隐孢子虫不同基因型P23基因的克隆及序列比较

为克隆隐孢子虫不同基因型子孢子表面抗原P23基因,比较其序列差异,提取上海地区分离的隐孢子虫鼠基因型(Cryptosporidiummouse genotype)、隐孢子虫兔基因型(Cryptosporidiumrabbit geno-type)、隐孢子虫猪基因型Ⅱ(Cryptosporidiumpig genotypeⅡ)总RNA,经RT-PCR扩增P23基因,克隆到pMD18-T载体中,进行序列测定,并与GenBank上下载的微小隐孢子虫(Cryptosporidium parvum)序列进行同源性比对。结果显示,从隐孢子虫3个基因型中均扩增出了P23基因。与微小隐孢子虫P23基因核苷酸序列比较,隐孢子虫鼠基因型、兔基因型、猪基因型ⅡP23基因同源性分别为97.6%、97.3%、97.3%,氨基酸序列同源性分别为97.3%、97.3%和96.4%。获得了隐孢子虫鼠基因型、兔基因型、猪基因型Ⅱ子孢子表面抗原P23基因。     

橡胶树PGAM基因的克隆及表达特性分析

从橡胶树中克隆并获得磷酸甘油酸变位酶基因（HbPGAM），该基因的cDNA序列全长1 559 bp，包含长度为1 260 bp的完整开放阅读框，编码一个由419个氨基酸残基组成的蛋白，氨基酸同源性分析发现，该蛋白含有保守的磷酸变构酶组氨酸磷酸酶结构域，属于磷酸甘油酸变位酶。生物信息学分析显示，HbPGAM蛋白无导肽酶切位点，不具有跨膜结构域且为亲水蛋白，该蛋白可能在细胞质中发挥作用。实时荧光定量PCR分析表明，HbPGAM基因在分析的7种组织中均表达，但在胶乳（产胶细胞乳管的细胞质）中的表达水平最高，且该基因在胶乳中的表达受割胶影响，推测其参与橡胶树胶乳再生过程。此外，HbPGAM基因表达受部分植物激素如乙烯利ET、植物生长素2,4-D及水杨酸SA调控，但调控不明显。结果说明，胶乳再生过程中HbPGAM对胶乳糖酵解起到一定的调控作用，为全面了解橡胶树PGAM家族奠定理论基础。     

Cloning and analysis of cross-intron genomic gene encoding ACO in Paeonia suffruticosa Andr.

The last step of ethylene biosynthesis in higher plants is to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). In our investigation, a cross-introns genomic DNA gene encoding ACC oxidase, PsgACO, was isolated from Paeonia suffruticosa. It revealed that PsgACO (FJ855434) has 1281 bases, containing four exons and three introns, encoding 312 amino acids. The four exons stretched from 1 to 105, 217 to 434, 592 to 925 and 1000 to 1281 bases. A splicing junction sequence of all the exon-introns conformed to the GT-AG rule in the cross-intron genomic DNA sequence of the ACO gene. PsgACO showed high homology to many characterized ACC oxidases both at the nucleic acid and amino acid levels. As well, twelve amino acid residues were conserved among many ACOs from other species. A phylogenetic tree analysis indicated that the amino acid of ACOs is quite conserved among the different eudicots. The phylogenetic tree showed that both the tree peony and herbaceous peony are quite isolated taxa. Bioinformatic analysis showed that the molecular weight of ACO is 35.29 kD, with a theoretical pI of 5.25. It is a non-secrete, stable hydrophilic protein, located in the cytoplasm.     

