A kind of gene recombination Corynesium fumigatus if01 GM and its application
A Corynebacterium and gene recombination technology, applied in the application, recombinant DNA technology, fungi and other directions, can solve the problems of genetically improved insect-producing fungal strains and other problems, and achieve good popularization and application value, fast insecticidal effect, and good The effect of continuous development promotion
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[0047] Example 1 Construction of recombinant plasmid
[0048] The target gene used in the present invention-the TLR7 gene of Bemisia tabaci is obtained from the sequence of the B. tabaci transcriptome (Unigene database: Unigene20701_B4592195), and its nucleotide sequence is shown in SEQ ID NO: 10.
[0049] The present invention transforms a partial fragment of the TLR7 gene of Bemisia tabaci (the nucleotide sequence is shown in SEQ ID NO. 1) into a Corynespora fumigatus strain to obtain a genetically recombinant Corynespora fumigatus strain. The specific steps are as follows:
[0050] 1. Obtain the target fragment
[0051] (1) The primers TLR7F and TLR7R of the target gene used in the design of the present invention, namely the TLR7 gene of whitefly.
[0052] The nucleotide sequence of the primer TLR7F is shown in SEQ ID NO.2. The nucleotide sequence of TLR7R is shown in SEQ ID NO.3.
[0053] (2) Extract the total RNA of Bemisia tabaci type B by Trizol method, and synthesize the first s...
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[0065] Example 2 Construction and screening of recombinant strains
[0066] 1. Obtain the protoplast of Corynespora fumigatus IfB01 strain
[0067] The Corynespora fumigatus IfB01 strain used has been disclosed in the patent application number 201210449949.0.
[0068] The steps to obtain protoplasts are as follows:
[0069] (1) The conidia of the IfB01 strain of Corynespora fumigatus was prepared with 0.05% (v / v) Tween-80 solution to a concentration of 10 8 A spore suspension of about 1 / mL.
[0070] (2) Inoculate 1 mL of the suspension into 100 mL of Potato Dextrose Liquid Medium (PD), and culture it with shaking at 150 rpm for 30 h at 25°C.
[0071] (3) Take the bacterial solution in a centrifuge tube, centrifuge at 5,000 rpm for 10 min, discard the supernatant, wash twice with 0.7 mol / L NaCl solution, and then centrifuge to obtain mycelium.
[0072] (4) Add 1% (w / v) snail enzyme and 2% (w / v) cellulase mixed enzymolysis solution, enzymolyse the mycelium, and filter the enzymolysis soluti...
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[0091] Example 3 Toxicity test of recombinant Corynespora fumigatus If01 GM
[0092] Under indoor conditions, the detached hibiscus leaves with 2nd instar B-type Bemisia tabaci nymphs (not less than 30 nymphs per leaf) were separated into 2.0×10 7 Conidia / mL, 1.0×10 7 Conidia / mL, 5.0×10 6 Conidia / mL, 2.5×10 6 Conidia / mL, 1.25×10 6 A concentration gradient of 1 conidia / mL recombinant Corynespora fumigatus If01 GM conidia suspension immersed the leaves in the conidia suspension for 10 s, dried them, and put them in a petri dish (wet filter paper at the bottom, wet cotton ball wrapped around the base of the petiole) , Then put the petri dish into the light 14:10 (L:D), the temperature is 25±1℃, and the experiment is repeated 3 times.
[0093] Observe and record the death of the nymphs every 2 days, calculate the mortality and the half-lethal concentration (LC 50 ) And half-lethal time (LT 50 ).
[0094] The results are shown in Table 1, Table 2, and Table 3. The results showed tha...
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