利用精密作图和DNA纤维荧光原位杂交把灯笼椒抗烟草花叶病毒属病毒L^3基因定位于含高度重复序列的类抗病基因簇的400kb区

我们用辣椒（Capsicum annuum）栽培种（已导入了灯笼椒Capsicum chinense L^3 基因）的种内F2代群体（2016株）和种间F2代群体（3391株）（由灯笼椒与Capsicum frutescence杂交产生）对灯笼椒抗烟草花叶病毒属病毒的L^3基因进行定位。通过L^3基因抗性紧密相关的AFLP分子标记的BAC文库的分析,揭示出番茄抗病同源基因12的存在。通过简并PCR技术,对来自35株不同辣椒的同源基因12的部分或全部编码序列进行克隆,且在种间组合中产生了17个遗传标记。图谱显示：L^3基因位于12同源基因标记IH1—04和BAC—end标记189D23M中间,L^3基因定位于包含两个不同BAC重叠群的区内,这两个不同的BAC重叠群分别由4个和1个无性系组成。DNA纤维荧光原位杂交揭示这两个重叠群被约30kb隔开。DNA纤维荧光原位杂交结果和BAC无性系的Southern杂交表明在高度重复序列中富集包含L^3基因位点的区。Southern杂交表明两个BAC重叠群包含多于十个的12同源基因拷贝体。相反,对于种间F2代群体,,重组后代没有结合位点,在种内F2代群体中,该结合位点存在于两个不同的BAC重叠群内,这两个不同的BAC重叠群分别由7个和2个无性系组成。而且,两个群体间结合位点分配的不同表明在含有L基因位点的区域连锁不平衡。     

Effects of two sex steroid hormones on early oogenesis in Japanese huchen (Hucho perryi)

The present study was carried out to investigate whether several sex steroid hormones are able to induce oogonial proliferation or initiation of meiotic division I in vitro. The results showed that 17β-estradiol (E2) and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DHP) were able to induce the DNA synthesis of ovarian germ cells. The percentage of oocytes in 17α, 20β-DHP treated fragments was significantly higher than that in the other treatments. From these results, we conclude that endogenous E2 and 17α, 20β-DHP synthesized in ovary play significant roles in the proliferation of oogonia and the initiation of meiotic division I in early oogenesis, respectively.     

Detection of an earliness per se quantitative trait locus in the proximal region of wheat chromosome 5AL

A homoeologous quantitative trait locus to that of eps5L on barley chromosome 5H was identified in a syntenic region of wheat chromosome 5A. Wheat single chromosome recombinant lines (SCRs) were developed from a cross between ‘Chinese Spring’(‘Cappelle-Desprez’ 5A) and ‘Chinese Spring’(Triticum spelta 5A), these were grown together with the parental controls under different vernalization and photoperiod regimes. The variation for ear emergence time accelerated heading induced by the T. spelta segment indicated an effect associated with the Xcdo412-Xbcd9 interval. Since no differences between the SCRs and controls in responses to vernalization and photoperiod treatments were detected, this effect was identified as an earliness per se gene, Q Eetocs-5 A.2, which may be homoeologous to the eps5L quantitative trait locus of barley. Xbcd926 has been found to be closely linked to the rice flowering time quantitative trait loci, QHd9a or FLTQ2, on chromosome 9, suggesting possible relationships among the quantitative trait loci across wheat, barley and rice genomes.     

Development of a Novel Staining Procedure for Visualizing the Gluten–Starch Matrix in Bread Dough and Cereal Products

A novel staining procedure has been developed to visualize the gluten– starch matrix in wheat flour dough. Dough samples mixed to the final stage were stained with 26 fluorescent reagents, and each stained sample was observed with three sets of fluorescence filters (blue, green, and red). Of all the combinations of reagents and filters, the combination of acid magenta and the blue fluorescent filter set was the most effective in distinguishing starch granules from gluten network structure. Its effectiveness was further demonstrated with gluten and starch granule samples, in which the contrast was clearer when observed with the blue fluorescent filter set than without any fluorescent filter. Visualizing the gluten–starch matrix in dough samples at four mixing stages with the same procedure resulted in clear identification of the changes in gluten network structure because of the differences in mixing stages. The same procedure also enabled us to distinguish starch from gluten in white salted noodles, baked cookies, and flour particles. The proposed procedure is quicker, simpler, and has a lower risk of altering the sample than other conventional ones, and it is expected to become a useful tool in cereal studies.     

Gene dosage effect of the wheat Wx alleles and their interaction on amylose synthesis in the endosperm

To find out gene dose effect of each of the three homoeologous Wx genes and their interaction on the production of granule-bound starch synthase (GBSS I) and amylose biosynthesis in the endosperm, Chinese Spring and its near-isogenic waxy types were crossed reciprocally and, obtained a plant population with varying doses of each Wx gene. The amount of GBSSI was increased linearly with increasing gene dose of either of Wxloci. In each of the three Wx loci, the change in amylose content was linear up to 3 doses, with a more potent capacity ofWx-B1a at any dose. Higher level of amylose production was observed in the reciprocal F₁ grains than the expected effect of dose/s of each gene or additive effect of different allelic combination by artificially blend starches which have amylose produced by equivalent number ofWx alleles to that of relevant F₁ cross. When Wx-B1a and Wx-A1a were combined, increase in amylase content was not in proportion to increase in gene dosage. The enhanced amylase synthesis was shown by 2-gene and 3-geneinteraction, indicating that not only type of the three Wx genes and its dose but the interaction among them have significant roles in determining the amylose content. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of the optimal concentration of several selective drugs useful for generating multi-transgenic porcine embryonic fibroblasts

Porcine embryonic fibroblasts (PEFs) are widely used as donor cells for somatic cell nuclear transfer (SCNT) in pigs. Transfection of PEFs with exogenous DNA is essential for producing genetically modified (GM; transgenic or knockout) pigs via SCNT. In this case, selectable markers are strictly required selecting and enriching stably transfected cells. The most frequently used selective drug for this purpose is a neomycin analogue (G418/geneticin); neo has been widely used as a selectable marker gene in the genomic manipulation of pigs. However, little is known about optimal concentrations of other selection drugs. This often hampers functional analysis of the porcine genome and development of individual GM pigs. This study explores the optimal concentrations of selective drugs, other than neomycin, that can be used for the selection of transfected PEFs. Porcine embryonic fibroblasts were incubated in media containing different concentrations of drugs for up to 10 days, to determine the optimal drug concentrations fatal for PEFs. The following concentrations were found to be optimal selective concentrations for use with PEFs: G418/geneticin, 400 μg/ml; blasticidin S, 8 μg/ml; hygromycin B, 40 μg/ml; puromycin, 2 μg/ml; and zeocin, 800 μg/ml. Repeated transfections with plasmids carrying selectable markers resulted in the generation of multidrug-resistant swine transfectants. Furthermore, these markers were found to be independent. The present information will be useful for the production of SCNT-mediated GM piglets that express multiple transgenes.     

Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein

We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.