Intron-mediated enhancement of heterologous gene expression in maize

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5 translated region but not when it was located upstream of the promoter or within the 3 untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity of mRNA.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

转Cry1Ac基因抗虫玉米Bt-799对田间节肢动物群落多样性的影响

【目的】安全评价是转基因品种研发的重要保障,为明确转基因玉米Bt-799在生物多样性影响方面的安全性,并为其在吉林春玉米区种植提供安全保证,开展了转基因玉米Bt-799对田间节肢动物群落多样性影响的研究。【方法】综合利用直接观察法和地面陷阱法,以多样性指数、均匀度指数、优势度指数等参数以及主要种群动态作为评价指标,系统研究转基因玉米Bt-799对田间节肢动物群落多样性的影响。【结果】转基因玉米Bt-799较之对应的非转基因对照郑58在田间节肢动物群落结构参数、主要种群动态等方面均无显著差异。【结论】转Cry1Ac基因玉米Bt-799在吉林省种植,不会对田间节肢动物群落多样性造成显著不良影响。     

Evaluation of five ab initio gene prediction programs for the discovery of maize genes

Five ab initio programs (FGENESH, GeneMark.hmm, GENSCAN, GlimmerR and Grail) were evaluated for their accuracy in predicting maize genes. Two of these programs, GeneMark.hmm and GENSCAN had been trained for maize; FGENESH had been trained for monocots (including maize), and the others had been trained for rice or Arabidopsis. Initial evaluations were conducted using eight maize genes (gl8a, pdc2, pdc3, rf2c, rf2d, rf2e1, rth1, and rth3) of which the sequences were not released to the public prior to conducting this evaluation. The significant advantage of this data set for this evaluation is that these genes could not have been included in the training sets of the prediction programs. FGENESH yielded the most accurate and GeneMark.hmm the second most accurate predictions. The five programs were used in conjunction with RT-PCR to identify and establish the structures of two new genes in the a1-sh2 interval of the maize genome. FGENESH, GeneMark.hmm and GENSCAN were tested on a larger data set consisting of maize assembled genomic islands (MAGIs) that had been aligned to ESTs. FGENESH, GeneMark.hmm and GENSCAN correctly predicted gene models in 773, 625, and 371 MAGIs, respectively, out of the 1353 MAGIs that comprise data set 2.these authors contributed equally to this work     

Streptomyces sp.NO1W98中杀黑星菌素的分离和生物合成基因簇的鉴定

[目的] 分离Streptomyces sp.NO1W98中的杀黑星菌素并鉴定其生物合成基因簇。[方法] 利用有机溶剂萃取法对Streptomyces sp.NO1W98放大规模发酵产物进行提取;以正向、反向色谱柱层析进行化合物的分离纯化;借助波谱学手段进行单体化合物的结构鉴定;采用Illumina Hiseq技术进行基因组序列测定,对得到的序列进行生物信息学分析、注释并定位杀黑星菌素的生物合成基因簇vtd,利用基于PCR-targeting的遗传操作系统构建vtd内相关基因的阻断突变株,同时利用pSET152AKE进行基因回补,并分析与野生菌株的发酵产物差异。[结果] 从NO1W98发酵产物提取物中初步分离鉴定了2个大环内酯类化合物杀黑星菌素A（1）和B（2）;NO1W98的基因组大小约为11.6 Mb,蕴涵49个次级代谢产物生物合成基因簇,其中scaffold 3上的Region 3.3可能负责杀黑星菌素的生物合成;基因阻断和回补实验初步鉴定了杀黑星菌素的生物合成基因簇,包含6个骨架基因、5个转运基因、2个调控基因以及9个后修饰基因。[结论] 杀黑星菌素的分离、结构鉴定和基因簇的鉴定以及生物合成途径的推导为其遗传改造和工程菌株的构建奠定了分子基础。     

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively.Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete.Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.     

Isolation and identification of a carbazole degradation gene cluster from Sphingomonas sp.JS1

The carbazole degrading bacterium JS1 was isolated from carbazole polluted soil and identified as Sphingomonas sp. bacterium based on its 16S rDNA gene. The car gene cluster located in the genome of JS1 was isolated by PCR and its presence verified by Southern hybridization. Sequence analysis of the car gene cluster showed that the arrangement of elements in JS1 was different from that of Pseudomons sp. CA10 and Nocardioides aromaticivorans IC177, but car gene cluster and neighboring regions were nearly identical to that of Sphingomonas sp. KA1 and Sphingomonas sp.GTIN11. Each element of the car gene cluster was expressed in E. coli upon IPTG induction. The amount of CaBb protein expressed was higher than CarBa and the ratio of these two proteins was 1:1.5. CarC expression level was detected using anti-CarC antibody. The result showed that carbazole degrading proteins were induced by the substrate carbazole. The quantity of CarC at 0.5 mg/ml carbazole was five times more than that at 0.1 mg/ml. Meiying Yang and Wenming Li have the equal contribution for this work.     

拟南芥At1g10300基因调节叶形和开花时间

核仁G蛋白1(Nucleolar G protein 1,NOG1)是一种高度保守的核仁GTP酶,在真核生物中广泛存在,参与60 S核糖体亚基前体的组装。在线虫中敲减NOG1的表达造成生长缓慢、虫体变小和寿命延长的表型,而过量表达NOG1则使线虫的寿命缩短。拟南芥的At1g10300基因注释为NOG1-2,但是其生物学功能还有待研究。该研究对其功能进行了初步研究,首先检测了该基因在拟南芥各个器官的表达情况。结果表明:该基因在7 d龄幼苗、茎生叶和花中均有表达,其中在花中表达量最高。获得了At1g10300基因的T-DNA插入突变体,发现在长日照条件下,At1g10300突变体植株的莲座紧凑,莲座叶片长宽比降低,但叶面积和植株高度与野生型相比无显著差异,表明其叶形发生改变;突变体植株的抽薹时间晚于野生型。荧光定量RT-PCR结果表明,突变体植株中开花促进因子FT、CO和GI的表达水平下调,而开花抑制因子FLC的表达水平上调。以上结果揭示At1g10300基因的突变影响了FT、CO、GI及FLC基因的表达,使植株出现晚花表型。     

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors

We have cloned and sequenced a wound-inducible cDNA clone designated WIP1 (for wound-induced protein) from maize coleoptiles. It was isolated by differential screening of a cDNA library prepared from excised maize coleoptile segments. The deduced amino acid sequence predicts a secretory, cysteine-rich protein of 102 residues with a calculated molecular mass of 11 kDa and a typical N-terminal signal sequence. The protein has about 30% identity with various Bowman-Birk type proteinase inhibitors. Most interestingly, it is novel in that it is double-headed with exclusive specificity for chymotrypsin. WIP1 is strongly wound-induced in contrast to other members of the Bowman-Birk proteinase inhibitor family, which occur in seeds and are regulated during development. The response is fast, similar to defenceinduced genes, and measurable as early as 30 min after wounding. Induction can also be evoked in the intact coleoptiles and the signal is systemically transmitted in the coleoptile to adjacent regions of the wounded area. Isolation and analysis of the corresponding genomic clone reveals that WIP1 contains an intron of 90 nucleotides.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.