Functional analysis of a reproductive organ predominant expressing promoter in cotton plants

Transgenic Bt insect-resistant cotton plants have high insect resistance in the early stage of development, but relatively low resistance in the late stage. Substituting a reproductive organ-specific promoter for the CaMV35S promoter presently being used could be an ideal solution. For the first time, the promoter sequence of ADP-ribosylation factor 1 (arf1) gene was isolated from Gossypium hirsutumY18 by means of inverse PCR. The sequencing result discovered the unique structure of the arf1 promoter, including four promoter-specific elements, the initiator, TATA box, CAAT box and GC box, and also an intron in 5′-untranslation region. Four plant expression vectors were constructed for functional analysis of the promoter. Based on the pBI121 plant expression vector, four truncated arf1 promoters took the place of the CaMV35S promoter. These vectors were different only in their promoter regions. They were introduced into cotton plants via pollen tube pathway. Histochemical GUS staining and fluorescence quantitative analyses were performed to examine the expression patterns of the GUS gene driven by the 4 arf1 truncated promoters in transgenic cotton plants respectively. The results showed that the arf1 promoter was a typical reproductive organ-specific promoter. Hopefully, the arf1 promoter can be a regulatory element for designing cotton reproductive organs with desired characteristics.     

Cloning Vectors for Rice

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.     

An Improved pPZP Vector for Agrobacterium-mediated Plant Transformation

We report a new and improved pPZP vector (pPZP3425) for efficient plant transformation. This vector is derived from the widely used pPZP100 series of binary Agrobacterium vectors. One disadvantage of these vectors is the use of chloramphenicol resistance for selection in Escherichia coli and Agrobacteria. We have therefore included a kanamycin resistance gene for selection in Agrobacterium. Furthermore, the strong 35S CaMV promoter driving the plant resistance gene has been replaced by the weaker nos promoter because it has been shown that the 35S promoter driving the plant resistance marker can lead to ectopic expression of the transgene. During replacement of the 35S promoter, the NcoI site within the plant resistance gene has been removed, and NcoI can now be used for cloning purposes within the expression cassette which consists of an intron-containing gus gene driven by a strong constitutive promoter (35S promoter with doubled enhancer plus omega-element as translational enhancer). Thus, a single vector can conveniently be used for two purposes: (1) for overexpression of proteins by replacing the gus gene by the coding sequence of choice and (2) for creation of promoter:gus fusions by substituting the constitutive promoter by any other promoter. We demonstrate the usefulness of this vector for cloning a promoter:gus fusion and in planta transformation of Arabidopsis.     

An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20–30 days after injection was proven by PCR analysis.     

Development of R4 Gateway Binary Vectors (R4pGWB) Enabling High-Throughput Promoter Swapping for Plant Research

We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction of chimeric genes between any promoter and any cDNA. The structure of R4pGWBs is almost the same as the promoterless type of improved pGWBs (ImpGWBs), except that the attR1 site is replaced with attR4, which enables tripartite recombination of these vectors with promoter- and cDNA-entry clones. While ImpGWBs are suitable for promoter analysis and constitutive expression of cDNAs in higher plants, R4pGWBs have a great advantage in expressing a cDNA under the regulation of desired promoters.     

Construction of two Gateway vectors for gene expression in fungi

We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana

We compared the organ specificity and the strength of different constitutive (CaMV-35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1, and barley leaf thionin BTH6 promoter) and one inducible promoter (soybean heat-shock promoter Gmshp17.3) in stably transformed Arabidopsis thaliana plants. For this purpose we constructed a set of plant expression vectors equipped with the different promoters. Using the uidA reporter gene we could show that the CaMV-35S promoter has the highest expression level which was enhanced two-to threefold by the addition of a translational enhancer (TMV omega element) without altering the organ specificity of the promoter. The barley leaf thionin promoter was almost inactive in the majority of lines whereas the ubiquitin promoter exhibited an intermediate strength. The heat-shock promoter was inducible up to 18-fold but absolute levels were lower than in the case of the ubiquitin promoter. Conclusive quantitative results for different organs and developmental stages were obtained by the analysis of 24 stably transformed lines per promoter construct.     

