IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF FLOWER AND POD WALL SPECIFIC PROMOTER FROM CHICKPEA FOR TISSUE SPECIFIC EXPRESSION OF TRANSGENE
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Date
2017-07
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AAU, Jorhat
Abstract
The transgene expression is, in part, a function of the promoter to which the coding region is fused. Constitutive over-expression of transgene occasionally interferes with normal growth and developmental processes in plants. Tissue-specific promoter can regulate transgene expression in a particular organ and developmental stage. In the present investigation, an attempt was made to isolate and characterize a flower and pod wall specific promoter from chickpea. The aim was to use the promoter to drive Bacillus thuringiensis (Bt) Cry gene in these organs of chickpea for enhanced resistance to a key pest, Helicoverpa armigera. For isolation of flower and pod wall specific promoter, a forward Suppression Subtractive hybridization (SSH) library was prepared using flower and pod wall (tester) and leaves (driver). Subtracted cDNAs were amplified, cloned and transformed into E. coli competent cells. In all, 226 clones of SSH library were sequenced and analyzed. After removing adaptors, vector sequences (<100bp) and low quality sequences, 179 high quality ESTs sequences were deposited in the NCBI GenBank database under the Accession numbers JZ923200-JZ923378. Based on CAP3 assembly of 179 ESTS, 126 genes comprised of 97 singletons and 29 contigs were computationally annotated. The mapping of 88.26% ESTs (158 out of 179 ESTs) was done based on Gene Ontology (GO) annotation, which distributed 751 GO terms into three categories: cellular location, molecular function and biological process. Within the biological process category, the 158 ESTs were classified into seven primary functional categories, including metabolic process (113; 28%), cellular process (103; 26%), single organism process (77; 19%), biological regulation (38; 9%), regulation of biological process (33; 8%), response to stimulus (22; 6%) and cellular component organization or biogenesis (17; 4%). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was also performed to understand functions and utilities of these ESTs in the biological system and a total of 45 ESTs involved in 49 different biological pathways were identified. Moreover, 67 ESTs were also identified encoding four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerese (2).
To identify the genes exclusively expressed in the flower and pod wall, the 179 EST sequences of the pod wall were searched using BLASTN of the Chickpea Transcriptome Database (CTDB) database to obtain CTBD ID. The CTDB IDs for pod wall ESTs were used to obtain the gene expression profile in the flower. The candidate genes were selected based on
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their high levels of expression in the flowers and pod wall. A total of eight (8) flower and pod wall specific genes were identified and subjected to quantitative PCR (qPCR) analysis. Of these, 3 genes (FHG: Floral homeotic AGAMOUS-like isoform X2, MADS1: MADS box transcription factor and CEP: chickpea expressed protein) showed significantly high levels of up-regulation in the flower and pod wall when compared with leaves. These are the differentially expressed genes in the chickpea pod wall that have been identified for the first time. In order to obtain a regulatory sequence of selected flower and pod wall specific genes, 1000 bp region upstream of the start codon of FHG and MADS1 gene was obtained by Genome Walking and subjected to in-silico analysis using PlantCARE promoter prediction database. Sequence analysis of these promoter regions revealed certain tissue specific cis-acting elements that may regulate transcript accumulation in the flower and pod wall. Finally, the isolated promoters were cloned in a binary vector, pBI121, harboring the GUS as a reporter gene in order to study the efficiency of the promoters. Thus, for the first time the transcript dynamics of the chickpea pod wall were revealed and the transcript profile demonstrated various differentially expressed genes in the pod wall, which may be participating in metabolic build up of not only the pod wall but also seeds. The transcript library was useful to identify two novel promoter of genes that exclusively expressed in the flower and pod wall. These information of pod wall transcripts and isolated promoter may be valuable for chickpea improvement.
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