Esterase gene estZ, esterase gene estZ encoded protein and application of esterase gene estZ
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An esterase and gene technology, applied in the esterase gene estZ and its encoded protein and application fields, can solve problems such as ecosystem damage and threats to human life and health, improve the quality of agricultural products, solve the problem of excessive pesticide residues, and increase additional value effect
Active Publication Date: 2015-06-10
SOUTH CHINA AGRI UNIV
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On the other hand, the residual pesticides in the environment are enriched through the food chain and finally enter the human body, directly threatening human life and health; in addition, less than 20% of the pesticides applied to the field by humans are intercepted by plants, and more than 80% enter the environment Among them, not only t...
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Embodiment 1
[0038] Example 1 Esterase gene est Z clone (see figure 1 )
[0039] S1. Construction of transposon mutant library
[0040] Utilizing the principle of transposon mariner random insertion mutation, using the method of parental strain hybridization, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) strain PAO1 and transposon strain E. coli S17-1(pBT20) was used as the parent for parental conjugation, and the transposon mariner was randomly inserted into the PAO1 genome, and the mutant strains were screened by using the gentamicin resistance gene on the transposon and whether a hydrolysis circle was generated during the degradation process. In this way, mutant strains with loss of degradation function were obtained.
[0042] Design left-wing nested specific primers and right-wing nested specific primers based on the known insert sequence of the transposon mariner, combined with random degenerate primers suitab...
Embodiment 2
[0045] Example 2 Esterase gene est Z Highly expressed in BL21 (pET28a(+)) (see figure 2 )
[0046] S1. Esterase gene est Z PCR amplification
[0047] With forward primer P1:GC TCTAGA TGCCGGCGCAAATAGCCAT (SEQ ID NO.3) and reverse primer P2: CG GAATTC ATGTCTTCTCAAAGGGC (SEQ ID NO.4) is a primer, using PCR method from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) the esterase gene was amplified in the total DNA of strain PAO1 est Z fragment.
[0048] PCR amplification system:
[0049]
[0050] PCR amplification program:
[0051] Pre-denaturation at 95 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min and 30 s, after 34 cycles, extension at 72 °C for 5 min, and finally stored at 16 °C .
[0052] S2. The PCR product was digested with BamH I and EcoR I
[0053] Enzyme cutting system:
[0054]
[0055] Placed in a 37 °C water bath for more than 2 h.
[0056] The digested p...
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Abstract
The invention discloses an esterase gene estZ, an esterase gene estZ encoded protein and application of the esterase gene estZ. The esterase gene estZ has a total length of 1,206bp, has a G and C content of 65.2% and has a nucleotide sequence as shown in SEQ ID NO. 1. The esterase gene estZ is an encoded product and contains 401 amino acids, and the sequence of the amino acids is shown in SEQ ID NO. 2. The esterase gene estZ is capable of catalyzing and degrading ester bond containing pesticides like beta cypermethrin, lambda cyhalothrin, deltamethrin, cypermethrin, fenpropathrin, bifenthrin, kresoxim-methyl, pyraclostrobin and azoxystrobin. An enzyme preparation produced from the esterase gene estZ can be used for removing pesticide residue in agricultural products such as vegetables, fruits, tea leaves, cotton and tobacco so that the quality of the agricultural products can be improved and the additional value of the agricultural products can be increased. The esterase gene estZ can be further used for producing genetically modified crops so that the problem of excessive pesticide residue in agricultural products can be solved and pollution-free green agricultural products can be produced. The esterase gene estZ can be also used for restoring pesticide contaminated soil so that the problem of environmental pollution can be solved and the ecological environment and the human health can be protected.
Description
technical field [0001] The present invention relates to the fields of applied environmental microorganisms and agriculture, more specifically, relates to an esterase gene est Z and its encoded proteins and applications. It is specifically used to remove pesticide residues in crops and produce pollution-free green agricultural products; at the same time, it can be used to restore natural environments such as soil and water bodies polluted by pesticides. Background technique [0002] The invention and use of pesticides have made great contributions to the survival and development of human beings, especially in agricultural production, crop disease and insect pest control, and weed removal. one of the essential conditions. According to statistics, through the prevention and control of pesticides in my country every year, about 58.45 million tons of grain, 1.015 million tons of cotton, and 2.285 million tons of oilseeds are saved, and the annual direct economic loss is about 8...
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