Samir V. Sawant

High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice

Abstract A synthetic gene coding for the surface glycoprotein (G protein) of rabies virus was strategically designed to achieve high-level expression in transgenic plants. The native signal peptide was replaced by that of the pathogenesis related protein, PR-S of Nicotiana tabacum. An endoplasmic reticulum retention signal was included at C-terminus of the G protein. Tobacco plants were genetically engineered by nuclear transformation. Selected transgenic lines expressed the chimeric G protein at 0.38% of the total soluble leaf protein. Mice immunized intraperitoneally with the G protein purified from tobacco leaf microsomal fraction elicited high level of immune response as compared to the inactivated commercial viral vaccine. The plant-derived G protein induced complete protective immunity in mice against intracerebral lethal challenge with live rabies virus. The results establish that plants can provide a safe and effective production system for the expression of immunoprotective rabies virus surface protein.

EST-derived SSR markers in Jatropha curcas L.: development, characterization, polymorphism, and transferability across the species/genera

A total of 12,080 sequences, including 5,851 transcriptome contigs developed at NBRI and 5,002 singlets and 1,227 contigs assembled from 13,201 expressed sequence tags (ESTs) of Jatropha curcas from National Center for Biotechnology Information database were used to search for simple sequence repeats (SSRs). Seven hundred and two sequences containing 786 SSRs with 93.4% simple and 7.6% compound repeat motifs were identified. Dinucleotide repeats (DNRs) were most abundant, followed by trinucleotide and tetranucleotide repeats. AG/CT was the most common motif (50.0%) followed by AT/AT (38.8%) and AC/GT (10.0%) among the DNRs. Four hundred and six primer pairs were designed out of the 702 SSR-containing sequences. Fifty randomly selected EST-SSR markers were amplified in 25 accessions collected from different geographical regions of India. Twenty-one SSR markers were polymorphic and with allele variation from two to four. Polymorphic information content value ranged between 0.04 and 0.61 with an average of 0.25 ± 0.16, indicating low to moderate level of informativeness within these EST-SSRs. The polymorphic markers showed 57.0% to 95.6% transferability among five species of Jatropha and 47.0% transferability across genera in Ricinus communis. Fifty-one alleles detected by the 21 polymorphic EST-SSRs were used to determine genetic relationships among 25 J. curcas accessions. Genetic similarity coefficient ranged from 0.44 to 0.94. The 25 accessions got grouped to three main clusters, comprising 10, 11, and four accessions. This is the first report of development of EST-SSRs in J. curcas and will be valuable resource for future genetical studies, like construction of linkage maps, diversity analysis, quantitative trait locus/association mapping, and molecular breeding of J. curcas.

Conserved nucleotide sequences in highly expressed genes in plants

Genes that code for proteins expressed at high and low levels in plants were classified into separate data sets. The two data sets were analysed to identify the conserved nucleotide sequences that may characterize genes with contrasting levels of expression. The AUG context that characterized the highly expressed genes is (A/C)N2AAN3(A/T)T(A/C) AACAATGGCTNCC(T/A)CNA(C/T)(A/C). The data set of highly expressed genes shows overrepresentation of codons for alanine at the second position and serine at the third and fourth positions after the translation initiation codon. The characteristic transcription initiation site in the highly expressed genes is CAN(A/C)(A/C)(C/A)C(C/A)N2A(C/A). The promoter region is characterized by two tandemly repeated TATA elements, sometimes with one and rarely with two point mutations in the highly expressed genes. Besides the two tandemly repeated TATA elements, the promoter context in the highly expressed genes is overrepresented by C, C and G at the -3, -1 and+9 positions respectively. The characteristic TATA motif in the highly expressed plant genes is (T/C)(T/A)N2TCACTATATATAG. Most of these features are not present in the genes ubiquitously expressed at low levels in plants.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Designing of an artificial expression cassette for the high-level expression of transgenes in plants

Abstract A dataset of highly expressed plant genes was developed from the nucleic acids sequence database. The characteristic features of the nucleotide sequences in TATA-box, transcription initiation, untranslated leader and translation initiation regions in the highly expressible genes in plants and the conserved sequences present 500 bp upstream of transcription initiation site were identified. These features were employed to theoretically design a ’minimal expression cassette’ and a promoter-upstream ’activation module.’ The ’minimal expression cassette’ was sufficient to express the gusA reporter gene in transient transformation of tobacco leaf. The context on the 3′ side of the initiator codon, conserved in a majority of the highly expressible genes, gave approximately a ninefold increase in the expression of β-glucuro- nidase. The artificially designed, upstream ’activation module’ enhanced gusA expression further by about 30-fold in transiently transformed tobacco leaves. A 450-bp-long complete expression cassette, containing both the ’minimal expression cassette’and the ’activation module’ expressed gusA at a high level in cotton leaves, potato tubers and cabbage stem also. In stably transformed tobacco plants, the ’complete expression cassette’ expressed gusA at levels higher than the native CaMV 35S promoter. Histological studies established that the ’complete expression cassette’ was expressed at a high level in different cell types in the roots, leaves, vascular tissues and flower parts of the transgenic tobacco plants. The results substantiate the functional validity of the features identified by us and demonstrate the potential of computational biology in designing artificial expression cassettes for applications in biotechnology.

