家蚕丝素基因pFb2.9限制性片段长度多态性研究

本研究利用丝素Ｈ链基因的结构基因ｐＦｂ２．９为探针，对家蚕基因组ＤＮＡ进行了限制性片段长度多态民生分析，并探讨了多态性与茧丝茸毛性状的关系。     

被子植物叶绿体基因及其表达

本文简要综述了叶绿体ＤＮＡ分子结构、叶绿体基因的功能及其表达产物。着重阐述了被子植物叶绿体ＤＮＡ与细胞质雄性不育的关系；借助ｃｐＤＮＡ限制性片段分析，许多学者认为由于ｃｐＤＮＡ分子的缺失、突变或插入，叶绿体ＤＮＡ可能涉及到ＣＭＳ的产生，为了得到ｃｐＤＮＡ在ＣＭＳ中起作用的肯定性结论和在分子水平上解释被子植物细胞质雄性不育的机理，作者提出了应该进一步采取的措施。     

RFLP在家蚕遗传和育种中的应用

限制性片段长度多态性（ＲＦＬＰ）是一种新的ＤＮＡ水平的遗传标记。与经典的形态标记相比，ＲＦＬＰ数量大，同一位点上等位基因多，能在任何组织、任何发育时期进行检测，相互之间为共显性，没有上位或多效效应。应用ＲＦＬＰ可以研究家蚕的分类和进化，提高育种过程中的选择效率，构建饱和的家蚕ＲＦＬＰ连锁图谱，进行抗病性、抗氟性、茧丝质性状的遗传分析和改良，以及分离和克隆重要的基因。ＲＦＬＰ技术将对家蚕遗传和育种工     

家蚕核多角体病毒Lef—1和DA26基因的克隆及部分序列分析

ｌｅｆ－１和ＤＡ２６基因分别位于ｅｇｔ基因的上游和下游．从ｐＵＡｃ－ｅｇｔ中分离出ＥｃｏＲＩ－ＸｂａＩ１．１ｋｂ的ｅｇｔ基因片段，进行缺口平移标记，获得探针。与ＢｍＮＰＶＤＮＡ经ＨｉｎｄⅢ酶切、电泳、转移至ＮＣ膜的ＤＮＡ进行Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交，发现ＢｍＮＰＶＤＮＡ的ＨｉｎｄⅢＤ片段呈阳性。从ＢｍＮＰＶ基因组中分离出１０．４ｋｂ的ＨｉｎｄⅢＤ片段，用ＥｃｏＲＩ酶切得到ＨｉｎｄⅢ－ＥｃｏＲＩ２．２ｋｂ、ＥｃｏＲＩ１．４ｋｂ和ＥｃｏＲＩ－ＨｉｎｄⅢ６．８ｋｂ３个片段，分别克隆于ｐＵＣ１９中，命名为ｐＵＨＥ２．２、ｐＵＥ１．４和ｐＵＥＨ６．８。经双链测序并与ＡｃＮＰＶ相关基因序列进行比较，发现ｌｅｆ－１基因被克隆于ｐＵＨＥ２．２中，ＤＡ２６基因克隆于ｐＵＥＨ６．８中。从所测定的ｌｅｆ－１和ＤＡ２６基因的启动子及５＇端部分序列来看，它们在ＡｃＮＰＶ和ＢｍＮＰＶ之间有极高的同源性。     

拟南芥菜LEAFY基因片段的分离及果树基因组中存在LEAFY同源基因…

利用ＰＣＲ技术从拟南芥菜基因组ＤＮＡ中扩增出一条长４１４ｂｐ的ＤＮＡ片段。该片段被克隆到ｐＢｌｕｅｓｃｒｉｐｔＳＫ（－）手ＥｃｏＲＶ位点上。序列分析结果证实扩增和克隆的ＤＮＡ片段是分生组织特征基因ＬＥＡＦＹ３＇端的部分片，不含内含子序列。结果表明，供试果树，基因组中均存在单拷贝的ＬＥＡＦＹ同源基因。     

谷胱甘肽还原酶基因的表达载体构建及对马铃薯的转化

 利用 ｐＧＲ２０２和 ｐＢｉｎ１９两种质粒经过 ｐＢＩ ２２２的改建，含有 ＣａＭＶ３５Ｓ启动子和 Ｎｏｓ终止子的 ＣＮ区ＤＮＡ片段与 ｐＢｉｎ１９的 Ｂｉｎ区 ＤＮＡ片段的不对称连接和目的基因 ＧＲ的 ｃＤＮＡ重组于真核表达双元载体三步亚克隆，筛选出一正向插入的谷胱甘肽还原酶基因的真核表达双元载体质粒ｐＢｉｎ－ＣＮ－ＧＲ。再利用此质粒转化的农杆菌ＬＢＡ４４０４进行马铃薯的遗传转化．并分化出卡那霉素抗性苗。过氧化物同工酶和酯酶同工酶谱带显示转化材料与对照之间存在显著的差异．表明转化材料的基因表达发生了变化。对转化影响因素的研究发现培养基的组成成分、选择压（Ｋｍ）添加浓度及加入时机是影响转化率的重要因素。     

