Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.     

Analyses in transgenic tobacco of the promoter from a Brassica napus gene highly expressed in the stigma

A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows 82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some transformants also showed relatively low GUS activity in the pollen. Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

Molecular characterization of an anther-specific gene from tobacco shows sequence similarity to a tapetum-specific gene from tomato

We have cloned and determined the DNA sequence of the cDNA of ntGRP15. The cDNA ntGRP15 represents an anther-specific, developmentally regulated gene from Nicotiana tabacum that encodes a glycine-rich protein. Northern analysis shows that the gene is specifically expressed in anthers and is stringently regulated during anther development. It appears only in anthers at the meiosis to free microspore stages of development. The encoded protein is small (12.2 kDa), has a 31% glycine content and contains a putative signal sequence. By both nucleotide and amino acid sequence alignment, the gene shows high sequence similarity to a gene previously isolated from Lycopersicon esculentum, namely, TomA92b9. High glycine content, presence of a signal sequence and similarity to the tomato TomA92b9 gene suggests the protein functions as a structural cell wall protein, possibly involved in pollen exine formation. Received: 14 September 1999 / Accepted: 24 September 1999     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

水稻谷蛋白启动子驱动下的ipt基因在转基因烟草中的表达

利用农杆菌系统介导 ,采用叶盘转化法 ,将在水稻谷蛋白启动子驱动下的外源ipt基因导入烟草植株中 ,经过抗生素筛选、PCR与测序分析检测出转基因植株。成熟的转基因烟草种子经过ELISA细胞分裂素试剂盒检测 ,发现iPAs含量为对照的 2 .43倍 ,此外 ,种子的重量也增加了 7.8%。     

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli β-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.     

Molecular characterization of two Brassica napus genes related to oleosins which are highly expressed in the tapetum

Two highly homologous Brassica napus flower cDNA clones, Sta 41-2 and Sta 41-9, were isolated and characterized. These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41-2 and Sta 41-9 proteins possessed characteristics similar to oleosins such as a polar N-terminal domain, a large relatively conserved hydrophobic domain and a long C-terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41-2 and Sta 41-9 proteins have a basic pl, lack a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.     

A Brassica S-locus related gene promoter directs expression in both pollen and pistil of tobacco

A portion (1.5 kb) of the promoter region of an S63 S-locus related (SLR) glycoprotein gene from the sporophytically self-incompatible species Brassica oleracea was inserted upstream of the β-glucuronidase (GUS) gene in binary vector pBI101.1. The resulting construct was then introduced into Nicotiana tabacum through Agrobacterium-mediated transformation. The expression pattern of GUS under the control of the S63 SLR promoter fragment was found to be similar to that already reported for expression of the GUS gene directed by an S-locus specific gene promoter in transgenic N. tabacum. Furthermore, this pattern of expression resembled more closely that reported for S-genes of the self-incompatible species Nicotiana alata, which has a gametophytic self-incompatibility system, than the predicted pattern of expression of S-genes in B. oleracea.     

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.     

Phenotypic alterations and component analysis of seed yield in transgenic Brassica napus plants expressing the tzs gene

Cytokinins play an important role in plant development. We investigated the possibility that the nopaline Ti plasmid gene ( tzs ) from Agrobacterium tumefaciens could encode a protein able to participate in plant cytokinin production and lead to alterations in plant phenotype as a result of the expression of endogenous tzs . tzs was placed under the control of a heat‐inducible promoter from the Zea mays hsp70 gene. The expression of this fused gene was examined in transgenic Brassica napus plants. The tzs gene, which encodes the enzyme dimethylallyl transferase, was used as a cytokinin biosynthetic gene. The expression of the tzs gene was monitored by RNA hybridization and analysis of cytokinin content. Overproduction of cytokinin was observed even when the plants had not been heat‐shocked, and the plants displayed a reduced root system, increased height and branching, and delayed flowering. In addition, a significant increase in seed yield was observed in the transgenic plants, accounted for by increased number of seeds per silique and seed weight. The results suggest that increased levels of cytokinins, through the expression of tzs , are correlated with growth rather than with differentiation processes.     

Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants

In this paper, we present the complete nucleotide sequence of the csp1 gene from Coffea arabica coding for the 11S-globulin seed storage protein. To investigate the sequences responsible for the regulated expression of this seed-specific coffee storage protein gene, about 1 kb of the 5'-upstream region from the csp1 gene was isolated using inverse polymerase chain reaction (IPCR) and then sequenced. Several DNA boxes were found in this coffee sequence that had similarity to those previously identified as being essential for grain (endosperm) specific expression in other plants. To study the ability of this sequence to direct grain-specific expression, the whole fragment, as well as a series of 5' deletions, was fused to the reporter gene β-glucuronidase (uidA) and analysed in transgenic Nicotiana tabacum plants. GUS measurements showed that all the deletions of the csp1 promoter directed the expression of the reporter gene in tobacco grain but not in the other tissues examined. GUS activities also revealed that the csp1 promoter constructs function as very strong promoters by comparison to the strength of the cauliflower mosaic virus (CaMV) 35S promoter. Therefore, this 11S promoter could represent a useful tool to change the expression of targeted genes in the grain of transgenic coffee plants.     

2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds.

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.