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61.
62.
为进一步分析H7N2禽流感病毒(AIV)分离株血凝素(HA)基因的特性,参照已发表序列设计了1对引物,采用RT-PCR获得了1条约1.7 kb的DNA片段,测序后进行了同源性比较、HA基因系统发育进化树分析和氨基酸编码分析.结果表明,所测的2个分离株的HA基因全长1 664 bp,编码除信号肽以外的HA蛋白的全部544个氨基酸,其中包括HA1的323个氨基酸,HA2的221个氨基酸.2个分离株HA基因核苷酸序列的同源性为99.4%;与GenBank中AIV标准株A/Afri.Star./Eng-Q/983/79(H7N1)的同源性最高,分别为99.4%和99.0%;与美国A/Chicken/NewYork/13142-5/94(H7N2)株同源性很低(仅65.0%),而与以色列、意大利H7N2 AIV的同源性较高(为96%~97%);2个分离株在HA基因进化树中均处于H7亚型AIV的欧亚群系分支内.推导氨基酸的序列分析表明,其HA蛋白裂解位点的氨基酸序列为-GR-GLF-,仅包含1个碱性氨基酸(R-)残基,符合低致病力AIV的基因特征. 相似文献
63.
《园艺学报》2003,19(5):622-626
AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue. 相似文献
64.
65.
Willem A. Man in 't Veld Arthur W.A.M. de Cock Elena Ilieva C. André Lévesque 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(1):51-62
Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2
83, Ldh-2
104, Idh-1
108, Idh-1
112, and Idh-2
98), whereas six different alleles were observed at four isozyme loci (Ldh-2
85, Ldh-2
100, Ldh-2
114, Idh-1
100, Idh-2
100, and Mdh-2
111) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow. 相似文献
66.
苏云金芽孢杆菌cry基因在大肠杆菌中表达产物和生物活性 总被引:4,自引:0,他引:4
研究了几种Bt cry基因于大肠杆菌(Escherichia coli)中表达产物在pH 10.0的50mmol/L碳酸钠和20mmol/L乙醇胺溶解液中的溶解性 ,发现同样的Cry蛋白在碳酸钠中的溶解度大于乙醇胺。通过胰蛋白酶消化 ,明确Cry1Ca7、Cry1Ia8酶解产物为 38kD多肽 ;Cry1Ie1、Cry1Cb2、Cry2Ab4酶解产物为 41kD多肽 ;Cry1Ac酶解产物为60kD多肽。采用FPLC层析方法对 6种原毒素及其酶解后得到的毒素多肽进行了分离纯化 ,比较了原毒素和毒素的杀虫活性的差异。其结果表明 ,Cry1Ac的原毒素和毒素对棉铃虫初孵幼虫的校正死亡率均为 100% ,Cry2Ab4的原毒素的毒力高于其酶解毒素。 相似文献
67.
苹果柱型基因Co的一个AFLP 标记的SCAR转换 总被引:15,自引:4,他引:15
将苹果柱型基因的一个AFLP 标记成功地转换成了简单实用的SCAR 标记。首先对AFLP 标记片段进行序列测定, 然后根据序列特点设计了两对特异引物CoA1/ CoA2 和CoA1/ CoA3 , 每条引物长20 bp。PCR 结果表明CoA1/ CoA2 可以扩增出216 bp 和148 bp 两条带, 其中216 bp 的为柱型性状的特征带; CoA1/CoA3 可以扩增出273 bp 和205 bp 的两条带, 其中273 bp 的为柱型性状的特征带。两对引物在杂交后代中扩增出的特征带与柱型性状的分离重组率都很低(CoA1/ CoA2 为6. 3 % ±2. 5 %; CoA1/ CoA3 为7. 3 % ±2. 6 %) , 所以它们都可以作为该SCAR 标记的特异引物所用。 相似文献
68.
69.
梨自交不亲和强度不同品种花柱S糖蛋白含量的差异 总被引:12,自引:2,他引:12
提取梨( Pyrus) 自交不亲和强度不同品种花柱可溶性蛋白质, 应用等电聚焦电泳法在凝胶板上分离出S 糖蛋白, 用图像解析法进行定量。结果表明, 花柱总S 糖蛋白含量因品种而异, 自交不亲和强、弱及自交亲和的品种每1 1048577;g 花柱可溶性蛋白中含总S 糖蛋白分别为1048578;1. 3 ng 、0. 7 ~ 0. 9 ng、 0. 7 ng 。不同的S 基因所表达的S 糖蛋白量差异显著, 即使是同一S 基因所对应的S 糖蛋白量也因品种而异。 相似文献
70.
ZHANG Yu-xia YU Lun-yin LIU Ming-qiu ZHANG Zheng-bin TANG Zhi-jiao XIA Dong WANG Ming 《园艺学报》2002,18(1):32-35
AIM:To investigate the protein expression of cyclin D2 and p16 in proliferation and differentiation of cultured cardiac myocytes.METHODS:One-day-old Sparague-Dawley rats were used. Cardiac myocytes(CM) were collected by a trypsin-dispersal method and cultured. Cell growth line and fluorescence activated cell sorting (FACS) were used to investigate the proliferation of CM. Ultra-thin sections were made to observe the ultrastructure of CM under transmission electron microscope. The expression of cyclin D2 and p16 in CM were measured using immunocytochemistry and image analysis.RESULTS:①Results of cell growth line and FACS analysis showed that cultured CM could proliferate in the first 3 cultured days, but the ability decreased quickly, concomitant with differentiation. CM was obseved quiescent in cell cycle three days later. The ultrastructure of CM showed the large amount of myofilaments and mitochondrion. ②The protein expression of cyclin D2 in 3,4,5 day CM group was 0.89 times(P<0.05),0.80 times (P<0.05) and 0.56 times (P<0.01) of that in 1 day group, respectively. The expression of p16 in CM was increased during the culture process, 2,3,4,5 day group were 1.63 times, 1.72 times, 1.99 times and 2.84 times (P<0.01) of that in 1 day group, respectively.CONCLUSION:Cultured neonatal rat cardiac myocytes could proliferate during the first 3 days after incubation, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D2 and p16 play the key roles in CM postnatal development. Downregulation of cyclin D2 and upregulation of p16 may induce CM differentiation. 相似文献