白菜型油菜DFR基因的克隆与序列分析

By using RACE (rapid amplification ofcDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. oleifera DC.. The gene, designated BcDFR here, is 1 722bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1311bp in length with a 1 158bp ORF as well as a 25bp 5‘ UTR and a 128bp 3‘ UTR. The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive homologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyses such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a quite potentially biofunctional gene. Its cloning enables us to further dissect the possible relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars.     

低吸油面包糠的制备工艺研究

[目的]制备低吸油率面包糠,以有效利用挂面面头,并为改进油炸食品质量提供新源料。[方法]以挂面面头为主要原料,以吸油率为考察指标,在单因素试验的基础上,进行4因素3水平正交试验,优化低吸油率面包糠的制备工艺,并比较分析了不同原料制备的面包糠的感官、吸油率、微观特性。[结果]低吸油率面包糠的适宜制备工艺条件为发酵温度28℃,发酵时间2.5 h,烘烤时间30 min,烘烤温度190℃,此条件下面包糠的吸油率为20.41%。面包糠的显微结构致密度与其吸油率高低密切相关。[结论]以挂面面头和面粉为主要原料制备低吸油率面包糠不仅拓展了挂面面头的应用市场,而且为油炸食品提供了新型的涂膜材料。     

兰州大尾羊心脏型脂肪酸结合蛋白（H-FABP） 基因克隆及其同源性比较

【目的】克隆兰州大尾羊心脏型肪酸结合蛋白（H-FABP）基因全长cDNA序列,为研究绵羊H-FABP生物学作用和生产应用提供理论依据。【方法】根据已知哺乳动物H-FABP基因 cDNA 序列,设计5''和3''特异引物,运用cDNA 末端快速扩增（RACE）技术获得兰州大尾羊H-FABP基因全长 cDNA 序列。【结果】 扩增获得兰州大尾羊5''端425 bp、3''端231 bp片段和 177 bp中间片段,拼接获得748 bp兰州大尾羊H-FABP基因全长cDNA 序列（GenBank登录号：JQ780322）。 兰州大尾羊H-FABP基因ORF长 402 bp,编码 133 个氨基酸。核苷酸序列分析显示兰州大尾羊H-FABP基因序列与大多数哺乳动物相似,但其第66位发生的碱基转换（T←→G）引起所编码的第22位天门冬氨酸（N）不同于其它所有物种的赖氨酸（K）。构建的基因进化树分析结果显示兰州大尾羊与山羊亲缘关系最近。预测兰州大尾羊H-FABP蛋白质的空间结构与山羊和牛H-FABP类似,由2个α螺旋和10个反向平行的β折叠组成,10 个折叠片围成一个桶状结构,疏水性残基位于桶内,用于结合脂肪酸。【结论】克隆了兰州大尾羊H-FABP基因,为进一步研究该基因的功能奠定了基础。     

应用气提原油稳定工艺提高轻烃回收率

对中原油田采油一厂文一联，文三联负压原油稳定装置存在的问题进行分析，利用ASPEN－PLUS软件对原油稳定塔内的平衡汽化，平衡冷凝过程进行模拟计算，确定量佳工艺参数，在此基础上对负压原油稳定装置进行了改造，介绍了履行后的气提原油稳定轻烃回收技术的作用的效益。     

食蚊鱼CYP19a基因的克隆与序列分析

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238~1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。     

Molecular Cloning and Characterization of a Novel Gene Involved in Fatty Acid Synthesis in Brassica napus L.

Based on the sequence of a novel expressed sequence tag (EST), the full-length cDNA of 1 017 nucleotides was cloned from Brassica napus cv. Xiangyou 15 through rapid amplification of cDNA ends (RACE). The gene was designated as Bnhol34 (HQ585980), encoding a protein of 338 amino acids. BLAST analysis showed no high degree of sequence identity to any known gene. The calculated molecular weight of the Bnhol34 protein was 36.23 kDa, and the theoretical isoelectric point was 8.74. The Bnhol34 was also cloned from a high oleic acid mutant 854-1 through homologous cloning. There was no difference between the two Bnhol34 genes. Bnhol34 was localized in a tissue-specific manner in B. napus, and its expression level was about eight-fold greater in Xiangyou 15 seeds than in 854-1. The promoter region sequences of Bnhol34 were then isolated from Xiangyou 15 and 854-1, and a 93-bp deletion was found to occur in the Bnhol34 promoter region of 854-1. Three abscisic acid-responsive cis-elements (ABRE) were identified in the promoter region of Xiangyou 15. Real-time PCR analyses revealed that exogenous abscisic acid increased Bnhol34 expression by about four-fold in Xiangyou 15 seeds, yet did not change Bnhol34 expression in 854-1. It appeared that Bnhol34 might be abscisic acid insensitive in 854-1.     

桥头跳车原因与防治措施

本文从路在沉降、施工方法和结构体系上对桥头跳车病害进行原因分析,提出减少病害防治措施。     

Relationship between three novel SNPs of BRCA1 and canine mammary tumors

The BRCA1 gene plays an important role in the development of human breast cancer, and recent research indicated that genetic variations of BRCA1 are also related to canine mammary tumors (CMTs). Here, using rapid amplification of cDNA ends (RACE), we cloned the 5′- and 3′-UTRs of BRCA1. By direct sequencing of the flanking sequences of the 5′- and 3′-UTRs of BRCA1, three previously unreported single-nucleotide polymorphisms (SNPs) were identified, two (−1228T >C, −1173C >T) in the putative promoter regions and one non-synonymous SNP (63449G >A) in exon 23. Compared with 16 normal samples, the sequences from 34 CMTs suggested that SNP (−1173C >T) was associated with the development of CMTs (odds ratio (OR)=2.57, 95% confidence interval (CI): 1.07–6.15).     

黄颡鱼DMRT1基因cDNA全长克隆及其表达分析

利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆了黄颡鱼DMRT1基因cDNA全长序列,并利用实时荧光定量RT-PCR技术对该基因在黄颡鱼成体不同组织及不同发育阶段的表达情况进行研究。结果表明,黄颡鱼DMRT1基因cDNA序列全长1 381bp,其中5′端非翻译区30bp,3′端非翻译区454bp[不包括poly(A)],开放阅读框885bp,编码295个氨基酸。氨基酸序列同源性分析表明,黄颡鱼DMRT1基因与革胡子鲶同源性最高(为81%),与黑鲷、虹鳟、斑马鱼、青鳉的同源性分别为60%、59%、64%和52%,与小鼠、人的同源性较低,分别为42%和44%。实时荧光定量RT-PCR分析表明:DMRT1基因在黄颡鱼胚胎发育阶段及胚后发育的1～51d仔鱼均有表达,且在胚后发育的第31天表达量最高;在成体,只在雄性精巢中特异性表达,其他组织均无表达,且性腺发育阶段的Ⅳ期精巢表达量最高,表明该基因可能在黄颡鱼雄性性腺的形成或功能维持上具有重要作用。