Pradeep Kumar Burma

Strategies for Development of Functionally Equivalent Promoters with Minimum Sequence Homology for Transgene Expression in Plants: cis-Elements in a Novel DNA Context versus Domain Swapping

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) “domain swapping,” wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using β-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.

The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-specific promoters, TA29 and A9. Several transformation vectors based on different promoter and marker gene combinations were developed and tested for their efficacy in generating agronomically viable male-sterile lines. Use of strong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-sterile plants at an extremely low frequency with most plants showing abnormalities in vegetative morphology, poor female fertility, low seed germination frequencies and/or distortion in segregation ratios of transgenes. Such abnormalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fragment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exhibited normal morphology, growth and seed set on backcrossing as observed for wild-type plants. Around 75% of single-copy events tested further also showed proper segregation of the marker gene/male-sterile phenotype among backcross progeny. Constructs based on the use of Spacer DNA fragments as insulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of agronomically viable male-sterile lines in crop plants and for cell/tissue ablation studies in general.

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5’ and 3’ RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (α-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.

Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

A two gene – two promoter system for enhanced expression of a restorer gene (barstar) and development of improved fertility restorer lines for hybrid seed production in crop plants

We report in this study, strategies for enhancing and extending tissue-specific expression of a restorer gene (barstar) and their use in the development of improved fertility restorer lines for transgenic male sterile (barnase) lines for hybrid seed production. In the first strategy, a chimeric promoter was developed by combining regulatory elements of two overlapping tapetum-specific promoters (A9 and TA29) by placing a 275bp fragment of the A9 promoter (minus the TATA box) upstream to a functional 330bp fragment of the TA29 promoter. Analysis of chimeric promoter activity using the gusA gene showed that it enhanced transgene expression levels in anthers of transgenic Brassica juncea (Indian oilseed mustard) plants by two- to three-fold over those obtained with either promoter alone but retained the temporal expression profile of the TA29 promoter. In the second strategy, the A9 and TA29 promoters were used to independently express two copies of the wild type barstar gene sequence within the same T-DNA borders. Alternatively, one of the promoters was used to express the wild type sequence and the other was used to drive expression of a codon-modified version of the barstar gene designed for enhanced and stable expression in dicot plants. The efficiencies of these constructs for fertility restoration was determined by retransforming a male sterile TA29-barnase line of B. juncea and analyzing the frequency and pollen viability of male fertile (restored) plants thus obtained. A significantly higher frequency of restored plants was obtained with constructs containing two transcription units of the barstar gene (92.9% for constructs with two copies of the wild type gene and 89.8% for constructs using the wild type and codon-modified sequences) as compared to that obtained with the chimeric promoter-barstar construct (77.8%) or the TA29-barstar construct (65.6%). Pollen viability assays on restored plants showed that the desirable extent of restoration is obtained in a higher proportion of male fertile plants generated using the construct containing a combination of the wild type and modified barstar genes. These observations were corroborated by genetic analysis of F1 progeny of crosses between various barstar lines and four different male sterile barnase lines, three of which could not be restored in earlier studies using TA29-barstar constructs. The simultaneous use of the wild type and modified barstar genes with the TA29 and A9 promoters was found to be the most effective method for fertility restoration with efficient restorers being identified for all the male sterile barnase lines tested, including those which could not be restored earlier. The two gene-two promoter strategy could be successfully deployed for enhancing and extending the tissue-specific as well as constitutive expression of other agronomically important transgenes.

High Frequency Regeneration of Brassica napus Varieties and Genetic Transformation of Stocks Containing Fertility Restorer Genes for Two Cytoplasmic Male Sterility Systems

Transformation and regeneration protocols for Brassica napus varieties adapted to agronomic conditions prevailing in India were standardized. Five different varieties of B. napus (BO-15, GSL-1, HNS-8, H-18, ISN706) and a hybrid of ISN706 and GSL-1 were tested for regeneration response. B. napus var GSL-1 showed a very high frequency of regeneration (98%). Regeneration response of some of the varieties was improved by alterations in the composition of media. Efficient transformation protocol was developed for the variety GSL-1 with a binary vector containing nptll and bar genes conferring resistance to kanamycin and herbicide phosphinothricin as selection marker and a gus-Int gene as a reporter. A new construct that contained Ds border, bar gene as integration marker and a mutant csr-1 gene conferring resistance to chlorsulfuron as an excision marker was developed for transposon-mediated gene tagging. This Ds-construct was introduced into B. napus genetic stock (ISN706 GSL-1 Rft/-, Rfp/-) harboring fertility restorer (Rf) genes for two different CMS systems namely ‘tour’ (Rft) and ‘polima’ (Rfp).

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

BackgroundA high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ), which enhances gene expression in tobacco and cotton.ResultsThe influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants.ConclusionssynJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.

A comparative analysis of green fluorescent protein and β-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, β-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

Retransformation of a male sterile barnase line with the barstar gene as an efficient alternative method to identify male sterile–restorer combinations for heterosis breeding

We report in this study, an improved method for identifying male sterile–restorer combinations using the barnase–barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line × tester cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66–90% of fertile retransformants (restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and ∼30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) × tester (barstar) crosses, wherein only two viable male sterile–restorer combinations were identified by screening 88 different cross-combinations. The retransformation strategy not only generated several independent restorers for a given male sterile line from a single transformation experiment but also identified potential restorers in the T0 generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T1 progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major limitation.

Delayed Trans-inactivation of Synthetic Domain A 35S Promoters by “Tobacco 271 Locus” due to Reduced Sequence Homology

Homology in the promoter region leads to transcriptional gene silencing (TGS). This could be circumvented by designing synthetic promoters which are functionally similar but divergent in their sequence. In the present study, we asked whether such synthetic promoters can escape trans-inactivation when challenged with a silenced copy of the wild-type prototype. We tested the activity of two synthetic cauliflower mosaic virus 35S (35S) domain A promoters, 2mA and 3mA, with only 35% and 41% sequence identity with the wild type 35S (wtA) in the presence of silencing locus 271 of Nicotiana tabacum that inactivates any 35S promoter driven transgene through TGS. Based on comparison of β-glucuronidase (gus) activity under the transcriptional control of wtA, 2mA, and 3mA in the presence and absence of the silencing locus, we demonstrate that the inactivation of the 2mA and 3mA promoters is significantly delayed in comparison to that of wtA although microhomology at the transcription factor binding sites eventually leads to the silencing of these synthetic promoters. We further postulate that it is possible to design functional synthetic promoters that can escape gene silencing in the background of silencing locus if the cis-elements are smaller and do not contain methylation prone CG and CNG.