Cv Pusa Research Articles

MicroRNA profiling of tomato leaf curl new delhi virus (tolcndv) infected tomato leaves indicates that deregulation of mir159/319 and mir172 might be linked with leaf curl disease

BackgroundTomato leaf curl virus (ToLCV), a constituent of the genus Begomovirus, infects tomato and other plants with a hallmark disease symptom of upward leaf curling. Since microRNAs (miRs) are known to control plants developmental processes, we evaluated the roles of miRNAs in Tomato leaf curl New Delhi virus (ToLCNDV) induced leaf curling.ResultsMicroarray analyses of miRNAs, isolated from the leaves of both healthy and ToLCNDV agroinfected tomato cv Pusa Ruby, revealed that ToLCNDV infection significantly deregulated various miRNAs representing ~13 different conserved families (e.g., miR319, miR172, etc.). The precursors of these miRNAs showed similar deregulated patterns, indicating that the transcription regulation of respective miRNA genes was perhaps the cause of deregulation. The expression levels of the miRNA-targeted genes were antagonistic with respect to the amount of corresponding miRNA. Such deregulation was tissue-specific in nature as no analogous misexpression was found in flowers. The accumulation of miR159/319 and miR172 was observed to increase with the days post inoculation (dpi) of ToLCNDV agroinfection in tomato cv Pusa Ruby. Similarly, these miRs were also induced in ToLCNDV agroinfected tomato cv JK Asha and chilli plants, both exhibiting leaf curl symptoms. Our results indicate that miR159/319 and miR172 might be associated with leaf curl symptoms. This report raises the possibility of using miRNA(s) as potential signature molecules for ToLCNDV infection.ConclusionsThe expression of several host miRNAs is affected in response to viral infection. The levels of the corresponding pre-miRs and the predicted targets were also deregulated. This change in miRNA expression levels was specific to leaf tissues and observed to be associated with disease progression. Thus, certain host miRs are likely indicator of viral infection and could be potentially employed to develop viral resistance strategies.

Fruit-specific Over-expression of LeEXP1 Gene in Tomato Alters Fruit Texture

Expansins are cellular proteins with diverse physiological functions. Expression of fruit-specific expansin gene in tomato is associated with fruit softening — a desirable trait from the processing point of view. In the present study, an expansin gene LeEXP1 was introduced via Agrobacterium tumefaciens in sense orientation under the control of a fruit-specific promoter LeACS4 with nptII gene as selection marker in Indian tomato cv Pusa Uphar. PCR detection and Southern blot analysis confirmed the integration of the transgene in the transformed tomato plants. RT-PCR and northern blot analysis using total RNA isolated from leaves and fruits confirmed over-expression of the LeEXP1 gene in transgenic fruits as compared to the wild type plants. Apart from the visual change in increased red colouration of fruits at different stages of ripening, overexpression of the LeEXP1 gene resulted in enhanced fruit softening, as determined by force required to rupture the fruit pericarp, in the transgenic fruits from breaker stage onwards as compared to the non-transformed wild type fruits. The results thus suggest an improvement in texture of the LeEXP1 over-expressing fruits, which might be useful for tomato processing industry.

Isolation and Characterization of Fruit-specific Promoters ACS4 and EXP1 from Tomato (Solanum lycopersicum L)

Promoter engineering in plants holds a great promise for understanding complexity of genetic regulatory system in response to specific internal and external cues and for crop improvement. In the present investigation, we report characterization of two fruit-specific promoters SIACS4 and SIEXP1 that were isolated from tomato (Solanum lycopersicum L cv Pusa Ruby). In silico analysis of the cloned promoter sequences revealed the presence of a seed-specific cis-element in SIACS4 and several putative seed, embryo and endosperm-specific cis-elements in SIEXP1 in addition to fruit-specific ethylene responsive regulatory elements. The fruit- and seed-specific expression of both the promoters was analyzed in transgenic tomato lines expressing the promoter:: GUS fusion constructs. The SIACS4 promoter (−1 to −373) showed GUS activity restricted specifically to flower buds and seeds in fruits. On the contrary, the SIEXP1 promoter (−1 to −769) showed high level of expression in seeds as compared to fruit tissues at different stages of fruit ripening. No GUS expression was observed in leaves satisfying the fruit-specific nature of both the promoters. Based on deletion analysis, minimal promoters SIACS4DL2 (−1 to −126) and SIEXP1DL1 (−1 to −254) were identified which can be used to drive tissue-specific expression of transgenes for introducing traits of agronomic importance such as resistance to fruit borer and for enhancing both nutritional and keeping quality of tomato fruits.

