Carrier of homozygous and dominant male sterility line, highly effective restoring line, and two-line method for breeding
A dominant and efficient technology applied in the field of plant gene factories
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Embodiment 1
[0022] Cloning of the sterile gene Barnase (BN) and its suppressor protein gene Barstar (BS)
[0023] (1) Cloning of nuclease BN gene
[0024] According to the data of Paddon, C.J et al., using BN-I 5'-CgggTACCATggCACAggTTATCAAC-3' and BN-II 5'-TCTAgAgCTCgAgTTATCTgATCTTTgTAAAgg-3' as primers, the genomic DNA of Bacillus amyloliquefaciens (preserved by the Institute of Microbiology, Chinese Academy of Sciences) digested with HindIII was The template was amplified by PCR, and the PCR conditions were denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 45 seconds, 52°C for 45 seconds, 72°C for 1 minute and 30 seconds, and extension at 72°C for 10 minutes. After the PCR product was digested with BamH I and SacI, it was connected to the pUC vector, transformed into E.coli JM109 (TaKaRa company) to obtain the vector pUC-BN with the BN gene, after sequencing, the nucleotide sequence of the BN gene is as follows: Shown in Seq.IDNo.1.
[0025] (2) Cloning of BN fragments for res...
Embodiment 2
[0030] Cloning of TA29, A9, GST27 promoters
[0031] (1) Cloning of tobacco anther-specific promoter TA29
[0032] According to the data of Seunick, J et al., PCR was carried out with PTA29-I 5′-CgggATCCATCTAgCTAAgTATAACTg-3′ and PTA29-II 5′-CATgCCATggTAgCTAATTTCTITAAg-3′ as primers, EcoRI and HindIII-digested genomic DNA of Nicotiana sativus as template Amplify. PCR conditions were denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 45 seconds, 54°C for 60 seconds, 72°C for 1 minute and 30 seconds, and extension at 72°C for 10 minutes. The specifically amplified DNA fragments are recovered. The sequence of TA29 is shown in Seq.ID No.5.
[0033] (2) Cloning of Arabidopsis anther-specific promoter A9
[0034] According to the Arabidopsis anther-specific expression promoter A9 sequence reported by Diane, L.H et al. (1993) and Wyatt, P et al. (1992), PA9-I 5'-gAgCTCgCggCCgCAAACAAACCATgTgTTACC-3' and PA9-II 5'-CTCgAgCCATggTAATTAgATACTATATTg -3' is the primer of A9; the ex...
Embodiment 3
[0038] Construction of a novel plant expression vector capable of chemically inducing homozygosity for male sterility in plants
[0039] (1) TA29-BN cassette:
[0040] 1) Digest pTvector-TA29 with BamH I and Nco I and recover the TA29 fragment, and connect it with the pUCBN plasmid vector that was also digested and recovered with BamH I and Nco I to form pUCTABN.
[0041] 2) Digest pUCTABN with BamHI and Sac I and recover the TA29BN small fragment, and connect it with the Rok II (Institute of Microbiology, Chinese Academy of Sciences) (with 35S) plasmid vector that is also digested with BamHI and Sac I to form Rok35sTN.
[0042] 3) Digest Rok35sTN with BamH I and Sac I and recover the small fragment of TA29BN (containing the Nos terminator), and pBin 19 (Institute of Microbiology, Chinese Academy of Sciences) (RB and LB) plasmids that were also digested with BamH I and Sac I and recovered The vector was ligated to form pB19TN.
[0043] (2)GST inducible cassette
[0044] 1) ...
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