A kind of multi-gene assembly vector system and its multi-gene assembly method

[0097] Construction of acceptance vector

[0098] S1. Construction of acceptance vectors pYL0380 and pYL1300H

[0099] Such as Figure 5 As shown in A, the sequence (EcoR I / Sac I / Kpn I / I-Ceu I / I-Ceu I / BamH I / PI-Psp I / Sal I / Pst I / loxP / I-Sce I / loxP1R / I-Ppo I / Hind III, image 3 B, this sequence is as shown in SEQ ID NO.4), after EcoR I and Hind III double enzyme digestion, connect into pCAMBIA0380 and pCAMBIA1300 plasmid (Australian CAMBIA company) through EcoR I and Hind III double enzyme digestion, obtain accepting vector pYL0380 and acceptor vector pYL1300H.

[0100] S2. Construction of acceptance vector pYLTAC380

[0101] Such as Figure 5 As shown in B, the TAC vector pYLTAC747 plasmid (disclosed in the literature "Lin et al., 2003. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci., 100:5962-5967") is Template, carry out PCR amplification with primer P1 / P2 (primer 5' end contains XbaI site), ...

[0107] Construction of supply vector I and supply vector II

[0108] S1. Construction of supply vector I plasmid pYL322d1

[0109] Such as Figure 6 As shown in A, use primers P7 / P8 (the 5' ends of the primers contain BssH II / EcoR V sites and Mlu I sites respectively) and P9 / P10 (the 5' ends of the primers contain BssHII and Mlu I sites respectively), and use Figure 5 The pYL1300H and pYLSV plasmids constructed in A (disclosed in the literature "Lin et al., 2003. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci., 100:5962-5967") were used as templates Perform PCR amplification to obtain BssHII / EcoR V / pBR322 replicon / Mlu I fragment and BssHII / Cm r / Mlu I fragment (insert fragment I). The intermediate vector d1-I was obtained by double digestion with BssH II and Mlu I and ligation; the commercially synthesized fragment containing BssH II site BssH II / loxP / PI-Sce I / loxP2R / MCS / loxP1L / I-Sce I / BssH II...

[0113] Construction of marker-free supply plasmid

[0114] Such as Figure 7 As shown, use long primers P15 / P16 (the 5' end of the primers contain Xba I / Asc I / attB1 sites and HindIII / Xba I / attB2 sites respectively), and use the supply vector II plasmid pYL322d2 as a template for PCR amplification to obtain Xba I / Asc I / attB1 / pBR322 Replicon / Amp r The backbone fragment of / attB2 / HindIII / Xba I was cut and self-ligated with Xba I to obtain the intermediate vector pYLMF-0.

[0115] Using the DNA of rice variety Zhonghua 11 as a template, use primers P17 / P18 to amplify the promoter PV4 of the anther-specific expression gene Villin 4 (gene number Os04g0604000) as fragment 1, and use primers P19 / P20 to use Escherichia coli NS3529 genomic DNA as a template Amplify the Cre gene coding region (sequence is the same as GenBank No.DQ340306) to obtain fragment 2, use primers P21 / P22 to amplify the Nos terminator Tnos from plasmid pCAMBIA1305 (CAMBIA company) as fragment 3, and use the abov...