eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2017-09-07 14 3 1211 1219 10.13005/bbra/2563 27827 article Sonia Sharma 1 Gurtej Singh 1 Sadiq Pasha Shaik 2 Gagandeep Singh 3 Sumangala Bhat 4 Gaurav Sharma 1 Department of Natural Sciences, Sant Baba Bhag Singh University, Jalandhar 144030, India. Division of Biotechnology, Indian Institute of Horticulture Research, Hesaraghatta, Bengaluru 560089, India. Department of Agriculture, Mata Gujri College, Fatehgarh Sahib 140407, India. Department of Biotechnology, University of Agricultural Sciences, Dharwad-580 005, India. Fungal diseases like early blight, late blight, fusarium wilt cause 30-40 per cent loss in fruit production. Form past decade many transgenic plants had been developed using genes encoding chitinases and glucanases with the objective of imparting fungal disease resistance. Since the genes encoding chitinase and glucanase act synergistically. The study was performed to construct plant transformation vector pRAGS carrying both ech42 and bgn under single T-DNA region. Initially, HindIII site at 5' end of earlier cloned bgn (T. harzianum) was removed using primers during reamplification of the gene. The amplicons were cloned into pTZ57R/T containing T overhangs at Eco321 site and transferred to E. coli DH5a and further to plant transformation vector pBI121 which was named as pRA121. In order to clone another gene (ech42) into pRA121, expression cassette from iHP vector was transferred to pRA121 and named as pRAG121. Further in order to gain XhoI site for cloning ech42 gene into pRAG121, ech42 (pSUM1) was cloned into pYES2/CT, named as pSAG1, ech42 from pSAG1 cloned with KpnI and XhoI in pRAG121 and named as pRAGS121. The vector constructed in the present study can be used to transform important crop plants to have enhanced resistance to fungal diseases. https://www.biotech-asia.org/vol14no3/gene-stacking-for-fungal-resistance-in-plant-transformation-vector/ Gene Stacking; Chitinase; Glucanase; Gene pyramiding; kb=Kilobase; bgn= beta glucanase; ech= Endochitinase; PCR= polymerase chain reaction