Chromium-Induced Modulation in the Antioxidant Defense System During Phenological Growth Stages of Indian Mustard

Chromium-induced modulation in the enzymes and metabolites of antioxidants was investigated at various phenological stages of Indian mustard (Brassica juncea (L.) Czern. & Coss. cv Pusa Jai Kisan)], grown with various levels of chromium (Cr) in pots under natural environmental conditions. Chromium accumulation in the root, stem and leaves increased with the advancement in the age of the plants. Growth of Indian mustard was not affected significantly by the supply of Cr upto the levels of 400 mg kg−1 soil. Activities of superoxide dismutase (SOD), ascorbate peroxide (APX), catalase (CAT), and glutathione reductase (GR) increased in the leaves of Cr-treated plants, when compared with control. High activities of antioxidant enzymes supported by high Cr concentrations in roots and aerial parts (except seeds) established the Indian mustard as a potential hyperaccumulator and a hypertolerant species to Cr stress. For this study, an edible crop was chosen intentionally so as to tap maximum benefit by remediating the contaminated site on one hand and getting uncontaminated seeds to raise the next generation, on the other.

Ontogenic variation in response of Brassica campestris L. to cadmium toxicity

Rapeseed (Brassica campestris L.) cv Pusa Gold plants, exposed to different cadmium (Cd) levels (0, 25, 50 and 100 mg kg−1 soil) in greenhouse, pot culture experiment, were analyzed with reference to distribution of metal, accumulation of biomass and the degree of growth stage Cd-sensitivity. A significant maximum decrease in plant biomass was observed at Cd-exposed flowering stage followed by pre-flowering and post-flowering stages. Activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) differentially increased; while, the concentrations of non-enzymatic antioxidants such as ascorbic acid (AsA) and glutathione (GSH) drastically decreased in plants exposed to Cd at various growth stages. However, the concentrations of GSH and AsA decreased maximally in plant groups exposed to Cd at their flowering stage. The maximum Cd-accumulation occurred in roots followed by leaves and stem. Various Cd levels inhibited also the contents of plant nutrients such as nitrogen (N), phosphorous (P), potassium (K) and sulfur (S) in leaves. The present endeavor hence concludes the existence of close relationships among growth parameters, Cd-sensitivity of phenological stages of the crop and the components of antioxidant system in rapeseed plants exposed at various growth stages.

Identification of hemizygous and homozygous transgenic rice plants in T1 generation by DNA blot analysis

Southern hybridization-based zygosity analysis was done in a transgenic rice plant (Oryza sativa L. cv Pusa Basmati 1) generated by Agrobacterium-mediated transformation with a rice chitinase (chi11) gene. A T0 plant with two unlinked T-DNA insertions (A and A′), was chosen for the application of Southern hybridization analysis to study genetic separation of the two loci by segregation and to identify the homozygous and hemizygous plants in T1- generation. The T1 plants showed differences in band intensities that reflected the homozygous and hemizygous status of each of the two integration events. The predictions of zygosity of T1 plants were confirmed by analyzing segregation in T2 plants. Southern hybridization analysis is demonstrated as a simple and effective method to distinguish hemizygous and homozygous plants in the T1 generation itself.