Comparative transcriptomic analysis of roots of contrasting Gossypium herbaceum genotypes revealing adaptation to drought

BackgroundRoot length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of Gossypium herbaceum that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation.ResultsThe variations in root length and growth were found among four genotypes of G.herbaceum when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols) were also highly expressed in Vagad and Gujcot-21.ConclusionOur analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these genes were very likely to be involved in drought stress signaling and adaptation, such as transmembrane nitrate transporter, alcohol dehydrogenase, pyruvate decarboxylase, sucrose synthase, and LEA. These results might serve as the basis for an in-depth genomics study of Gossypium herbaceum, including a comparative transcriptome analysis and the selection of genes for root traits and drought tolerance.

The TATA-Box Sequence in the Basal Promoter Contributes to Determining Light-Dependent Gene Expression in Plants

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T7 or A8→C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants.

Development and characterization of genomic and expressed SSRs for levant cotton ( Gossypium herbaceum L.)

Four microsatellite-enriched genomic libraries for CA(15), GA(15), AAG(8) and ATG(8) repeats and transcriptome sequences of five cDNA libraries of Gossypium herbaceum were explored to develop simple sequence repeat (SSR) markers. A total of 428 unique clones from repeat enriched genomic libraries were mined for 584 genomic SSRs (gSSRs). In addition, 99,780 unigenes from transcriptome sequencing were explored for 8,900 SSR containing sequences with 12,471 expressed SSRs. The present study adds 1,970 expressed SSRs and 263 gSSRs to the public domain for the use of genetic studies of cotton. When 150 gSSRs and 50 expressed SSRs were tested on a panel of four species of cotton, 68 gSSRs and 12 expressed SSRs revealed polymorphism. These 200 SSRs were further deployed on 15 genotypes of levant cotton for the genetic diversity assessment. This is the first report on the successful use of repeat enriched genomic library and expressed sequence database for microsatellite markers development in G. herbaceum.

Effect of copy number and spacing of the ACGT and GTcis elements on transient expression of minimal promoter in plants

A variety of cis-acting DNA sequences regulate gene expression from basal promoter. In this study, two types of regulatory motifs, called ACGT and GT elements were placed at different distances from TATA -box to examine their affect on reporter gene expression in transient tran s-formation of tobacco leaf. The ACGT core motif is re-cognized by transcription factors of the bZIP family. The core ACGT element occurs at different relative positions in one or more copies upstream of the minimal promoter region. Protein-DNA interaction studies have shown that sequences flanking the ACGT core affect bZIP protein binding specificity. The bZIP transcription factors regu-late a variety of processes like pathogen defence (Zhou et al. 2000), light (Weisshaar et al. 1991) and flower deve -lopment (Chuang et al. 1999). The GT elements have a core sequence with T and A, preceeded by one to two G nucleotides on the 5 ′ side (Zhou 1999). Though hig h de-generacy makes it difficult to identify them by sequence search, a variety of such sequences are present in the genes encoding diverse functions like light regulation (Dehesh et al. 1990), tissue specificity (Villain et al. 1994) and pathogen defence ( Buchel et al. 1996). These are recognized by a family of transcription regulatory proteins called GT-factors (Buchel et al. 1999). The dif-ferences in binding affinity and the nature of transcrip-tional complex formed on promoters have been related to differ ences in regulatory functions of the GT sequence motifs (Puente et al. 1996). The GT elements are usually present in tandem repeats within a relatively long pro-moter region. An increase in spacing between two ele-ments by as little as 2 bp can dramatically reduce the transcript level (Gilmartin and Chua 1990) as in the case of rbcS -3A , suggesting critical sequence requirements for the activation of gene expression. This study aims at establishing the activation behaviour of these two ele-ments, when placed upstream of a minimal promoter in one or two copies, at different positions from the TATA -box.

Large‐scale resource development in Gossypium hirsutum L. by 454 sequencing of genic‐enriched libraries from six diverse genotypes

The sequence information has been proved to be an essential genomic resource in case of crop plants for their genetic improvement and better utilization by humans. To dissect the Gossypium hirsutum genome for large-scale development of genomic resources, we adopted hypomethylated restriction-based genomic enrichment strategy to sequence six diverse genotypes. Approximately 5.2-Gb data (more than 18.36 million reads) was generated which, after assembly, represents nearly 1.27-Gb genomic sequences. We predicted a total of 93,363 gene models (21,399 full length) and identified 35,923 gene models which were validated against already sequenced plant genomes. A total of 1,093 transcription factor-encoding genes, 3,135 promoter sequences and 78 miRNA (including 17 newly identified in Gossypium) were predicted. We identified significant no. of molecular markers including 47,093 novel simple sequence repeats and 66,364 novel single nucleotide polymorphisms. In addition, we developed NBRI-Comprehensive Cotton Genomics database, a web resource to provide access of cotton-related genomic resources developed at NBRI. This study contributes considerable amount of genomic resources and suggests a potential role of genic-enriched sequencing in genomic resource development for orphan crop plants.