分子生物学新方法在根瘤菌分类中的应用

随着分子生物学技术的发展,一些全新的方法应用于根瘤菌分类,使根瘤菌类的研究得到了迅速发展,从过去的传统分类过渡到以遗传特征和系统发育为主要依据的现代系统分类。研究在参考大量文献的基础上,概述了全细胞可溶性蛋白电泳,多位点酶永金ＤＮＡＧ＋Ｃ摩尔百分含量测定ＤＮＡ－ＤＮＡ杂交,限制性片段长度多态性分析,随机扩增ＤＮＡ片段的多态性,基因外回文重复序列ＰＣＲ扩增技术及１６ＳｒＤＮＡ序列的分析确定根瘤菌系统     

中国家蚕抗菌肽基因的PCR扩增,克隆和序列测定

本实验采用ＰＣＲ技术，扩增中国家蚕抗菌肽ｃｅｃｒｏｐｉｎＢ基因片段，并以ＰＵＣ１９质粒为载体，将该基因片段转化到大肠杆菌中。从筛选得到的阳性克隆中分离出ｃｅｃｒｏｐｉｎＢ基因，测定其序列，并由此推导出氨基酸序列。结果表明：（１）抗菌肽ｃｅｃｒｏｐｉｎＢ基因片段长为８６６ｂｐ，包括７３ｂｐ的ＥｘｏｎＩ，５５６ｂｐ的Ｉｎｔｒｏｎ，８７ｂｐ的ＥｘｏｎⅡ和１５０ｂｐ的３’端非编码区。其中，非内含子部分的碱基序列与已克隆的日本家蚕的两个ｃｅｒｏｐｉｎＢｃＤＮＡ序列比较，同源性分别为８７．５％和９５％；（２）推导出的成熟抗菌肽由３７个氨基酸残基组成，其氨基酸序列与日本家蚕和天蚕的氨基酸序列相比较，同源性为９１．９％和８０．６％。     

防御素基因的化学合成及克隆

将防御素基因分成４个寡聚核苷酸片段，采用固相亚磷酰胺法在ＤＮＡ合成仪上化学合成。片段２和４分别用Ｔ＿４多聚核苷酸激酶将５′端磷酸化，４个片段等量混合，用Ｔ＿４ＤＮＡ连接酶连成一个１０９ｂｐ的双股ＤＮＡ片段，其两侧为ＢａｍＨＩ和ＳａｌⅠ粘性末端。把它与经上述双酶切的质粒ｐＵＣ１９连接，转化大肠杆菌ＤＨＳα。转化株经含Ｘｇａｌ青霉素平板初筛，质粒酶切分析以及ＰＣＲ专一性扩增，证实已获得完整的防御素基因克隆。为进一步表达防御素提供了材料．     

猪ESR基因一个外显子DNA片段的分子克隆

参考人、鸡,鼠的ＥＳＲ基因序列,设计一对引物,采用ＰＣＲ技术扩增出猪的ＥＳＲ基因一段ＤＮＡ片段,将该片段克隆到ｐＧＥＭ－Ｔ Ｅａｓｙ质粒载体中,重组质粒经ＰＣＲ鉴定和酶切分析确定克隆成功,经测序结果分析确定该片段为猪ＥＳＲ基因与其他动物第４号外显子相对应的一段ＤＮＡ片段,大小为３３２ｂｐ。     

Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers.

Recent studies have suggested the existence of a tumor suppressor gene located at chromosome region 5q21. DNA probes from this region were used to study a panel of sporadic colorectal carcinomas. One of these probes, cosmid 5.71, detected a somatically rearranged restriction fragment in the DNA from a single tumor. Further analysis of the 5.71 cosmid revealed two regions that were highly conserved in rodent DNA. These sequences were used to identify a gene, MCC (mutated in colorectal cancer), which encodes an 829-amino acid protein with a short region of similarity to the G protein-coupled m3 muscarinic acetylcholine receptor. The rearrangement in the tumor disrupted the coding region of the MCC gene. Moreover, two colorectal tumors were found with somatically acquired point mutations in MCC that resulted in amino acid substitutions. MCC is thus a candidate for the putative colorectal tumor suppressor gene located at 5q21. Further studies will be required to determine whether the gene is mutated in other sporadic tumors or in the germ line of patients with an inherited predisposition to colonic tumorigenesis.     

