樱桃品种DFR基因多态性与果皮颜色的相关性。【方法】通过DNA序列分析,以不同果皮颜色的10个甜樱桃品种（Prunus avium L.）为材料,检测DFR基因的多态性。根据多态性出现的位点设计特异引物,通过PCR扩增检测70个甜樱桃品种DFR基因的多态性。【结果】获得甜樱桃DFR基因约1 kb的片段,测序结果用BLAST分析发现,其核苷酸序列与樱桃李（Prunus cerasifera）的核苷酸相似性为80 %,预测的氨基酸序列与已知的甜樱桃（P. avium）DFR氨基酸序列相似性达99 %。该片段由3个外显子和3个内含子组成,2个多态性位点分别在内含子2和内含子3上。在黄色、黄底红晕和红色果皮3个组的70个甜樱桃品种中发现3个单倍型,共5种单倍型组合。通过SAS 9.0软件分析发现,所检测到的DFR基因的等位基因频率在黄底红晕果皮品种组和红色品种果皮组中的分布无显著差异。【结论】本研究在甜樱桃DFR基因座上得到2个差异位点,分别在内含子2和内含子3内,其优势基因频率依次为：0.864和0.679;但未发现DFR基因内含子2和内含子3的多态性与果皮颜色之间存在直接关系。     

藏绵羊基因OLA-DQA2第2外显子多态性分析

 【目的】针对藏绵羊腐蹄病所带来的死亡现象,选择与该病相关的基因OLA-DQA2为研究对象,分析群体中该基因的遗传多态性及变异特征,为进一步寻求抗病分子育种提供依据。【方法】采用PCR-SSCP检测216只表型正常和患病藏绵羊OLA-DQA2基因第2外显子的多态性,克隆、测序群体内变异产生的各等位基因序列,构建系统发育树以明确藏绵羊OLA-DQA2基因等位基因之间的遗传关系。【结果】藏绵羊DQA2基因第2外显子中表现了15个基因型。发现藏绵羊群体中OLA-DQA2基因第2外显子有8个新等位基因,分别命名为OLA-DQA2*H、*I、*J、*K、*L、*M、*N和*O,使绵羊基因库中该座位的等位基因数量从23个增加到31个。序列分析中发现71个核苷酸多态位点,这些多态位点主要由点突变形成,其中转换39个(占54.9%),颠换23个(占32.4%)。【结论】藏绵羊DQA2基因第2外显子具有丰富的遗传多态性,群体中可能蕴藏着更多的抗性遗传资源。     

Suppression of human colorectal carcinoma cell growth by wild-type p53

Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.     

Variable number of tandem repeat (VNTR) markers for human gene mapping

A large collection of good genetic markers is needed to map the genes that cause human genetic diseases. Although nearly 400 polymorphic DNA markers for human chromosomes have been described, the majority have only two alleles and are thus uninformative for analysis of genetic linkage in many families. A few known marker systems, however, detect loci that respond to restriction enzyme cleavage by producing a fragment that can have many different lengths. This polymorphism is due to variation in the number of tandem repeats of a short DNA sequence. Because most individuals will be heterozygous at such loci, these markers will provide linkage information in almost all families. Ten oligomeric sequences derived from the tandem repeat regions of the myoglobin gene, the zeta-globin pseudogene, the insulin gene, and the X-gene region of hepatitis B virus, were used to develop a series of single-copy probes. These probes revealed new, highly polymorphic genetic loci whose allele sizes reflected variation in the number of tandem repeats.     

