4.3. Construction of SlMBP21-RNAi and SlMBP21-SlMADS1-RNAi Vectors and Plant Transformation

In order to down-regulate SlMBP21 expression, a RNAi vector was generated using PHANNIBAL and pBIN19 vector. A 381 bp specific DNA fragment of SlMBP21 was amplified using SlMBP21-i-F/R primers (Table S1). After being purified, the amplified fragments were digested with the estriction enzyme Kpn I/Xho I and Hind III/Xba I, respectively. Then, the SlMBP21-RNAi vector (Figure S3A) was generated using the same method following our previous study described [30].

In order to repress the expression of SlMBP21 and SlMADS1 simultaneously in tomato, the double RNAi vector of SlMBP21 and SlMADS1 was constructed using pDH51, PHANNIBAL and pBIN19 vector. Firstly, the 381 bp SlMBP21-specific DNA fragments and the 515 bp SlMADS1 specific DNA fragments were amplified with the primers SlMBP21-1(p)-F/SlMBP21-1(X)-R and SlMADS1-1(X)-F/SlMADS1-1(B)-R (Table S1), respectively. Secondly, after being digested with pst I and Xba I, the purified SlMBP21 fragments were linked into the pDH51 plasmid at the pst I and Xba I restriction site to generate the pDH51-SlMBP21 vector. Thirdly, the digested SlMADS1 fragments using estriction enzyme Xba I and BamH I were cloned into the pDH51-SlMBP21 plasmid at the Xba I/BamH I restriction site to yield pDH51-SlMBP21-SlMADS1. Fourthly, the START DNA polymerase (Takara) was used to amplify the tandem fragment of SlMBP21 and SlMADS1 using the primers SlMBP21-2(K, H)-F/SlMADS1-2(X, B)-R (Table S1). Fifthly, the purified tandem fragments of SlMBP21-SlMADS1 were digested by Kpn I/Xho I and Hind III/BamH I and linked into the pHANNIBAL vector at the Kpn I/Xho I and the Hind III/BamH I restriction site, respectively. At last, the double-stranded RNA expression unit, possessing the cauliflower mosaic virus 35S promoter, the sense-orientated SlMBP21-SlMADS1 tandem fragment, PDK intron, the antisense-orientated SlMBP21-SlMADS1 tandem fragment, and the OCS terminator, was digested with Spe I/Sac I and cloned into pBIN19 vector to generate the Pbin19-SlMBP21-SlMADS1 vector (Figure S3B).

The obtained binary plasmids of SlMBP21 and SlMBP21-SlMADS1 were transferred into Agrobacterium tumefaciens (strain LBA4404), respectively. After that, the transformation of tomato cotyledon explants mediated by Agrobacterium tumefaciens was performed to obtain SlMBP21-RNAi lines and SlMBP21-SlMADS1-RNAi lines [82]. The tissue culture plants were screened by kanamycin and the PCR reactions were carried out to select the positive transgenic plants with the NPTII-F/R primers (Table S1).