Transformation of Intron hairpin RNAi (ihpRNAi vector) of ERD15 Gene into Tobacco
Keywords:ERD15, GENE Silencing, ihpRNAi, Intron Hairpin RNA Vector
ERD15 (Early Responsive to Dehydration 15) is a negative regulator of abscisic acid (ABA) responses which includes rapid activation or response to drought and freeze as well as stomatal closure regulation. It is widely involved in the process of gene transcription and drought tolerance in plants. The objective of this study was to utilize the high-throughput system for making hairpin RNA (hpRNA) constructs of ERD15 gene fragment using one tube restriction-ligation approach. The cloning of the 609 bp full-length synthetic ERD15 gene
silencing (GenBank accession number MN816266) flanked with BsaI restriction sites at both sense and antisense using the pRNAi-GG (Golden Gate) based on the Golden Gate (GG) cloning had been successfully constructed and named the synthetic gene pRNAi-ERD15 construct. For Agrobacterium-mediated transformation, the PCR analysis results using 35S CaMV (reverse) and Nos (forward) primers showed that twenty four percent (4 out of 17 calluses) were transformed plants. The ERD15 gene sequence of the transformed tobacco plant when compared with the nucleotide database in GanBank exhibited sequence similarity to that of ERD15 gene. In conclusion, we succeeded in constructing ihpRNAi plasmid construct of the ERD15 gene to be used for plant gene transformation with any other tolerant traits in plants
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