Herbicide resistant gene and application in plant breeding
A technology of herbicides, plant cells, applied in the field of botany, which can solve the problem of insufficient tolerance to resist herbicide treatments
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Embodiment 1
[0327] Example 1 Generation of PaHPPD-M3 Mutant and Screening for Resistance to Herbicides
[0328] 1.1 Random mutagenesis and sequencing using error-prone PCR methods
[0329] By using the error-prone PCR method, several putative mutant colonies were obtained and cultured on LBT (LB+0.1% tyrosine) medium according to their ability to produce brown pigment during tyrosine metabolism , to evaluate the activity of HPPD.
[0330] Only a few putative mutants were able to produce brown pigment on LBT medium, and the putative mutants with HPPD activity were then sequenced and analyzed. Sequence analysis showed that selected mutants had mutations at different nucleic acid positions (Table 2). Compared with wild-type PaHPPD, six (M1, M2, M3, M4, M5, M7) mutants showed new amino acid substitutions in their polypeptides, while M6 only showed the base substitution G396A but no amino acid changes ( figure 1 ).
[0331] Table 2: PaHPPD mutants obtained by random mutagenesis (ep-PCR)
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Embodiment 2
[0336] Example 2 Generation of HPPD-DS18 Mutant and Screening for Resistance to Herbicides
[0337] The PaHPPD-M3 mutant obtained in Example 1 is shuffled with a DNA shuffling system to produce more HPPD mutants. And several putative mutant colonies were obtained. Sequencing analysis of the second-generation mutants (or Ds I mutants) with active HPPD revealed the mutation results (Table 3), and found that all these mutants also had a unique base mutation or amino acid substitution in the PaHPPD-M3 mutant .
[0338] Mutants with unique mutations or amino acid substitutions (DS7, DS8, DS18, DS23, and DS44) were compared with wild-type PaHPPD in the presence of triketone herbicides (sulcotrione, mesotrione, and tembotrione). The activity of HPPD was compared with the first generation mutant PaHPPD-M3.
[0339] The result is as Figure 4 and Figure 5 As shown, compared with the first-generation mutants (PaHPPD-M3) of all the second-generation mutants produced by the first ro...
Embodiment 3
[0342] Example 3 Generation of HPPD-DS73 Mutant and Screening for Resistance to Herbicides
[0343] Round 2 DNA shuffling mutants (PaHPPD-DS73) with active HPPD were cloned and screened for their amount of pigmentation in the presence of 100 μM HPPD inhibitors (sulcotrione, mesotrione, and tembotrione) The magnitude of herbicide resistance. It was observed that, in the presence of 100 μM HPPD inhibitors (sulcotrione, mesotrione and tembotrione), PaHPPD-DS73 showed (or retained) pigmentation in LBT medium ( Figure 6 ). Sequence analysis revealed a conservative mutation of the PaHPPD-M3 mutant in the PaHPPD-DS73 sequence and an additional amino acid substitution of alanine at 156 with valine (A156V) (Table E).
[0344] The HPPD activities of WT, PaHPPD-M3, PaHPPD-DS18 and PaHPPD-DS73 were qualitatively and quantitatively analyzed at 405 nm wavelength in the presence of 100 μM triketone herbicides (sulcotrione, mesotrione and tembotrione) , indicating that PaHPPD-M3, PaHPPD-D...
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