Plants having increased tolerance to herbicides
一种除草剂、植物的技术，应用在植物肽、植物细胞、植物产品等方向
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Embodiment 1
[0899] Example 1: Cloning of the gene encoding HPPD
[0900] (A) Cloning Arabidopsis HPPD
[0901] Using primers HuJ101 and HuJ102 (Table 5), the partial coding sequence of Arabidopsis AtHPPD (SEQ ID No: 52) was amplified from Arabidopsis cDNA by standard PCR technique.
[0902] Table 5: PCR primers (SEQ ID NO: 70, 71) for AtHPPD amplification
[0903] Primer name
Primer sequence (5'→3')
HuJ101
GGCCACCAAAACGCCG
HuJ102
TCATCCCACTAACTGTTTGGCTTC
[0904] According to the manufacturer's instructions, in the carrier (Invitrogen, Carlsbad, USA) to clone PCR products. The resulting plasmid was isolated from E. coli TOP10 by performing a mini-plasmid preparation Confirmed by DNA sequencing to encode N-terminal plus His 6 Label the AtHPPD expression cassette.
[0905] (B) Cloning of Chlamydomonas reinhardtii HPPD1
[0906] The Chlamydomonas reinhardtii HPPD1 (CrHPPD1) coding sequence (SEQ ID No: 54) was codon optimized for expressi...
Embodiment 2
[0935] Example 2 : Heterologous expression and purification of recombinant HPPD enzyme
[0936] The recombinant HPPD enzyme was produced and overexpressed in E. coli. Chemically competent BL21 (DE3) cells (Invitrogen, Carlsbad, USA) were used (see Example 1) or transformed with other expression vectors according to the manufacturer's instructions.
[0937] Transformed cells were incubated for 6 hours at 37°C in autoinduction medium (ZYM5052 supplemented with 100 μg / ml ampicillin) followed by 24 hours at 25°C.
[0938] in OD 600 (optical density at 600nm) 8 to 12, cells were harvested by centrifugation (8000xg). Cell pellets were resuspended in lysis buffer (50 mM sodium phosphate buffer, 0.5 M NaCl, 10 mM imidazole, pH 7.0) supplemented with EDTA-free intact protease inhibitor cocktail (Roche-Diagnostics) and homogenized using an AvestinPress. The homogenate was clarified by centrifugation (40,000 xg). Added His was purified by affinity chromatography on a ProtinoNi-...
Embodiment 3
[0939] Embodiment 3: the assay method of HPPD activity
[0940] HPPD from 4-hydroxyphenylpyruvate (4-HPP) and O 2 Produces homogentisic acid and CO 2 . The activity assay for HPPD is based on reverse phase HPLC analysis of homogentisate.
[0941] The assay mixture may contain 150 mM potassium phosphate buffer pH 7.0, 50 mM L-ascorbic acid, 100 μM catalase (Sigma-Aldrich), 1 μM FeSO in a total volume of 505 μl 4 and 0.2 units of purified HPPD enzyme. 1 unit is defined as the amount of enzyme required to produce 1 nmol of HGA per minute at 20 °C.
[0942] After 30 minutes of pre-incubation, the reaction was initiated by adding 4-HPP to a final concentration of 0.05 mM. The reaction was allowed to proceed for 45 minutes at room temperature. The reaction was stopped by adding 50 μl of 4.5M phosphoric acid. Samples were filtered using a 0.2 μM pore size PVDF filter unit.
[0943] Use 90% 20mM NaH 2 PO 4 5 μl of the clarified sample was analyzed on a UPLCHSST3 column (pa...
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