黑龙江省稻瘟病菌无毒基因AVR-Pib、AVR-Pik和AvrPiz-t的检测与分析

【目的】检测黑龙江省稻瘟病菌（Magnaporthe oryzae）无毒基因AVR-Pib、AVR-Pik和AvrPiz-t在不同地区、年份间流行菌株的分布情况与变异机制,了解其等位基因的致病表型,为黑龙江省抗瘟品种布局提供参考依据。【方法】利用NCBI中公布的无毒基因序列对3个无毒基因AVR-Pib、AVR-Pik和AvrPiz-t的基因全长和编码序列（coding sequence,CDS）分别设计特异性引物,将2016年和2017年采自黑龙江省不同地区335个稻瘟病菌单孢分离菌株的DNA进行PCR扩增,通过琼脂糖凝胶电泳检测分析,并挑选不同带型和不同地区代表菌株的PCR产物进行测序。测序结果与相应无毒基因序列进行碱基与氨基酸序列的比较分析,并利用水稻抗性单基因系,对不同变异类型的稻瘟病菌菌株进行致病型测定。【结果】在PCR电泳检测中AVR-Pib、AVR-Pik和AvrPiz-t均出现特异性条带,说明这3个无毒基因在黑龙江省均有分布,并以不同分布频率与突变类型出现。3个无毒基因平均扩增频率分别为75.52％、87.16％和85.67％。其中AVR-Pib通过电泳检测与PCR产物测序检测出4种带型（无带、高带、中高带与低带）和5种变异类型AVR-Pib（1-1、1-2、2、3、3-1）,基因型AVR-Pib-1-1、AVR-Pib-1-2、AVR-Pib-2和AVR-Pib-3-1为新发现的变异类型,其中基因型AVR-Pib-1-1和AVR-Pib-1-2均为转座子Pot2的插入,但插入位点不同;基因型AVR-Pib-2在阅读框上游存在小片段的插入;基因型AVR-Pib-3-1碱基序列与原序列比对有4处差异,即32（C/G）35（T/A）36（T/A）38（T/A）,导致氨基酸翻译提前终止。对检测到的5种等位基因型进行致病型测定,分析发现除正常基因型AVR-Pib-3外,其他变异基因型无毒功能均已丧失。而AVR-Pik经PCR产物测序检测出7种等位基因型,AVR-Pik（D、A、B、C、E、F、F2）,碱基序列的变化均导致氨基酸错义突变,7种等位基因型均已见报道。无毒基因AvrPiz-t通过电泳检测和PCR产物测序分析,发现2种带型（高带和正常带型）与4种基因型AvrPiz-t（A、B、C、D）,其中AvrPiz-t-A为正常基因型;基因型AvrPiz-t-B在191位处有碱基A的插入,导致氨基酸翻译提前终止;基因型AvrPiz-t-C为新发现的等位基因型,其特点在于与A类型存在17（T/C）和19位处碱基C的插入,发生移码突变翻译提前终止;高带型对应的无毒基因型AvrPiz-t-D经测序验证为Pot3转座子的插入。4种基因型菌株分别接种水稻单基因系发现,AvrPiz-t-A可以被Piz-t识别表现无毒,AvrPiz-t（B、C、D）均不能被Piz-t所识别而表现为有毒性。【结论】黑龙江省稻瘟病菌无毒基因AVR-Pib、AVR-Pik和AvrPiz-t分布较为广泛,且变异类型丰富,研究结果可为选育和推广携带相应抗病基因的水稻品种提供参考。     

‘河西绒山羊’MHC-DQB2基因第3外显子多态性及其与流产性状的关联分析

采用PCR-SSCP方法检测431只‘河西绒山羊’MHC-DQB2基因第3外显子的多态性,测序并分析群体内突变产生的等位基因.结果表明:MHC-DQB2基因第3外显子有15个基因型、8个等位基因、35个变异位点;χ2适合性检验结果表明,该群体处于Hardy-Weinberg平衡状态(P>0.05);该位点多态信息含量为0.838 5,杂合度为0.317 9,有效等位基因数为6.899 8;等位基因平均遗传距离为0.048 8;经NJ树分析发现,各等位基因为同源序列;经流产数据统计发现,基因型CF中无流产个体,且在其198位插入1个碱基C.说明‘河西绒山羊’MHC-DQB2基因第3外显子具有丰富的遗传多态性;牛和羊的MHC-DQB2外显子3最初是由2个等位基因突变分化而来,而同物种的绵羊与山羊在该位点有很高的同源性;基因型CF可能与流产性状有关.     

Detection and Analysis of Magnaporthe oryzae Avirulence Genes AVR-Pib,AVR-Pik and AvrPiz-t in Heilongjiang Province

