家鸡2个Dmrt基因的序列分析

Dmrt基因编码的蛋白质是一个具DNA结合能力的转录调控因子,其DM结构域在不同进化类型的生物中具有相当的保守性.通过简并PCR克隆技术,扩增和克隆了家鸡基因组中的DM结构域,经序列分析,获得了两个具有不同DM序列的克隆,分别命名为GgDmrt2和GgDmrt5.联机与GenBank中不同进化地位动物的Dmrt基因进行聚类分析,结果显示,具有DM结构域的基因家族在两栖类、爬行类、鸟类中高度保守,并且存在着许多成员基因.     

Identification of Chinese alligators (Alligator sinensis) meat by diagnostic PCR of the mitochondrial cytochrome b gene

With the commercial farming and exploitation of Chinese alligators (Alligator sinensis), illegal and inappropriately labeled Chinese alligator meat has appeared in markets. To prevent the illegal hunting and commerce for Chinese alligators, it will be important to develop an expedient and practical method for the identification of Chinese alligator meat. In this study, a pair of the species-specific PCR primers (Alli-M and Alli-R) was designed using sequence variations of mitochondrial DNA (mtDNA) cytochrome b gene between Chinese alligators and other crocodilians. By the multiplex PCR of using the species-specific primers and 12S rRNA universal primers L1091 and H1478, 31 samples (27 meat samples, 4 skin samples) were identified. The result of amplification displayed that only the fresh and the cooked meat samples from the Chinese alligator could be amplified with two bands. We also present a case of identification of a crocodilian body part found in a local market using the newly developed primers. The specific primers designed in this study could be widely used for the rapid and accurate identification of not only alligator meat but also other commercial products from Chinese alligator.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

Application of the TRAP technique to lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) genotyping

Summary To demonstrate the applicability of the target region amplification polymorphism (TRAP) marker technique to lettuce genotyping, we fingerprinted 53 lettuce (Lactuca sativa L.) cultivars and six wild accessions (three from each of the two wild species, L. saligna L. and L. serriola L.). Seven hundred and sixty-nine fragments from 50 to 900 bp in length were amplified in 10 PCR reactions using 10 fixed primers in combination with four fluorescent labeled arbitrary primers. Three hundred and eighty-eight of these fragments were polymorphic among the 59 Lactuca entries and 107 fragments were polymorphic among the 53 lettuce cultivars and the six wild accessions; 251 fragments were present only in the wild species. These markers not only discriminated all cultivars, but also revealed the evolutionary relationship among the three species: L. sativa, the cultivated species, is more closely related to L. serriola than to L. saligna. Cluster analysis grouped the cultivars by horticultural types with a few exceptions. These results are consistent with previous findings using RFLP, AFLP, and SAMPL markers. The TRAP markers revealed significant differences in genetic variability among horticultural types, measured by the average genetic similarity among the cultivars of the same type. Within the sample set, the leaf type and butterhead types possessed relatively high genetic variability, the iceberg types had moderate variability and the romaine types had the lowest variability. The genetic behavior of TRAP markers was assessed with a mapping population of 45 recombinant inbred lines (RILs) derived from an interspecific cross between L. serriola and L. sativa. Almost all the markers segregated in the expected 1:1 Mendelian ratio and are being incorporated into the existing lettuce linkage maps. Our results indicate that the TRAP markers can provide a powerful technique for fingerprinting lettuce cultivars. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (～5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype‐specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species‐specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.     

不同引物RT-PCR方法检测猪繁殖与呼吸综合征病毒的比较研究

用3对分别针对猪繁殖与呼吸综合征病毒(PRRSV)的ORF7、ORF5和ORF5的PCR引物N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2进行RT-PCR,检测PRRSV,从其敏感性、特异性和临床样品检出率等方面进行比较,在此基础上进一步建立一步法RT-PCR检测方法。结果显示:3对引物对PRRSV均有很高的特异性;应用N1/N2引物病毒最低检测量为7.9 TC ID50,而AdGP5.1/AdGP5.2引物和RFLP5.1/RFLP5.2引物PCR最低检测量为79 TC ID50;运用N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物分别进行RT-PCR扩增检测临床样品,PRRSV检出率分别为28/48、27/48和25/48,且用AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物检测的阳性样品,用N1/N2引物检测也都呈阳性。运用N1/N2引物,通过一步法RT-PCR成功地从PRRSV S1株中扩增出374 bp的目的基因片段。结果表明,用N1/N2引物扩增PRRSV目的基因,其敏感性和临床样品检出率更高,更适合临床样品PRRSV的检测。     