Enhanced Induction of Vir Genes Results in the Improvement of Agrobacterium - Mediated Transformation of Eggplant

Induction of Agrobacterium vir genes is one of the basic requirements for T-DNA transfer and integration into plant genome. Here we study the vir gene induction by various explant types of eggplant in order to develop a transformation protocol with improved efficiency using binary vector constructs - harbouring a hygromycin phosphotransferase gene (hpt) as a selection marker and a gfp:gus fusion gene as a reporter. A protocol for efficient Agrobacterium-mediated transformation of eggplant (Solanum melongena L cv Pusa Purple Long) has been developed by optimizing factors. Leaf, cotyledon and hypocotyl explants were tested for their ability to induce Agrobacterium vir-genes using a VirE:lacZ fusion construct and were shown to be poor inducers of the same. Addition of 100 µM acetosyringone during infection and co-cultivation steps of transformation could enhance the vir gene induction as well as a 2–3 fold increase in transformation frequency. Transformed explants showed the expression of reporter genes gus and gfp. The transgenics were analysed by peR and Southern blot hybridization, and were shown to have T-DNA integrated into their genome. The data suggest that eggplant is a relatively poor inducer of Agrobacterium vir genes, probably due to minimal phenolic production, and by modulating vir gene induction using phenolics like acetosyringone eggplant transformation can be improved.

Nitrogen Use Efficiency, 15N Balance, and Nitrogen Losses in Flooded Rice in an Inceptisol

The low efficiency of fertilizer nitrogen (N) in flooded rice is mainly attributed to the losses of nitrogen taking place from rice field through ammonia volatilization, denitrification, leaching, and runoff. A field experiment was conducted during the wet season of 1994 and 1995 on a sandy loam soil (Typic Ustochrept). Objective was to study nitrogen use efficiency and 15N balance in rice (CV Pusa Basmati-1) and nitrogen losses in flooded rice fields through ammonia volatilization, denitrification, and leaching as influenced by sources and levels of N. Rice was grown with three sources of nitrogen, i.e., urea, urea+coated calcium carbide (CCC), and urea+farmyard manure (FYM) each at three levels, i.e., 60, 120, and 180 kg N ha−1. The results revealed that 50% of urea N could be supplemented through FYM for higher N use efficiency. The variety Pusa Basmati‐1 responded only up to 120 kg N ha−1. Application of N, half as urea and half as FYM, resulted in the highest fertilizer N recovery by the crop (30.6%), the highest retention of fertilizer N in soil (39.1%), and the minimum unaccounted‐for fertilizer N (30.3%). Application of coated calcium carbide along with urea significantly improved the fertilizer N recovery by the crop (24.4%) and reduced the unaccounted for fertilizer‐N (47.6%) over that of sole application of urea. The direct measurements of nitrogen losses were less than the unaccounted‐for fertilizer N. Ammonia volatilization was the major pathway of N loss from the rice field. Application of coated calcium carbide along with urea reduced denitrification and leaching loss of N but enhanced the volatilization loss of ammonia over the sole urea application.

Solanum torvum, as a useful source of resistance against bacterial and fungal diseases for improvement of eggplant ( S. melongena L.)

Accessions of Solanum torvum SW, a wild relative of eggplant, were collected in Java Island, Indonesia, and assessed for morphology, fertility, levels of resistance against both Ralstonia solanacearum and Fusarium oxysporum f. sp. melongenae, as well as the genetic diversity by using RAPD and I-SSR markers. When grown to maturity in the field in Bogor, Indonesia, plants were very vigorous and showed a high polymorphism in all characters examined among the accessions, except for the stem diameter and the number of branches. The relatively high heterogeneity in morphology and fertility could not be related to the geographical origin of the accessions surveyed, and was not corroborated by genetic analysis. By using RAPD markers, 8.4% polymorphism were detected in two genotypes (BML and BG1) among the 11 accessions analysed, whereas screening with I-SSR markers has resulted in 3.3% polymorphism in three accessions, BG1, CN1 and CN2. This therefore gives a global 4.7% polymorphism, individualising only four accessions among the 11 tested. Twenty nine accessions of S. torvum and eggplant, cv Pusa Purple Long used as positive control, were evaluated for resistance to R. solanacearum (race 1 biovar 3, strain T926) in the greenhouse in Bogor. All inoculated plants of eggplant died within 2 weeks. Bacterial wilt symptoms occurred on lower leaves of S. torvum plants tested without however causing any plant death. The presence of bacteria, serologically detected in roots of symptomless plants, suggested S. torvum to be tolerant to R. solanacearum. A sample of 10 accessions was shown to be highly resistant to an Italian isolate of F. oxysporum f. sp. Melongena, while the cultivated eggplant, line Tina used as positive control, died within 2 weeks after inoculation. The low levels of genetic diversity of Javanese accessions of S. torvum and their relatively homogeneous response in tolerance and resistance to bacterial and fungal wilts are discussed, respectively.