【Objective】The objective of this study is to investigate the distribution and variation mechanism of avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t in Magnaporthe oryzae strains from different regions and years in Heilongjiang Province, to understand the pathogenic phenotypes of different avirulence gene alleles, and to provide a reference for utilization and distribution of resistance cultivars in Heilongjiang Province.【Method】Based on the avirulence gene sequences published in NCBI, specific primers were designed to amplify full length and the coding sequence (CDS) regions of three genes, respectively. From 2016 to 2017, 335 M. oryzae strains in different regions of Heilongjiang Province were collected and isolated, and their DNA was PCR-amplified using avirulence genes primers and analyzed by agarose gel electrophoresis. The PCR products with different band patterns and from representative strains of different regions were selected for sequencing. The sequencing results were compared with the corresponding avirulence gene sequences for base and amino acid. The pathogenic phenotype of M. oryzae strains with different variant types was determined based on the rice resistance to single-gene lines.【Result】The specific bands of AVR-Pib, AVR-Pik and AvrPiz-t were detected in PCR detection and appeared in different distribution frequencies and mutation types, indicating that these 3 avirulence genes were all distributed in Heilongjiang Province. The average amplification frequency of the 3 avirulence genes was 75.52%, 87.16% and 85.67%, respectively. Among them, 4 types of band (bandless, high band, mid to high band and low band) of AVR-Pib were detected by electrophoresis analysis and 5 variant types AVR-Pib (1-1, 1-2, 2, 3, 3-1) were detected by PCR product sequencing. The genotypes AVR-Pib-1-1, AVR-Pib-1-2, AVR-Pib-2 and AVR-Pib-3-1 are newly discovered variant types, of which genotypes AVR-Pib-1-1 and AVR-Pib-1-2 are insertions of transposon Pot2 but with different insertion sites. The genotype AVR-Pib-2 has a small fragment insertion in the upstream of CDS region. The genotype AVR-Pib-3-1 base sequence has 4 differences from the original sequence, namely 32 (C/G) 35 (T/A) 36 (T/A) 38 (T/A), and the amino acid translation was terminated prematurely. Pathogenic analysis showed that except for the normal genotype AVR-Pib-3, the other alleles lost their avirulence functions. Seven AVR-Pik alleles (D, A, B, C, E, F, F2) were detected after PCR product sequencing, and the alterations in the nucleotide sequences of these alleles all resulted in amino acid missense mutations. The 7 AVR-Pik alleles have been reported previously. The avirulence gene AvrPiz-t was analyzed by electrophoresis and sequencing of PCR products, and 2 types of band (high band and normal band type) and 4 genotypes of AvrPiz-t (A, B, C, D) were revealed. Among them, AvrPiz-t-A is the original genotype, while AvrPiz-t-B has a base A insertion at position 191, causing premature termination of amino acid translation. Genotype AvrPiz-t-C is a newly discovered allelic type, characterized by the presence of a nucleotide variation at position 17 (T/C) and the insertion of base C at position 19 compared with type A, leading to the frameshift mutation and premature translation termination. The high band type avirulent genotype AvrPiz-t-D was sequenced and verified as having an insertion of the Pot3 transposon. Rice single-gene lines infection showed that the strains with AvrPiz-t-A were avirulent to Piz-t line due to Piz-t recognition, whereas the strains with AvrPiz-t (B, C, D) were virulent to Piz-t line due to lost the ability recognized by Piz-t.【Conclusion】The avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t of M. oryzae in Heilongjiang Province are widely distributed and the types of variation are abundant. The results of this study can provide a reference for breeding and popularizing rice cultivars with corresponding disease-resistance genes.     

动物组蛋白H3K4三甲基化转移酶MLL3的生物信息学分析

[目的]对动物组蛋白H3K4三甲基化转移酶MLL3进行生物信息学分析。[方法]利用生物信息学的方法,对小鼠MLL3的基因结构、氨基酸序列、系统进化树、染色体定位和共线性等问题进行分析。[结果]编码合成的小鼠MLL3蛋白质一级结构包括7个锌指结构域、1个HMG-box(高迁移率族蛋白)、1个FYRN(N-末端富含苯丙氨酸或酪氨酸区域)、1个FYRC(C-末端富含苯丙氨酸或酪氨酸区域)、1个SET域和1个postSET域;从序列对比和同源性上发现,该研究中的19种动物都基本上具有这些结构,说明这些结构在进化上是相对保守的,其中SET域具有高度的保守性,是维持组蛋白甲基化酶活性所必须的;从系统发生上看,19种动物在进化树上的位置与其分类地位相一致;在共线性分析中,虽然小鼠和人的MLL3基因位于不同的染色体上,但其上游和下游具有相同的基因,说明小鼠和人的MLL3基因具有共线性。[结论]不仅揭示了MLL3的核苷酸序列及其氨基酸序列的一级结构,为以后研究其高级结构和蛋白质的功能奠定了基础;同时也为后期进行小鼠MLL3基因的引物设计、启动子分析、基因的克隆、定位和表达的调控模式研究奠定了基础。     

美康寿系列茶的研制

研制出美康寿1、2、3号茶,成品经品质审评、成份检测、安全试验和临床观察,表明该茶保持了茶叶应有风味,不含合成物质、安全可靠、保健效果良好,长期饮用,有利于人体健康。     

Construction of a Full-Length cDNA Library of Gossypium hirsutum L.and Identification of Two MADS-Box Genes

A full-length normalized cDNA library for the flower development stages of short-season cotton(Gossypium hirsutum L.)(CCRI36)was constructed.A total of 3 421 clones were randomly selected for sequencing,with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences.Clustering the 3 175 high-quality expressed sequence tags(ESTs)resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons.Comparative analyses indicated that 913(43.6%)of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information.In addition,763(36.4%)of the unigenes were functionally classified using Gene Ontology hierarchy.Through EST alignment and the screening method,the full-length cDNA of two MADS-box genes viz.,GhMADS11 and GhMADS12 were acquired.These genes may play a role in flower development,Phylogenetic analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes,respectively.The expression of both GhMADS11 and GhMADS12,genes was high in reproductive organs.In floral organs,GhMADS11 expression was high in petals(whorl2)and ovules,while GhMADS12 expression was high in petals(whorl2)and stamens(whorl3).Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification.The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.