毛白杨纤维素合酶基因PtCesA_4的克隆、表达及单核苷酸多态性分析

组合利用生物信息学和RT-PCR方法,首次从毛白杨未成熟木质部cDNA中分离出PtCesA4cDNA全长,并进行测序和序列分析,结果表明克隆的毛白杨PtCesA4 cDNA片段总长为3 757 bp,基因内部含有完整的开放阅读框架,大小为3 129 bp,可编码长度为1 042个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列与拟南芥AtCesA4、水稻OsCesA,和火炬松PtCesA2的蛋白质氨基酸序列同源性分别为80.3%,78.9%和75.6%.组织特异性Realtime-PCR结果显示,PtCesA4基因在杨树根、茎、叶片和顶端分生组织中均有表达,但其表达模式却不同:PtCesA4在成熟叶片、未成熟木质部和成熟木质部中表达丰度最高,在根部和顶端分生组织表达丰度中等,在树皮和韧皮部有少量表达,在形成层中表达丰度最低.在此基础上,组合利用MEGA3.1和DnaSP4.50.4软件对毛白杨40株基因型个体的PtCesA4序列进行比对和分析,检测到153个单核苷酸多态性(sinsh nueleotide polymorphism,SNP)位点,SNP频率为1/35 bp,多样性指数π/0.005 02.其中51个是常见SNPs,102个为罕见SNPs.在这些SNPs中,有118个属于转换,35个属于颠换.在外显子区域,共检测到69个SNP位点,其中59个为同义突变,10个为错义突变.对PtCesA4基因内SNPs进行的连锁不平衡分析结果显示,随着核苷酸序列长度的增加,SNPs的连锁不平衡在基因内部迅速衰退,因此,在毛白杨中,基于PtCesA4基因的连锁不平衡作图是可行的,而基于整个杨树基因组的连锁不平衡作图是不可行的.也是不必要的.研究结果为毛白杨PtCesA4基因的连锁不平衡作图及其基因辅助毛白杨木材纤维性状的分子育种提供了理论依据.     

小叶杨GA20氧化酶基因的克隆、表达及单核苷酸多态性分析

利用RT-PCR方法,首次从小叶杨未成熟木质部cDNA中分离出PsGA20Ox cDNA全长及其基因组DNA,并进行测序和序列分析.结果表明:克隆的小叶杨PsGA20Ox cDNA片段总长为1 403 bp,基因内部含有完整的开放阅读框架,大小为1 155 bp,编码区可编码长度为385个从残基,所推导的蛋白质氨基酸序列与拟南芥和水稻GA20Ox蛋白的同源性分别为66.0%和57.0%.组织特异性RT-PCR结果显示,PsGA20Ox基因在杨树茎、叶片和顶端分生组织中均有表达,但其表达模式却不同:PsGA20Ox在成熟木质部中表达丰度最高,在未成熟木质部和嫩叶中表达丰度较高,在顶端分生组织中有少量表达,在形成层组织中表达丰度极低.在此基础上,组合利用MEGA4.0和DnaSP4.50.4软件对小叶杨36株基因型个体的PsGA20Ox基因组DNA序列进行比对和分析,检测到49个单核苷酸多态性(SNP)位点,频率为1/35 bp.其中,15个是常见SNPs,34个为罕见SNPs.在这些SNPs中,37个属于转换,12个属于颠换.在外显子区域,共检测到26个SNP位点,其中23个为同义突变,3个为错义突变.对PsGA20Ox基因内SNPs进行的连锁不平衡分析表明,随着核苷酸序列长度的增加,SNPs的连锁不平衡在基因内部就迅速衰退,因此,在小叶杨中,基于候选基因的连锁不平衡作图是可行的,而基于整个杨树基因组的连锁不平衡作图是不可行的,也是不必要的.     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)     

Characterization of a New Barley Mild Mosaic Virus Pathotype in France

In March 2002 in a French field, severe mosaic symptoms appeared on plants of the barley cultivar Tokyo with the rym5 locus controlling resistance to all European strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV). Electron microscopic examination revealed that the disease symptoms were associated with the presence of flexuous particles which resemble bymoviruses. From these observations and after enzyme-linked immunosorbent assay analysis it was first determined that the plants could be infected by BaMMV and BaYMV. Mechanical transmission of these viruses to the barley cultivar Magie susceptible to both viruses was only possible for BaMMV. This new pathotype (BaMMV-Sil) from Sillery (Marne Department, 51, France), in contrast to another mechanically transmitted French BaMMV isolate (BaMMV-MF), could be transmitted mechanically to two barley cultivars (Tokyo, Misato Golden), Arachis hypogaea, Datura stramonium and Lactuca sativa. BaMMV-Sil was indistinguishable from three BaMMV isolates from Germany (G), Japan (Ka1) and France (PF) by monoclonal antibodies in ELISA while the Japanese isolate (Na1) and BaMMV-MF were distinguishable from all. The sequence of the 3-terminal region of BaMMV-Sil RNA1 was determined. Comparison with previously published sequence data of capsid proteins indicated that BaMMV-Sil was closely related to BaMMV-Ka1, BaMMV-G and another German isolate (BaMMV-ASL1). Resistance-breaking BaMMV strains able to infect cultivars carrying the rym5 locus have also been described in Japan (BaMMV-Na1) and Korea (BaMMV-Kor). No specific amino acid differences were detected between the capsid proteins of BaMMV-Sil, BaMMV-Na1, BaMMV-Kor and those BaMMV isolates that do not overcome the rym5 resistance gene. These results indicate that BaMMV-Sil is a new pathotype of BaMMV in France and suggests that the capsid protein is not the determining factor of the pathogenicity towards the resistance gene rym5.     

