Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

Cholera toxin B (CTB) subunit is a well-characterized antigen against cholera. Transgenic plants can offer an inexpensive and safe source of edible CTB vaccine and may be one of the best candidates for the production of plant vaccines. The present study aimed to develop transgenic tomato expressing CTB protein, especially in the ripening tomato fruit under the control of the tomato fruit-specific E8 promoter by using Agrobacterium-mediated transformation. Transgenic plants were selected using PCR and Southern blot analysis. Exogenous protein extracted from leaf, stem, and fruit tissues of transgenic plants was detected by ELISA and Western blot analysis, showing specific expression in the ripening fruit, with the highest amount of CTB protein being 0.081% of total soluble protein. Gavage of mice with ripe transgenic tomato fruits induced both serum and mucosal CTB specific antibodies. These results demonstrate the immunogenicity of the CTB protein in transgenic tomato and provide a considerable basis for exploring the utilization of CTB in the development of tomato-based edible vaccine against cholera. The rCTB antigen resulted in much lower antibody titers than an equal amount of exgenous CTB in trangenic fruits, suggesting the protective effect of the fibrous tissue of the fruit to the exogenous CTB protein against the degradation of protease in the digestive tracts of mice. Xiao-Ling Jiang and Zhu-Mei He contributed equally to this work.     

In vivo tracing of canonical Wnt signaling in Xenopus tadpoles by means of an inducible transgenic reporter tool

The canonical Wnt pathway is recurrently used during embryogenesis and adult life. To track the cellular output of Wnt signaling in a living organism, we designed a hormone-inducible Wnt responsive system, capable to dynamically and specifically report Wnt pathway activities through eGFP expression. In contrast to previous in vivo reporters, our system essentially avoids interference of consecutive signals by remaining dormant until addition of hormone, which makes it a valuable tool to map canonical Wnt signaling in post-embryonic stages. Transgenic Xenopus laevis embryos were analyzed revealing at tadpole stage in specific tissues and organs cell populations with high Wnt pathway activity.     

以黑丝羽乌骨鸡为供体制作家鸡嵌合体的研究

以黑丝羽乌骨鸡（BS）为供体,自来航鸡（WL）为受体,进行了BCs嵌合体制作技术研究。结果表明：（1）“黑羽”对“白羽”、“丝羽”对“片羽”为完全隐性,可以在供体与嵌合体测交中,后代是否出现这些特征作为种系嵌合体判断依据。（2）供体的其它表型,对受体属于完全或不完全显性,可以作为体细胞嵌合体判断依据。（3）通过改进嵌合体制作技术,嵌合体雏鸡的出壳率为40％（29／73）,其中据羽色判断的嵌合体率为18％（13／73）;以黑羽为依据选择体细胞嵌合体雏鸡,10只饲养至720d,其中60％（6／10）的嵌合体外观基本不变（其余的换羽后褪去黑羽）;嵌合体鸡与供体测交,以黑羽、灰羽和丝羽判断,8只嵌合体鸡的种系传递率分别为2．5％-71．4％、5．5％～14．3％以及1．7％～10．5％。首次利用BS鸡资源,建立了多表现型嵌合体模型,为家鸡嵌合体技术深入研究提供了方便的检测方法。     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco

Summary A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm. The mature seed thus consists of a seed coat of one rigid cell layer, and a degenerated embryo. The genetic ablation technique has thus contributed to the establishment of the sequence of events and elucidation of the role of different cell lineages and tissues in seed development.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.