Methods and Compositions for Expressing Multiple Genes in Plants by Alternate Splicing of a Polycistronic Message

The present invention provides a method and composition for expression of multiple genes from a polycistronic message in transgenic plants using genetic elements derived from the peanut chlorotic streak caulimovirus promoter-leader sequence and antisense sequence of PC1SV ORF VII. Also provided are compositions and methods for intron-mediated enhanced and regulated expression of genes in transgenic plants

An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis Thaliana is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications

GUS expression in transgenic tobacco and <i>Arabidopsis</i> plants generated for constructs pSiM24 and pSiM24-GUS.

<p>(A) GUS enzymatic activity of pSiM24-GUS transgenic tobacco (second generation, 3 weeks old) lines was measured in whole plant, leaf, stem and root tissues. Soluble protein extracts isolated from different tissues of plants were used for GUS assay along with the wild-type plants (C). The data represent means ± S.D. of four second generation individuals from one line for each tissue (n = 4). The values significantly differ between control and transgenic plants at P<0.01 based on Student's <i>t</i>-test. T1, T2 and T3: Representative transgenic lines generated by agrobacterium-mediated plant transformation procedure; Tb1, Tb2 and Tb3: Representative transgenic lines generated by biolistic plant transformation procedure. (B) GUS enzymatic activity of pSiM24-GUS transgenic <i>Arabidopsis</i> (second generation, two weeks old) lines was measured in whole plant (WP), leaf (L), stem (S) and root (R) tissues. Soluble protein extracts isolated from different tissues of plants were used for GUS assay along with the wild-type plants (C). The data represent means ± S.D. of four second generation individuals from one line for each tissue (n = 4). The values significantly differ between control and transgenic plants at P<0.01 based on Student's <i>t</i>-test. T1, T2 and T3: Representative transgenic lines generated by floral-dip plant transformation procedure.</p

Transient expression of pSiM24-GFP in tobacco protoplasts and onion epidermal cells.

<p>(A) Protoplasts were transfected with plasmids pSiM24 (Control) and pSiM24-GFP (having GFP reporter gene). Transformation efficiencies were determined by analyzing the protoplasts with fluorescence after incubation for overnight. Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of tobacco protoplasts are presented. Scale bar, 92 µm. (B) Fluorescent, bright-field and superimposed (bright-field and green fluorescent) confocal laser scanning micrographs of onion epidermal cells bombarded with pSiM24-GFP construct DNA-loaded gold particles are presented. Control represents onion epidermal cells with pSiM24 visualized under CLSM. Scale bar, 100 µm.</p

Schematic presentation of binary vector pSiM24.

<p>The backbone structure of binary vector pSiM24 (7081-bp) containing the modified full-length transcript promoter (M24) of the <i>Mirabilis mosaic virus</i>, which directs the coding sequences of the gene of interest; left T-DNA and right T-DNA borders (Left T-DNA, Right T-DNA); selection marker genes (KanR, neomycin phosphotransferase II, nptII) directed by nopaline synthase promoter (Nos promoter); terminator sequences of ribulose bisphosphate carboxylase small subunits (3′rbcSE9); nopaline synthase terminator (Nos terminator); multiple cloning sites (first MCS, second MCS and third MCS) with various restriction sites; replicon unit pRK2 oriV; trfA gene for agrobacterium; ColE1 origin of replication for <i>E. coli</i>; and ‘bla’ AmpR gene for resistance to ampicillin.</p

Transformation frequencies of <i>Escherichia coli</i> strain TB1 for pSiM24 binary vector.

<p>The bacteria were transformed with equal molar amounts of each of the plasmid DNA. Statistical analysis of the data was performed adopting one way ANOVA analysis (using GraphPad Prism version 5.01) and presented as the means ± S.D. A <i>P</i> value of less than 0.05 was considered significant indicated by different superscript letters.</p

Transformation frequencies of <i>Agrobacterium tumefaciens</i> strain GV3850 for pSiM24 binary vector.

<p>The bacteria were transformed with equal molar amounts of each of the plasmid DNA. Statistical analysis of the data was performed adopting one way ANOVA analysis (using GraphPad Prism version 5.01) and presented as the means ± S.D. A <i>P</i> value of less than 0.05 was considered significant indicated by different superscript letters.</p

GUS expression analysis of pSiM24-GUS and pKCaMV35S-GUS in transient and transgenic systems.

<p>(A) Representative transient GUS expression levels from agrobacterium infiltration assays in <i>N. benthamiana</i> leaves are shown for pKCaMV35S-GUS and pSiM24-GUS constructs. GUS was detected histochemically. Control represents pSiM24 without GUS reporter gene. (B) Relative expression of GUS specific transcripts was measured in whole transgenic <i>Arabidopsis</i> (second generation, two weeks old; Ar-CaMV35S with pKCaMV35S and Ar-pSiM24 with pSiM24) and tobacco (second generation, three weeks old; Tob-CaMV35S with pKCaMV35S and Tob-pSiM24 with pSiM24) plants. The data represent relative expression of GUS transcript ± S.D. of four independent lines (n = 4) for each construct in which five plants per line were analyzed. The values significantly differ between control and transgenic plants at P<0.01 based on Student's <i>t</i>-test.</p

Binary Ti vectors pSiM24 produced higher plasmid DNA yields in <i>Escherichia coli</i> strain TB1 over pCAMBIA.

<p>Three single colonies of each plasmid constructs were grown for 16 hrs in 25 ml LB media with 100 mg/L ampicillin (for pSiM24) or 50 mg/L kanamycin (for pCAMBIA) or 15 mg/L tetracycline (for pKM24KH). Plasmid DNA was purified using QIA Midiprep columns. DNA yields represent the average of three independent samples. Statistical analysis of the data was performed adopting one way ANOVA analysis (using GraphPad Prism version 5.01) and presented as the means ± S.D. A <i>P</i> value of less than 0.05 was considered significant indicated by different superscript letters.</p

GUS expression in flowers of transgenic tobacco and <i>Arabidopsis</i> plants generated for constructs pSiM24 and pSiM24-GUS.

<p>Histological GUS staining in different floral tissues of untransformed (control), pSiM24 and pSiM24-GUS plants. Histological GUS staining shows strong GUS expression in all floral tissues of transgenic pSiM24-GUS <i>Arabidopsis</i> (upper panel) and tobacco (lower panel) plants.</p