Tobacco Plant Axillary Bud Related Gene Cloning and RNAi Expression Vector Constructing and Transforming

In order to breed tobacco plant without axillary bud growing after topping, the fragment DNA sequence of tobacco axillary bud related gene (named as TLR gene) was cloned from flue-cured tobacco cv. K326 basing on homology gene cloning method, and its sequence was analyzed with BLAST search and Genebank homology comparison, it was found that that gene had high identity with LS gene of Lycopersicum esculentum, and had certain identity with MOC gene of Oryza sativa and LAS gene of Arabidopsis thaliana. The reverse and forward fragments (640 bp) in the reading frame of TLR gene were configured into right and left sides of intron in intermediated RNAi vector pHANNIBAL, respectively. After being cut by NotI' a fragment of about 4300 bp was recovered and inserted into a binary plasmid pART27, plant expression plasmid pHANNIBAL-TLR-PART27 with inverted repeated sequence containing TLR was successfully constructed. The pHANNIBAL-TLR-PART27 was introduced into Agrobacterium tumefaciens LBA4404, then transformed cells in tobacco leaf, but no transgenic plant was obtained, it was suspected that the expression of TLR gene was restrained.