Cloning of y3 gene encoding a tobacco mosaic virus inhibitor from Coprinus comatus and transformation to Nicotiana tabacum

Abstract:

Abstract: 【Objective】Since protein Y3 encoded by gene y3 reduced TMV activity, we intended to clone the full length of gene y3 in order to study gene y3 and its inhibitory function against TMV in in vivo conditions.【Methods】We amplified the unknown 5′- terminal cDNA sequence of gene y3 with 5′-RACE and using 5′- Full RACE Core Set (TaKaRa), and then the full length of gene y3 was obtained by RT-PCR amplification of total RNA of Coprinus comatus. The expression plasmid of y3 was constructed on the basis of gene y3 and transformed into Nicotiana tabacum via agrobacterium- mediation. 【Results】We obtained the full length of y3 successfully. It consisted of 534 bps including one ORF that encodes 130 amino acid residues. The cDNA sequence and its deduced amino acid sequence, which were obtained in this study, showed high similarity (up to 94%) to known fragment of gene y3 sequence. According to the full sequence, we cloned gene y3 and constructed the expression plasmids. The plasmids were constructed via inserting gene y3 sequence, CaMV 35S promoter, and NOS terminator, which were obtained from pBI121, at the MCS of pCAMBIA1301. The plasmids were used for gene transformation, resulting in transgenic Nicotiana tabacum plantlets. Gene y3 transcription was characterized by Northern blot analysis. Inoculation tests of the transgenic plantlets with TMV demonstrated inhibitory activity against TMV. 【Conclusion】Gene y3 transcription was expressed in transgenic tobacco plants. Gene y3 demonstrated some inhibitory function against TMV in vivo. The full length of gene y3 obtained in this study and its expression in Nicotiana tabacum plantlets could provide background information and benefits for future study on gene y3.