Cloning and Sequencing of cnf1 from Escherichia coli Incriminated in Mink and Bovine Colibacillosis

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.     

Detection of Fowl Adenovirus Associated With Hydropericardium Hepatitis Syndrome by a Polymerase Chain Reaction

Hydropericardium hepatitis syndrome HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction PCR) was developed to detect the fowl adenovirus FAV) associated with HHS. The virus from infectedl ivers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.     

IL-1B-511 single nucleotide polymorphism and gastric atrophy in high prevalence regions of gastric cancer in China

AIM: To study the relationship between IL-1B-511 single nucleotide polymorphism,H.pylori infection,and gastric atrophy in high prevalent (Shanxi) region in China.METHODS: Five hundred healthy volunteers from Shanxi Province of China were recruited in this study,which were divided into five subgroups according to age,namely age 20-29,30-39,40-49,50-59 and >60 years,respectively.Genomic DNA was extracted from peripheral blood.IL-1B-511 single nucleotide polymorphism was analyzed by PCR-RFLP.Serum pepsinogen was used as a biomarker of gastric atrophy.Serum pepsinogen Ⅰ (PGⅠ),pepsinogen Ⅱ (PGⅡ) and anti-H.pylori IgG were determined by an ELISA assay.RESULTS: The mean serum PGⅠ concentration and ratio of PGⅠ /PGⅡ decreased gradually with increasing age,and were lower in subjects with IL-1B-511 T/T genotype and H.pylori infection than those without H.pylori infection in each subgroup,respectively (All P<0.05).Multiple linear regression showed that IL-1B-511 T/T genotype,age and Hp infection were significantly associated with gastric atrophy (P<0.05,P<0.05 and P<0.01,respectively).CONCLUSION: Gastric atrophy is closely correlated with IL-1B-511 T/T genotype,age,H.pylori infection in high prevalence region of gastric cancer in China.The risk of gastric atrophy is significantly increased for the IL-1B-511 T/T genotype with Hp infection.     

单核苷酸多态性的研究进展

染色体的某个位点上存在单个碱基的变化 ,称为单核苷酸多态性 ,即SNP。SNP是继限制性片段长度多态性和微卫星多态性以后 ,近年来出现的第三代遗传标记 ,广泛用于基因定位、克隆和遗传多样性研究。本文综合介绍了单核苷酸多态性的遗传特性、功能、研究意义及其在畜牧业中的应用     

猪伪狂病病毒粤A株gE基因的克隆与序列分析

参考GenBank中伪狂犬病病毒(PRV)gE基因的序列设计了1对引物,对PRV粤A株gE基因进行了PCR扩增,PCR产物克隆到PMD18-T载体.对重组质粒进行限制性内切酶分析和基因测序,证实了克隆片段的可靠性,并与2株不同来源的毒株进行了同源性分析和比较.     

超高压处理对南美白对虾在冷藏过程中贮藏特性的影响

该文以不同的超高压条件(200,400,600,700MPa)对南美白对虾进行处理,通过考察与比较不同压力处理组冷藏过程中感官、微生物以及各化学指标的变化情况,揭示超高压技术对南美白对虾冷藏特性的影响。结果表明:超高压处理可以有效地杀灭南美白对虾中绝大多数微生物,抑制贮藏过程中挥发性盐基氮的积累,延缓pH值的变化,从而延长南美白对虾的货架期,且处理压力越高延长效果越显著;超高压处理会给鲜虾带来不同程度上的煮熟虾的风味;400MPa和600MPa处理使虾在冷藏过程的黑变提前,而700MPa处理可以完全抑制南美白对虾黑变现象的发生;超高压能够改变腺苷三磷酸(ATP)及其代谢产物的代谢情况,但不影响腺苷酸(AMP)的代谢途径。因此超高压处理可以改善南美白对虾在冷藏过程中的品质,对南美白对虾冷藏具有潜在的应用价值。