黑麦1R染色体的微切微克隆研究

显微分离出黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体,用ＣｏｈｅｓｉｖｅａｄａｐｔｅｒｓｓｉｎｇｌｅｐｒｉｍｅｒＰＣＲ（ＣＡＳＰＰＣＲ）方法进行体外扩增,以ＤＩＧ１１ｄＵＴＰ标记扩增产物为探针,进行Ｓｏｕｔｈｅｒｎ分子杂交,结果表明扩增产物来自黑麦１Ｒ染色体。用１／１０体积的连接物转化Ｅ．ｃｏｌｉＤＨ５α,获得１００００多个重组菌落。经酶切分析,克隆子的插入片段为２５０～５００ｂｐ,为进一步筛选１Ｒ染色体的分子标记打下了基础     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

利用微分离黑麦IR染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（Ｓｅｃａｌｅｃｅｒｅａｌｅ．Ｌ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡＰＣＲ）经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用ＩＲ染色体的第二轮扩增产物,黑麦基因组ＤＮＡ,ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的ＩＲ当色体体我扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总     

黑麦2R染色体的显微分离与回收

建立了利用显微担任技术分离植物单个染色体的方法。以黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌｅ Ｌ）为材料,以其标准染色体组型图为依据,识别出黑麦含抗病基因的１Ｒ染色体。经显微操作,将单条１Ｒ染色体放入Ｅｐ－ｐｅｎｄｏｆ管中。研究表明,用α－溴萘饱和液对细胞进行预处理,可快速鉴别出黑麦１Ｒ染色体。采用去壁低渗制片技术,可明显地改善显微分离意境染色体的条件。     

黑麦1R染色体的显微分离与回收

建立了利用显微操作技术分离植物单个染色体的方法。以黑麦(Secale cereale L.)为材料,以其标准染色体组型图为依据,识别出黑麦含抗病基因的1R染色体。经显微操作,将单条1R染色体放入Ep-pendorf管中。研究表明,用α-溴奈饱和液对细胞进行预处理,可快速鉴别出黑麦1R染色体。采用去壁低渗制片技术,可明显地改善显微分离单染色体的条件。     

黑麦1R染色体的显微分离、体外扩增及扩增产物的鉴定

借助Leitz显微操作器,在国产倒置显微镜下(400×)用玻璃针对处于有丝分裂中期的黑麦根尖细胞中的1R染色体成功地进行了分离。分离出来的1R染色体转入0.5 ml的Eppendorf管中,用蛋白酶K处理,把DNA释放出来;经Sau3A酶切,再与人工合成的Sau3A连接头连接;以连接头的一条链的核苷酸顺序片段为引物对DNA酶切片段进行了PCR扩增。琼脂糖凝胶电泳显示扩增产物的长度大约为300~1000 bp。以生物素分别标记的黑麦总体DNA和小麦rDNA为探针进行斑点杂交,结果表明PCR扩增产物确实来源于黑麦的1R染色体DNA。这个方法为构建黑麦1R染色体亚基因组文库和筛选1R染色体特异性探针奠定了基础。     

小麦背景中黑麦1R染色体的遗传变异

运用细胞遗传学方法鉴定了来源于中国春×Ｍ２７（１Ｒ／１Ｄ代换系）的花粉植株和Ｆ２单株染色体组成。发现７个花粉植株中出现７种染色体组成变异类型,每株呈现一类变异;而２７个Ｆ２单株中,存在１１种染色体组成变异类型,变异频率仅为３７．０％,低于花粉植株。花粉植株群体中,观察到一个能稳定向后代传递的小麦／１Ｒ小片段易位,但Ｆ２群体中未检测到小麦／黑麦易位。表明常规染色体工程结合花药培养是有计划、有目的实现异源染色体小片段向小麦转移的简便、高效、快速途径。     