利用差异显示技术克隆小麦抗白粉病相关基因的研究

对小麦—簇毛麦抗病易位系进行差异显示分析,以期获得小麦抗白粉病基因的分子克隆。根据已克隆植物抗病基因的保守域,设计了简并引物,与锚定引物组合,对抗病诱导的小麦抗白粉病易位系进行了差异显示分析,共获得10个差异片段,其中两个片段R3-1和8C1.3-6Northern杂交为阳性。将这两个片段克隆后进行了测序,序列分析结果为两个新序列,GenBank登录号分别为AF498271和AF498272。R3-1没有发现同源性较高的植物基因序列,发现8C1.3-6与Sphenostylisstenocarpa 类几丁质酶基因有同源性。     

斑茅NBS-LRR类抗病基因同源序列的克隆与分析

根据已知NBS-LRR抗病基因[含有核苷酸结合位点(NBS)和富亮氨酸重复(LRR)的胞内受体蛋白基因1NBS结构域蛋白质的保守序列,设计简并引物,对斑茅基因组进行体外扩增,获得了对应区段的DNA片段,回收、克隆这些特异片段,测序分析,共获得8个片段序列.序列分析发现其中7个编号分别为RGA-Q1、RGA-Q2、RGA-Q3、RGA-Q4、RGA-Q5、RGA-Q6和RGA-Q7的片段推导的氨基酸序列均具有典型的NBS结构域,即Ploop(GGVGKTr)、Kinase-2a(VLDDVW)、Kinase-3a(GSR/KILVTTR)及疏水结构域HD(hydrophobic domain).它们在NCBI上的登录号为EU685828、EU685829、EU685830、EU685831、EU685832、EU685833和EU685834.这些抗病基因同源片段(RGA)与已经克隆的N、L6、RPS2和胧等11个抗病基因在氨基酸水平上的同源性为2.3%～39.8%.可进一步用作斑茅抗病候选基因的分子筛选及遗传图谱的构建.     

从混合线虫样品和植物组织中直接检测香蕉穿孔线虫的ITS-PCR方法

 【目的】建立直接从多种线虫混合样品以及香蕉和红掌根组织中检测鉴定香蕉穿孔线虫的PCR方法。【方法】使用Primer Premier 5.0在香蕉穿孔线虫的ITS区设计1对特异性引物,运用PCR技术对目的线虫DNA进行特异性检测。【结果】通过对17种24个种群线虫的检测表明,所设计的引物只能从香蕉穿孔线虫种群中特异扩增出rDNA-ITS片段,产物大小为518 bp。利用该特异引物以及建立的DNA提取方法和PCR体系,可以直接从香蕉穿孔线虫与短体线虫、螺旋线虫、肾状线虫、根结线虫、茎线虫、矮化线虫、纽带线虫、丝尾垫刃线虫、滑刃线虫和小杆线虫混合样品中特异扩增出目的线虫的rDNA-ITS片段,并可以分别从混合有不少于3条香蕉穿孔线虫的2 cm香蕉或红掌根组织（约0.1 g）中特异检测出目的线虫的DNA片段。【结论】本研究设计的特异性引物以及建立的DNA提取方法和PCR体系可直接从多种线虫混合的样品以及香蕉和红掌根组织中快速检测鉴定出香蕉穿孔线虫。     

西藏自治区那曲县草地退化空间差异分析

在分析生态特征区域差异基础上,通过计算草地退化的相对动态度和退化草地的图形斑块特征指标,探索西藏自治区那曲县草地退化的空间分异规律,并提出相应的生态建设和经济发展建议。