日本大耳白兔TLR3部分cDNA克隆及mRNA在组织中的分布

采用RT—PCR技术从日本大耳白兔的脾脏组织克隆出兔的Toll样受体基因3（命名为。TLR3），并对其mRNA在兔的17种组织中的分布情况进行了检测。结果显示，RTLR3核苷酸序列与GenBank中登载的穴兔（Oryctolagus cuniculus）TLR3序列（登录号：NM_001082219）相似性为100％；兔的胸腺、脾脏、睾丸等14种组织中检测出TLR3 mRNA的转录产物，而在骨骼、胃和皮肤中未能发现。推导的氨基酸序列同源性比对表明，兔TLR3与人、野猪、牛、猫、褐鼠等动物的同源性最高，为80％-85％；与原鸡同源性次之，为61％；与草鱼、绿河豚等同源性在45％～48％之间；系统进化树分析表明，兔TLR3与马的亲缘关系最接近，与猪、人／绵羊、牛、猩猩／猫的亲缘关系依次渐远，与鼠的亲缘关系最远。可见，TLR3在不同种属动物及不同组织中所起的作用具有生物多样性。     

Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by γ-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion (InDel) markers C1-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene D10 within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the D10 allele in gsor23 revealed that the base cytosine (C) at the 404^th position in the coding region was deleted, which would cause frameshift mutation after the 134^th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 (CCD8), a key enzyme involved in the biosynthesis of the new plant hormone strigolactones (SLs). After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene D10 was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.     

黄海黄杆菌YS-9412-130低温碱性蛋白酶的基因克隆和序列测定

黄海黄杆菌YS－9412－130具有稳定的分泌低温碱性蛋白酶的能力。将其染色体DNA用Sau3AⅠ部分酶切后，低熔点琼脂糖回收2－10Kb的DNA片段，用Klenow大片段酶半补齐，与用SalⅠ酶切且半补齐的质粒pUC19连续后，转化E.Coli.JM109，构建基因文库。且酪蛋白平板法和酶联免疫吸附（ELISA）的活性筛选方法从基因文库中筛选到一株产海洋低温碱性酶的阳性克隆（命名为pHH1）。序列测定分析表明，此重组质粒包含有长度为768bp低温碱性蛋白酶基因的完整的开放读码框架（ORF）和上游基因调控序列。此片段编码由256个氨基酸组成的酶，计算分子量为83000Dal。通过Southern杂交证实了此片段来自于黄海黄杆菌YS－9412－130基因组DNA。     

鳙小卫星DNA的克隆

鳙基因组ＤＮＡ 经内切酶Ｓａｕ３ＡⅠ完全酶切后，与Ｂａｍ ＨⅠ－ ｐＵＣ１８ 质粒连接，转化ＤＨ５α后，获得９３３ 个重组体。用人源小卫星探针３３ ．１５ 对重组体进行原位杂交筛选，获得１４ 个阳性克隆，取其中的１ 个阳性克隆（ 即小卫星ｐＢＣ１７４） 作探针，与５ 尾鳙和４ 尾鲢基因组ＤＮＡ 杂交，能产生具有高度变异性的、个体特异性的ＤＮＡ 指纹图，表明克隆的鳙小卫星ｐＢＣ１７４ 可用于鱼类ＤＮＡ 指纹分析。     

BBTV两个株系DNA组分1的克隆及序列分析

 对在生物学特性特别是寄主范围上存在明显差异的NSP株系和NS株系的代表分离物(广州天河分离物和高州分离物)的DNA组分1进行了克隆和序列分析,结果表明:2个代表分离物的DNA组分1全序列、ORF及其编码的氨基酸序列的变异率分别为3.2%、3.1%和2.8%,从而进一步确证了可以用寄主范围来作上述2个株系的鉴定。此外,这2个株系的DNA组分1、ORF及其编码的氨基酸序列分别与肖火根等报道的中国(广东)分离物、以及亚洲组和南太平洋组各分离物进行比较,结果表明:NS株系与肖火根等报道的中国(广东)分离物的亲缘关系很密切;这2个株系与亚洲组各分离物的差异均较小,而与南太平洋组各分离物的差异均较大,它们应属亚洲组。     

可供利用的植物基因克隆技术

植物基因工程研究的首要目标是把特定的靶基因从植物基因组或其它生物基因组中分离出来，只要把基因分离并克隆，才有可能了解其遗传信息，然后进行分子操作。本文主树常用图谱克隆基因技术及克隆基因；转座子标鉴克隆基因技术及克隆基因；文库克隆基因技术；基于PCR技术克隆基因；人工合成基因技术进行了综述。