染色体特异DNA文库的构建与鉴定

为构建人类21号染色体特异DNA文库, 以应用于人类遗传疾病的鉴定和研究, 文章采用循环温度梯度法溶解释放微分离的人外周血细胞21号染色体DNA, 将其进行简并寡核苷酸引物PCR(Degenerate oligo nucleotide primer-PCR, DOP-PCR)扩增后, 利用100~500 bp和500~2 000 bp分段回收纯化的两种不同片段大小的DOP-PCR产物构建染色体特异DNA文库, 并分别采用荧光原位杂交(Florescence in situ hybridization, FISH)和斑点杂交对DOP-PCR产物的来源和随机取样的文库克隆进行检测以评估所构建DNA文库的特异性。结果表明: 循环温度梯度法能有效溶解释放微分离的21号染色体DNA; 通过对DOP-PCR产物的分段回收纯化和克隆, 增加了大片段DNA的连接效率; 利用FISH技术和斑点杂交双重鉴定实验证明了文库的特异性, 从而构建了21号染色体特异的DNA文库, 并建立了构建染色体特异DNA文库及检测其特异性的方法, 为21号染色相关遗传疾病的鉴定和研究奠定了基础。     

Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Studies on Microdissection and Microcloning of the Rye Chromosome 1R

Two 1R chromosomes of Secale cereale L. were isolated from one metaphase cell by means of chromosome micro-isolation, and the chromosomal DNA was amplified adopting the cohesive adapters single primer polymerase chain reaction (CASP-PCR) technique. The CASP-PCR products were labeled as probes. The results of Southern blot hybridization confirmed that the CASP- PCR products derived from the chromosome IR were homologous with the genomic DNA of S. cereale. The clones of PCR products were obtained with high efficiency. Over 10 000 recombinant clones were obtained from one-tenth of the ligation mixture which was transferred into the competent E. coli DH5a. The size of the inserted fragments of clones ranged from 250 bp to 500 bp. This research has established the foundation for further selection of chromosome 1R markers.     

柱穗山羊草2C染色体诱发中国春小麦背景中黑麦1R染色体结构变异的研究

当柱穗山羊草(Aegilops cylindrica Host．)2C染色体单体添加到普通小麦品种中国春和以中国春为背景的派生系时,减数分裂时,不含2C染色体的配子会发生染色体结构变异。为了制备一套黑麦1R染色体缺失系以用于定位黑麦1R染色体上的控制重要农艺性状的基因,把一条2C染色体导人到小黑麦1R二体附加系(21″ 1R″)中,然后让这些个体(21″ 1R″ 2C′,2n=45)自交,以便产生1R染色体结构变异体。实验共检测了345粒F,种子,83粒种子带有结构变异的黑麦1R染色体(24．1％)。通过C分带和原位杂交检测,对来自于23株F2的46个F3植株所带有的异常1R染色体进行了归类：其中1RL端体为39．1％,1RL等臂染色体为2．2％,1RL易位系为32．6％。1RS端体为4．3％,1RS等臂染色体为4．3％,切点在长臂上的缺失体为2．2％。在6．5％的植株中同时含有2种类型的1R染色体结构变异。其余8．7％带有异常1R染色体的个体因为没有原位杂交结果而无法判断是属于哪种类型。已获得的1R结构变异株将有可能进一步发展成为一套可用于定位黑麦1R染色体上重要功能基因的遗传材料。另外,还探讨了综合应用细胞学和分子标记方法鉴定易位染色体中小麦染色体片段的尝试,并对所获结果进行了讨论。     

黑麦6R抗白粉病基因向小麦的渗进与鉴定

为了将黑麦６Ｒ染色体上抗小麦白粉病的基因导入小麦,选用了一个６Ｒ／６Ｄ代换系Ｍ２４为亲本之一,分别与小麦栽培品种和第６部分同源群缺体系杂交,杂种出现６Ｒ或／或６Ａ,６Ｂ,６Ｄ单,双或三单体等各种情况,取其花药进行培养,共获得２４１个再生植株,对其中３２个抗白粉病的花粉植株经染色体计数,Ｃ－分带,基因组原位杂交,同工酶等电聚焦电泳和或／ＲＦＬＰ分子标记检测,发现有６株仍保持为６Ｒ／６Ｄ代换系,有１０     

Microdissection and Recovery of 1R Chromosome in Rye ( Secale cereale )

The technique of mieromanipulaion has been used to establish a system of single chromosome mierodissection. According to the standard karyotype of rye ( Secale cereale L. ), 1R chromosome carrying disease-resistant gene has been identified, microdissected and transferred into Ep-pendorf tube using mieromanipulator. The results showed that IR chromosome in cells pretreated with α-bromonaphthalene could be identified quickly and the efficiency of microdissection was greatly improved when the technique of wall degradation and hypotonic treatment was applied.     

