番茄抗病基因Cf9的3′UTR区含有内含子序列

从番茄（ＬｙｃｏｐｅｒｓｉｃｏｎｅｓｃｕｌｅｎｔｕｍＭｉｌ．）抗病品种“中杂９号”基因组ＤＮＡ中扩增并克隆了番茄抗叶霉病基因Ｃｆ９。序列分析结果表明,该基因全长２７５１ｂｐ,含有一个编码８６３个氨基酸的开放阅读框架。在该基因的３′ＵＴＲ区发现了一段未曾报道的内含子序列,长１１５ｂｐ,它所处的位置与番茄另一个抗叶霉病基因Ｃｆ２内含子的位置相似,其５′和３′边界序列为一重复序列,ＴＣＣＡＧＧ（Ｔ）ＡＴＴＣ,并与Ｃｆ２基因内含子边界序列高度同源。与已报道的Ｃｆ９的ｃＤＮＡ序列相比,它的第３７１位的核苷酸Ｔ突变成了Ｃ,从而使其编码蛋白的ＬＲＲ区第１２１位的亮氨酸突变为脯氨酸。     

应用FISH技术鉴定一个小冰麦易位系

以生物素（ｂｉｏｔｉｎ１６ｄＵＴＰ）标记的天蓝冰草（Ａｇｒｏｐｙｒｏｎｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）Ｐ．Ｂ．＝Ｅｌｙｔｒｉｇｉａｉｎｔｅｒｍｅｄｉａ（Ｈｏｓｔ）Ｎｅｖｓｋｉ＝Ｔｈｉｎｏｐｙｒｕｍｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）ＢａｒｋｗｏｒｔｈａｎｄＤｅｗｅｙ）染色体组ＤＮＡ为探针,普通小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）“中国春”ＤＮＡ为封闭,用荧光原位杂交（ＦＩＳＨ）技术对小冰麦３３号进行检测。结果表明：在一对染色体的端部显现出绿色荧光,这说明小冰麦３３号携带外源基因的冰草染色体片段位于小麦染色体端部,而且易位的染色体片段是较小的。从ＤＮＡ水平直接证明小冰麦３３是一个冰草染色体片段易位到小麦染色体端部的易位系。     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Variation Within and Between Phytophthora Species from Rubber and Citrus Trees in China, Determined by Polymerase Chain Reaction Using RAPDs

Variation among 39 isolates of Phytophthora of six morphological species (P. citrophthora. P. parasitka, P. capsici, P. palmivora and P. meadii. from rubber and citrus trees, and P. colocasiae from taro) was studied using random amplified polymorphic DNA (RAPD) analysis. Ten randomly-chosen 10-mer primers were used. Generally, the banding patterns were similar within species and different between species, but no one primer was able to distinguish all six species from one another. Cluster analysis on pooled data from all the primers gave six groups of isolates corresponding to the six morphological species. The group corresponding to P. citrophthora was divided further into subgroups that were related to host species and geographical location. This work confirmed the existing morphological classification of Phytophthora isolates from rubber and citrus trees in tropical China and showed the validity of using RAPDs to study the taxonomy of Phytophthora.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

Horizontal transmission versus vertical inheritance of P elements in Drosophila and Scaptomyza: has the M-type subfamily spread from East Asia?

Polymerase chain reaction (PCR) screening for P elements was carried out in 77 species with a primer set highly specific for the M-type subfamily. In the course of this search M-type elements were detected in 29 species: In the melanogaster (subgroups montium and rhopaloa ) and obscura species groups of Drosophila (25 out of 71 species examined), and in the genus Scaptomyza (four out of six species). M-type elements are present in all species of the montium subgroup investigated so far (21), but occur only sporadically in other groups. Within the montium subgroup 20 species possess only incomplete copies, only one species ( D. lacteicornis ) harbours apparently full-sized elements. In contrast, outside the montium subgroup almost all species with M-type elements carry full-sized copies suggesting transpositional activity, at least in the recent past. The interior section of the full-sized M-type element of D. lacteicornis was partially sequenced (936 bp). In addition, the complete sequences of four internally deleted M-type elements of D. lacteicornis, D. rufa, D. quadraria , and D. triauraria were determined. Sequence comparisons (including sequence data from previous investigations) revealed striking discrepancies between P element phylogeny and the cladogenesis of their host species. Among several possible pathways for interspecific transfers of M-type elements, we favour the hypothesis assuming the invasion of Scaptomyza as well as the obscura group species of Drosophila via independent transmission routes originating from Asian species of the montium subgroup of Drosophila . The logical geographic scenario for these events would be East-Asia.     

Use of PCR for detection of subpatent infections of lizard malaria: implications for epizootiology

The estimated prevalence of a malaria parasite, Plasmodium mexicanum , of western fence lizards, Sceloporus occidentalis , was compared using two techniques: microscopic examination of blood smears, and nested PCR amplification of the 18S small subunit rRNA gene. Two sites in northern California, USA were investigated, one with known long-term high prevalence of the parasite (30% by blood smear scanning), and one with low prevalence (6%). The nested PCR readily detected very low-level infections (< 1 parasite per 10 000 erythrocytes); such infections are often subpatent by normal microscopic examination. False negatives (scored as not infected after scanning the blood smear, but found infected via PCR) were rare at both sites (4% at the high-prevalence site, 6% at the low-prevalence site). However, a greater proportion of infections was detected only by PCR at the low-prevalence site (50% vs. 9%). If 50% of the infections sustain very weak parasitaemia where lizards are rarely infected, this would accord with hypotheses that predict that parasites should reduce infection growth when transmission is uncommon. The study demonstrates that PCR is a powerful tool to detect very low-level malarial infections in vertebrate hosts, including those with nucleated erythrocytes.     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

AC—PCR法检测连云港海域贝类HAV

为了解连云港海域贝类甲型肝炎病毒（ＨＡＶ）污染状况,证实其在本地区甲肝传播中的媒介地位,我们应用抗体捕捉聚合酶链反应（ＡＣ／ＰＣＲ）检测市售贝类的ＨＡＶ,结果报告如下：材料和方法１贝类样品于１９９６年春、秋二季采集新浦区、连云区和赣榆县等地水产品市场...     

肾综合征出血热病毒基因检测及分型的研究

根据流行于我国的两型ＨＦＲＳＶ代表株汉滩型７６１１８株及汉城型Ｒ２２株Ｍ节段的核酸序列,设计两型共同引物,建立了逆转录－聚合酶链反应（ＲＴＰＣＲ）方法,检测３９株从不同地区、不同宿主分离的ＨＦＲＳＶ感染鼠脑及细胞培养物;同时还建立了捕捉ＥＬＩＳＡ法（ｃＥＬＩＳＡ）,检测了３９株中的３６株,每份样本设复孔,以Ｐ／Ｎ≥２．１０且Ｐ－Ｎ≥０．１０者判为阳性。ＲＴＰＣＲ及ｃＥＬＩＳＡ两法的检出率分别为９７．６％与８２．４％,二者符合率８４．６％。此外,对ＲＴＰＣＲ产物进行酶切分型,３８份扩增产物中的１５份可被ＡｌｕＩ切开。根据所获酶切图谱的差异,可分为汉滩型及汉城型两型,显示了酶切分型的潜在价值