Optimization of Ionic and Chelated Iron and Its Interaction with Disodium Ethylenediaminetetraacetic Acid for Enhancement of Plant Regeneration in Rice (Oryza sativa L)

Interactive effects of Fe2(SO4)3 and Na2EDTA on callus induction and plant regeneration in indica rice (Oryza sativa cv Pusa Basmati-1) was investigated. Callus induction and subsequent plant regeneration from seed was obtained on MS medium supplemented with 11.31 µM 2,4-D and 2.68 µM NAA + 8.87 µM BAP respectively. Both the callus induction and the plant regeneration media were supplemented with different levels of Fe2(SO4)3, Na2EDTA and their combinations. Callus induction, its morphogenic potential, average number of regenerated plants as well as the appearance of the regenerants was influenced by the levels of Fe2(SO4)3 and Na2EDTA. Embryogenic callus could be induced in the presence of Fe2(SO4)3 / Na2EDTA. Iron was essential for plant regeneration. Non-chelated iron could induce callus formation as well as plant regeneration, yet the chelated form was more suitable. A higher level of Na2EDTA in the regeneration medium was detrimental. Differential requirement of Fe2(SO4)3 at induction and regeneration level was observed. Method of medium preparation affected the regeneration response. Nearly three fold increase in the number of regenerated plants was achieved with 0.05 mM Fe2(SO4)3 + 0.1 mM Na2EDTA at callus induction and 0.1 mM Fe2(SO4)3+ 0.1 mM Na2EDTA at plant regeneration and standard method of autoclaving.

Molecular Variability in the Replicase Gene of Viruses Causing Tomato Leaf Curl Disease in India

Tomato Leaf Curl Virus (ToLCV)-infected samples collected from four different ecological zones of India: Northern zone, NewDelhi (IARI) [ToLCNDV-IARI]; Central zone, Jabalpur [ToLCV-Jb], Raipur [ToLCV-Rp] and Southern zone, Dharwad [ToLCV-Dh] were maintained by whitefly transmission into tomato cv Pusa Early Dwarf. Replicase gene coding for replication initiator protein (Rep) was amplified by PCR using gene specific primers, yielding an amplicon of 1086 bp for all the isolates. Sequence analysis of the rep amplicons of all the Indian isolates of ToLCV causing the disease clearly shows the presence oftwo subgroups irrespective of their geographical evolution. Isolates belonging to subgroup I comprising of tomato leaf curl virus-New Delhi (ToLCV-NDe) having bipartite genome are conserved within themselves and showed 94–95% nucleotide sequence homology. Second subgroup comprising of tomato leaf curl virus-Bangalore (ToLCBV) having monopartite genome and conserved among themselves, but showed only 73-75% homology with the subgroup I. In phylogenetic tree analysis, ToLCV-NDe appeared in a different cluster than ToLCV-Ban. All the four isolates used in this study fall in ToLCV-NDe group based on their replicase gene sequences. The specificity determinant located at N terminal suggests ToLCV-NDe and ToLCV-Rp as mild isolates.

