Investigation of miRNA-induced gene silencing technique in petunia
An MIGS construct designed to silencing CHS genes was introduced into Petunia plants. Petals of opening flower buds of a transgenic line showed CHS gene silencing phonotype was used to extract total RNA. One microgram high-quality total RNA of MIGS-chs transgenic petals was ligated to 5’- and 3’- adaptors, reverse transcribed, and amplified by PCR (12 cycles) using a TruseqTM Small RNA sample prep Kit (Illumina) according to the manufacturer’s protocol. Small RNA library was purified by electrophoresis on a 6% Novex TBE PAGE gel (Invitrogen). After quantified by TBS380 (Picogreen), nucleotide sequence of amplified cDNA was analyzed using Illumina Hiseq2500 (1× 51bp read length).