玉米胚胎发生后期丰富蛋白基因的启动子克隆与功能验证

该研究在生物信息学分析的基础上,克隆玉米胚胎发生后期丰富蛋白基因(MGL3)的启动子序列(pMGL3),进行非生物逆境应答元件分析以及实时定量PCR验证其非生物逆境胁迫响应特性,构建了pMGL3启动子驱动报告基因(GUS)表达载体,基因枪法转化玉米愈伤组织,通过GUS染色验证pMGL3启动子在非生物逆境胁迫下的驱动活性。再根据启动子序列分析结果,去除不同的顺式作用元件,构建不同长度pMGL3启动子驱动报告基因GUS表达载体,农杆菌介导法转化烟草叶盘,以确定pMGL3启动子的最短活性序列。结果显示:pMGL3启动子长1 554bp,存在多种与非生物逆境胁迫应答相关的调控元件,在干旱、高盐、低温胁迫及脱落酸、乙烯诱导下驱动MGL3基因增量表达,用以驱动GUS基因转化玉米愈伤组织,在高渗、高盐、低温胁迫及脱落酸诱导下具有驱动活性,且截短至325bp仍可保持驱动活性。研究表明,pMGL3启动子的确有非生物逆境诱导启动活性,进一步验证其作用机理后可运用于玉米抗逆转基因研究。     

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors

Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.

Significance and Impact of the Study

A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.     

家蚕核型多角体病毒lef-11基因RNA干扰策略的优化

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)lef-11基因作为杆状病毒高度保守的晚期表达因子之一,对病毒晚期基因的表达极其重要。因此对lef-11基因进行有效RNA干扰将提高宿主细胞对BmNPV的抗性。【方法】本文基于经典的sh-RNA-loop骨架及家蚕内源性的miRNA骨架,构建以BmNPV lef-11基因为靶标的干扰载体pIZ-shRNA1、pIZ-shRNA2和pIZ-Ds RedamiR279;分别构建基于A4、IE1、IE1-295、IE2、IE2-339、hr3A4和hsp70启动子驱动的干扰载体,用以评价不同启动子驱动的干扰载体的抗病毒效果,并对干扰载体进行优化。【结果】首先,本文通过比较miRNA-based和shRNA-based RNAi载体对同一靶基因同一位点的干扰效率,发现pIZ-Ds Red-amiR279对BmNPV lef11基因的干扰效率超过90%,远优于shRNA-based RNAi载体的干扰效果;其次,通过对干扰载体进行优化,发现IE1启动子的效果最优,由其驱动的pIZ-Ds Red-milef11干扰载体也是本研究中的最优干扰载体,对病毒的增殖抑制效果最为明显。【结论】对目的基因的干扰效果并非启动子的活性越高、miRNA积累得越多就越好,只有综合考虑干扰片段的选择、启动子的活性以及靶基因自身的功能等多方面的因素,才能提高干扰效率,达到干扰目的。     

Evaluation of agrobacterium-mediated transformation of agricus bisporus using a range of promoters linked to hygromycin resistance

There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.     

Heterologous expression of human interleukin-6 in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK24 using novel secretory expression vectors

Streptomyces is an attractive host for heterologous protein secretion. To further optimize its expression capacity, better expression vectors will be helpful. Here, based on pSGL1, a high copy number plasmid present in Streptomyces globisporus C-1027, we constructed a series of novel E. coli-Streptomyces shuttle expression vectors pIMB2–4. These vectors, which are compatible with pIJ-derived vectors, contain the strong promoter ermE*p and signal sequence SP _MelC1 of the first ORF of melanin operon in S. antibiotics (pIMB2), SP _CagA of C-1027 apoprotein in S. globisporus C-1027 (pIMB3 and pIMB4). Using these vectors, human interleukin-6 (IL-6) could successfully be expressed and secreted using S. lividans TK24 as host. Furthermore, replacement of a rare leucine codon TTA with CTG in SP _CagA enhanced IL-6 production.     

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Summary We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.