猪生长激素基因(cDNA)的分子克隆

采用 Watson 方法由猪垂体总 mRNA 反转录合成了总 cDNA,合成产率约为12.6%。将总 cDNA 克隆到质粒 pUC19中后,转化大肠杆菌 JM107得到约2500个重组子克隆。用猪生长激素探针进行菌落原位杂交筛选得到2个阳性克隆。对这两个克隆进行多种酶切分析的结果表明,其中质粒 pWH101插入片段长度为830bp 左右,带有全长猪生长激素基因及部分5′端和3′端非编码区。用 Sanger 法分析了此基因5′端203个 bp 的序列,结果除确证己克隆了猪生长激素基因(cDNA)外,还查明了此基因5′端非编码区46bp 的序列。     

Gene encoding the beta subunit of S100 protein is on chromosome 21: implications for Down syndrome

S100 protein is a calcium-binding protein found predominantly in the vertebrate nervous system. Genomic and complementary DNA probes were used in conjunction with a panel of rodent-human somatic cell hybrids to assign the gene for the beta subunit of S100 protein to the distal half of the long arm of human chromosome 21. This gene was identified as a candidate sequence which, when expressed in the trisomic state, may underlie the neurologic disturbances in Down syndrome.     

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA

The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.     

A new probe for the diagnosis of myotonic muscular dystrophy

Myotonic muscular dystrophy (DM) is the most common muscular dystrophy, affecting adults as well as children. It is inherited as an autosomal dominant trait and is characterized by variable expressivity and late age-of-onset. Linkage studies have established the locus on chromosome 19. In order to identify tightly linked probes for diagnosis as well as to define in detail the DM gene region, chromosome 19 libraries were constructed and screened for restriction fragment length polymorphisms tightly linked to DM. A genomic clone, LDR152 (D19S19), was isolated that is tightly linked to DM; recombination fraction = 0.0 (95% confidence limits 0.0-0.03); lod score, 15.4.     

细胞色素b基因标记绵羊种内亲缘关系的初步研究

[目的]从群体的角度,初步探讨细胞色素b（Cytb）基因标记绵羊种内亲缘关系。[方法]用PCR方法扩增出滩羊和洼地绵羊2个地方绵羊品种共112个个体的线粒体DNA（m tDNA）Cytb基因,用限制性内切酶EcoRΙ对其进行限制性长度多态性（RFLP）分析。[结果]在56个滩羊样品中,有51个个体检测到1个酶切位点,5个个体没有检测到切点,呈现出2种限制性态型。在56个洼地绵羊样品中均能检测到酶切位点,表现为1种限制性态型。[结论]受试绵羊品种线粒体DNA多态性较贫乏,且m tDNA Cytb基因在绵羊品种内、品种间都有很强的保守性。因此,Cytb基因标记绵羊种内亲缘关系有一定的局限性。     

Cloning of breakpoints of a chromosome translocation identifies the AN2 locus

Chromosome translocations involving 11p13 have been associated with familial aniridia in two kindreds highlighting the chromosomal localization of the AN2 locus. This locus is also part of the WAGR complex (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation). In one kindred, the translocation is associated with a deletion, and probes for this region were used to identify and clone the breakpoints of the translocation in the second kindred. Comparison of phage restriction maps exclude the presence of any sizable deletion in this case. Sequences at the chromosome 11 breakpoint are conserved in multiple species, suggesting that the translocation falls within the AN2 gene.     

水稻通用叶绿体表达载体的构建

本研究建立了以水稻叶绿体来源的trnA、trnI为同源重组片段,水稻叶绿体来源的Prrn为启动子,烟草叶绿体来源的Tps-bA为终止子,EPSP为筛选标记基因,GFP为外源基因,并含有两个多克隆位点(MCS)的"水稻通用叶绿体表达载体":pBAC823-NdeI-trnA-ApaI-XhoI-Prrn-AgeI-SalI-GFP-KpnI-SmaI-PacI-tpsba--HindIII-Prrn-EPSP-tpsba-EcoRI-trnI-NotI-pBAC823,并将其命名为pBAC8234。酶切试验结果证明,载体pBAC8234上GFP基因两侧设计的酶切位点为单克隆酶切位点,可用于后续实验。     

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.     

鸡γ-干扰素与新城疫病毒F蛋白抗原表位融合基因的构建与表达

[目的]研究鸡γ-干扰素（ChIFN-γ）的免疫佐剂功能,构建ChIFN-γ与新城疫F蛋白抗原表位（NDV F2-3）的融合基因,并进行原核表达。[方法]以头尾相连的方式构建鸡γ-干扰素基因与NDVF2-3的pET-32a重组质粒,经PCR扩增、双酶切和测序鉴定后,重组子在E.coliBL21细胞进行IPTG诱导融合蛋白表达,采用SDS-PAGE和Western blot方法分别检测表达的产物。[结果]获得了726 bpChIFN-γ与NDVF2-3融合基因,经原核表达,融合蛋白分子量约为35 000,能与相应的抗体结合。[结论]ChIFN-γ与NDVF2-3串联基因在原核细胞中能有效表达,且融合蛋白具有一定的免疫活性。