Isolation and Characterization of Two Protein Isoforms with Antiviral Activity from Chenopodium album L Leaves

Two antiviral proteins (AVPs) named CAP-I and CAP-II isolated and purified from the leaves of Chenopodium album cv Pusa Bathua 1 were found to inhibit tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) infection on their respective host plants. The molecular weight of both the AVPs was found to be 24 kD. They were devoid of carbohydrate moiety and were highly basic with pI ∼10.2. However, they differed with respect to amino acid composition and N-terminal sequence. They also differed with respect to IC50 values, and CAP-I was found to be 2.5 fold more effective than CAP-II in inhibiting viral infection.

Cloning, Sequencing and Characterization of FAE1 Genes of Brassica juncea cv Pusa Bold and the Mutant fae1 of B. rapa cv Tobin

Level of erucic acid (22:1), the major storage fatty acid of oil seed Brassica, is controlled by the activity of the Fatty Acid Elongation1 (FAE1) gene. Southern hybridization revealed the presence of two FAE1 genes in B. juncea. The two FAE1 genes of B. juncea and the mutant fae1 of B. rapa cv Tobin were isolated from genomic libraries of the respective species and sequenced. The two BjFAE1 gene sequences shared more than 98% homology and contained ORF capable of coding for 509/510 amino acid polypeptides. One of the FAE1 genes of B. juncea was found to be nearly identical (99.6%) to the mutant formof B. rapa suggesting its origin from the later species. Comparison of the sequences generated with one another and with other FAE1 sequences in the database revealed that substitution of C233 (cysteine) with G (glycine) might be responsible for the loss of enzyme activity in B. rapa cv Tobin.

Isolation and Characterization of Ripening-related Expansin cDNA from Tomato

Among different ripening-related genes, expansins are unique due to their non-enzymatic nature and their ability to stimulate cell wall disassembly by reversible disruption of hydrogen bonds at cellulose-hemicellulose interface. In the present study, LeExp1 cDNA from an Indian tomato cv Pusa Sheetal has been cloned and sequenced. The LeExp1 gene expression was analyzed in vegetative tissues, flowers and fruit tissues at different stages of ripening. The gene was found to express only in fruit tissues from breaker stage onwards. The transcript level progressively increased beyond the breaker stage and achieved a maximum level at the pink and red ripe stages of fruit development. The nucleotide and predicted amino acid sequences of the LeExp1 cDNA were compared with ripening-related expansins from others species and revealed high degree of conservation.

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1RifR. The resulting A. tumefaciens strain C58C1RifR (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1RifR (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T0 plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.

Dual purpose summer legumes and zinc fertilization for improving productivity and zinc utilization in aromatic hybrid rice (Oryza sativa)

Field experiments were conducted during 2007-08 at New Delhi, to study the effect of dual purpose summer le- gumes and zinc (Zn) fertilization on productivity and Zn-use efficiency of aromatic hybrid rice cv Pusa Rice Hybrid 10 (PRH 10). The experiment was carried out in split plot design with 3 treatments of legume residue in main plots and seven treatments of Zn fertilization in sub-plots. The tallest plant height, number of tillers/m2, dry matter accumulation/hill, leaf area index (LAI), yield attributes and grain yield of aromatic hybrid rice was recorded with incorporation of cowpea residue followed by mungbean residue. Zinc fertilization in aromatic hybrid rice increased growth parameters, yield attributes, grain and straw yields. Application of 2% ZEU (ZnSO .7H O) was found to be 4 2 the best in terms of all the yield attributes, grain yield of aromatic hybrid rice, 2% ZEU (ZnO) was found very close to it. In respect to Zn concentrations in grain and straw of aromatic hybrid rice, 2% ZEU (ZnSO .7H O) recorded 4 2 higher Zn concentrations in grain as well as in straw of aromatic hybrid rice. The highest agronomic efficiency (AE) was obtained with the application of 2% ZEU (ZnSO .7H O). Physiological efficiency (PE) and Zn harvest index 4 2 (ZnHI) of applied Zn were also significantly influenced due to dual purpose summer legumes and various Zn fertili- zation treatments applied in aromatic hybrid rice. Cooking quality parameters were significantly improved by le- gume residue incorporation and with the application of ZEU. The highest net returns and B:C ratios of the legume- rice sequence were recorded with the legume residue incorporation and with the application of ZEU in the aro- matic hybrid rice. Thus, growing of cowpea or mungbean as dual purpose summer legumes along with Zn fertiliza- tion as 2% ZEU either with ZnSO .7H O or ZnO to aromatic hybrid rice led to higher productivity, cooking quality 4 2 and Zn-use efficiency in aromatic hybrid rice and higher net returns of this sequence.