弥漫大B细胞淋巴瘤1号染色体的基因表达分析

目的:探讨弥漫大B细胞淋巴瘤(Diffuse Large B-Cell Lymphoma,DLBCL)中1号染色体基因表达情况。方法:采用激光显微切割技术分离临床DLBCL病人淋巴结标本中的淋巴细胞,提取淋巴细胞的mRNA并与表达谱芯片杂交,通过信号扫描、处理后获得表达基因杂交信号强度。每基因设11-20对探针。杂交信号与错配探针对比,扣除背景值后,使用Wilcoxon符号秩和检验选取与错配杂交信号有显著差异的基因作为分析结果(P=0.05)。然后随机选取四个检测到的基因,使用PCR方法检验基因芯片结果的可靠性。结果:成功地从快速冷冻保存的DLBCL标本中提取RNA。使用表达谱芯片进行研究,发现了共316条1号染色体编码的基因在DLBCL细胞中表达。根据胞内定位,基因功能和基因所属的代谢通路三种分类方法对所得基因进行分类分析。基因表达密度分析显示DLBCL中1号染色体上的基因表达情况与编码基因分布情况存在统计学差异。结论:使用表达谱芯片研究了DLBCL中1号染色体上的基因表达情况。     

Identification and Characterization of Single‐Nucleotide Polymorphisms in MCH‐R1 and MCH‐R2*

Objective: To identify and functionally characterize single‐nucleotide polymorphisms (SNPs) in melanin‐concentrating hormone (MCH)‐R1 and ‐R2. Research Methods and Procedures: The entire coding regions and intron/exon splice junction regions of MCH‐R1 and MCH‐R2 were sequenced from anonymous white (n = 45) and African‐American (n = 46) individuals. DNA was analyzed, and SNPs were identified using Phred, Phrap, and Consed software. DNA constructs containing MCH‐R1 and MCH‐R2 SNPs were generated and expressed in CHO cells. The effect of the SNPs in MCH‐R1 and MCH‐R2 were assessed in receptor binding assays and functional assays measuring changes in intracellular cAMP and Ca²⁺ levels. Results: We identified 12 SNPs in the MCH‐R1 gene. Two of these SNPs are in coding regions, and one produces an arginine‐for‐glycine substitution at residue 34 in the MCH‐R1 sequence. This SNP is present at a minor allele frequency of 15% in the African‐American population tested in this study. We identified eight SNPs in the MCH‐R2 gene. Four of these SNPs are in coding regions, and two produce amino acid substitutions. Lysine substitutes for arginine at residue 63 of the African‐American population, and glutamine substitutes for arginine at residue 152 in whites (minor allele frequency of 2% for both SNPs). No changes in receptor binding or functional signaling were observed with the SNP mutations in MCH‐R1 or MCH‐R2. Discussion: These data indicate that potential therapeutics designed to act at the MCH receptor are unlikely to have altered effects in subpopulations that express variant forms of MCH‐R1 or MCH‐R2.     

黑麦6R染色体在小麦背景中的传递

带有６Ｒ的配子传递率普通显著下降。６Ｒ通过雌配子的传递率为８．８％,通过雄配子的传递率为１０．３％,通过花药培养的传递率较高,为２３．３％,但均低于理论值。进一步分析各种配子经花药离体培养的传递率,发现附加型提高最多,正常型次之,缺失型减少最多,代换型次之,说明离体培养对各种配子具有选择作用。另外,还观察到６Ｒ单体附加,６Ｒ＾Ｌ端体单附加和６Ｒ＾Ｌ等臂单附加自交传递率差异不显著。对于影响传递率的各