Abstract：pFGC5941 is a widely used vector for RNA interference of plant genes, but its spacer is too long, and the restriction sites between the promoter and the spacer are not enough. In this study, a novel spacer based on the 2nd intron of Brassica napus PAP2 (BnPAP2I2) was used to substitute the original PhChsA spacer of pFGC5941, and an AatⅡ site was introduced between the promoter and the spacer, yielding an improved vector pFGC5941M. An RNAi fragment BTT1Ⅰ targeted to Brassica transparent testa 1 (TT1) gene family was cloned. Its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites using NcoⅠ+ AatⅡand BamHⅠ+ XbaⅠ double digestions, respectively. The resulted recombinant vector was pFGC5941M-BTT1Ⅰ(simplified as pBTT1I), which was verified by multiple-PCR checking and successfully transformed into Agrobacterium tumefaciens LBA4404 to form engineering strain. Construction of pBTT1Ⅰ will promote the clarification of the mechanism of development and pigment deposition in the seed coat and molecular breeding of yellow seed trait in Brassica.