Isolation and Characterization of a Novel S-Adenosyl-L-Methionine Synthetase cDNA from Rice (Oryza sativa L cv Pusa Basmati 1)

An expression assay of randomly selected cDNAs resulted in the isolation of a novel cDNA (OSISAM) of SAM synthetase from rice. Nucleotide sequence data show that the cDNA is similar to a genomic clone (pRSAM1) from rice in the coding region but differs in the 5’ untranslated region (UTR) that is unique to this cDNA. The RNase protection experiments and search of EST database confirmed the presence of such novel cDNA. Northern analysis shows that the gene is differentially expressed in various tissues. The transcript abundance also increases when plants are stressed with salt, drought, ABA, injury, osmotic- or heat-shock, but it is not influenced by cold stress.

Induction of microspore embryos in a CMS line of Brassica juncea and formation of the androgenic plantlets

Anther culture of two wide hybrids (Diplotaxis erucoides × Brassica campestris) × B. juncea and (D. berthautii × B. campestris) × B. juncea, their CMS lines and the parent species elicited a range of responses highlighting the importance of the genotype. Androgenesis was expressed in cultured anthers of CMS (D. erucoides) B. juncea (22.8%), in restored pollen fertile plants of this CMS line (1.66%), and in the parent, B. juncea cv Pusa Bold (13.02%). AgNO3 was essential for androgenic response in the CMS lines, and it markedly increased the frequency of androgenesis in the cultivated species. Multiple crops of microspore embryos were obtained from responsive anthers of CMS plants in anther recultures. As high as92% microspore embryos of the CMS line germinated on basal B5medium and formed normal plantlets.

Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium

The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing β-glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency. The effects of some factors like the bacterial density and inclusion of sorbitol in the medium on the co-culture and transformation have been evaluated; the procedure for selection and regeneration from transformed calli was found to be critical. Furthermore, co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations. Regeneration medium supplemented with 3% maltose was found to be better for regeneration as compared to 3% sucrose. The transformed calli were subjected to three cycles of regeneration, thus converting a higher number of transformation events into regenerants. The selected calli as well as leaf sections and roots of the transformants were GUS positive. The stable integration of the transgene was confirmed by polymerase chain reaction and Southern blot analysis of the transformants. Interestingly, the presence of three additional vir genes in supervirulent plasmid pTOK233 was not required for transformation as transformation was successful with non-supervirulent plasmid pJB90GI, although the transformation and regeneration frequency was higher with the former. This effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency.

Sequence Analysis of a Plastidial Omega-3 Desaturase Gene from Brassica juncea

A genomic clone (λg2) encoding a plastidial omega-3 desaturase has been isolated from a genomic library of Brassica juncea cv Pusa Bold and sequenced after subcloning. A continuous stretch of 3866 by of the clone sequenced showed homology with omega-3 desaturase from other plant sources but maximum homology was with that of a plastidial omega-3 fad 7 gene. This sequence designated as Bjgfad7 includes 1381 by 5’ untranslated region, 273 by 3’ untranslated region along with an open reading frame of 1290 by interrupted by 7 introns. The 5’ untranslated region contains putative cis elements responsible for seed specificity, light responsive and stress-inducible expression besides minimal promoter elements.