Use of insecticidal protein

Abstract

Related is a use of an insecticidal protein. The insecticidal protein may be used to control Ostrinia furnacalis (Hubern). A method for controlling Ostrinia furnacalis (Hubern) includes: allowing the Ostrinia furnacalis (Hubern) to be at least in contact with an ACe1 protein. In the present application, the ACe1 protein that can kill the Ostrinia furnacalis (Hubern) is produced in bacteria and/or a plant body to control the Ostrinia furnacalis (Hubern).

Claims

1. A method for controlling Ostrinia furnacalis (Hubern), comprising allowing the Ostrinia furnacalis (Hubern) to be at least in contact with an ACe1 protein; preferably, the ACe1 protein is present in a host cell that produces at least the Ace1 protein, and the Ostrinia furnacalis (Hubern) is in contact with at least the Ace1 protein by ingesting the host cell; and more preferably, the Ace1 protein is present in bacteria or a transgenic plant that produces at least the ACe1 protein, the Ostrinia furnacalis (Hubern) is in contact with at least the ACe1 protein by ingesting the bacterium or a tissue of the transgenic plant, and after contacting, the growth of the Ostrinia furnacalis (Hubern) is inhibited and/or death is caused, so as to achieve the control of the damage of the Ostrinia furnacalis (Hubern) to plants.

2. The method for controlling Ostrinia furnacalis (Hubern) according to claim 1, wherein the transgenic plant is corn or sorghum.

3. The method for controlling Ostrinia furnacalis (Hubern) according to claim 1, wherein the tissue of the transgenic plant is a root, a leaf, a stem, a tassel, an ear, an anther, or a filament.

4. The method for controlling Ostrinia furnacalis (Hubern) according to claim 1, wherein the ACe1 protein is an ACe1_3 protein, an ACe1_4 protein, an ACe1_5 protein, ACe1_6 protein, an ACe1_8 protein, an ACe1_9 protein, an ACe1_10 protein, an ACe1_11 protein, an ACe1_12 protein, an ACe1_13 protein, an ACe1_14 protein, an ACe1_15 protein, an ACe1_16 protein, an Ace1_17 protein, an ACe1_18 protein, an ACe1_19 protein, an ACe1_20 protein, or an ACe1_21 protein.

5. The method for controlling Ostrinia furnacalis (Hubern) according to claim 4, wherein the ACe1 protein has an amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:18.

6. The method for controlling Ostrinia furnacalis (Hubern) according to claim 4, wherein the ACe1 protein has an amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO: 36; and the ACe1 protein has a nucleotide sequence in the transgenic plant that are shown in any one of SEQ ID NO:43 to SEQ ID NO:60.

7. The method for controlling Ostrinia furnacalis (Hubern) according to claim 6, wherein the transgenic plant further comprises at least one second nucleotide different from the nucleotide encoding the ACe1 protein.

8. The method for controlling Ostrinia furnacalis (Hubern) according to claim 7, wherein the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, lectin, α-amylase, or a peroxidase.

9. The method for controlling Ostrinia furnacalis (Hubern) according to claim 7, wherein the second nucleotide encodes a Cry1Ab protein, a Cry2Ab protein, or a Cry1A.105 protein.

10. The method for controlling Ostrinia furnacalis (Hubern) according to claim 9, wherein the Cry1Ab protein, the Cry2Ab protein, or the Cry1A.105 protein has an amino acid sequence shown in SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, respectively.

11. The method for controlling Ostrinia furnacalis (Hubern) according to claim 7, wherein the second nucleotide has a nucleotide sequence shown in SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.

12. The method for controlling Ostrinia furnacalis (Hubern) according to claim 7, wherein the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.

13. A method of producing a plant for controlling Ostrinia furnacalis (Hubern), comprising introducing a polynucleotide sequence encoding an ACe1 protein into a genome of the plant.

14. The method of producing a plant for controlling Ostrinia furnacalis (Hubern) according to claim 13, wherein the polynucleotide sequence of the ACe1 protein is shown in any one of SEQ ID NO:43 to SEQ ID NO:60.

15. The method of producing a plant for controlling Ostrinia furnacalis (Hubern) according to claim 13, wherein the ACh1 protein has an amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO: 36.

16. A method of producing a plant seed for controlling Ostrinia furnacalis (Hubern), comprising hybridizing a first plant obtained by the method according to claim 13 with a second plant, so as to produce a seed containing a polynucleotide sequence encoding an ACe1 protein.

17. A method of cultivating a plant for controlling Ostrinia furnacalis (Hubern), comprising: planting at least one plant seed, wherein a genome of the plant seed comprises a polynucleotide sequence encoding an ACe1 protein; growing the plant seed into a plant; and growing the plant under conditions that the Ostrinia furnacalis (Hubern) is artificially inoculated and/or the hazard of the Ostrinia furnacalis (Hubern) naturally occurs, and harvesting a plant that has an attenuated plant damage and/or has an increased plant yield compared with other plants that do not have the polynucleotide sequences encoding the ACe1 protein.

18. The method of cultivating a plant for controlling Ostrinia furnacalis (Hubern) according to claim 17, wherein the polynucleotide sequence of the ACe1 protein is shown in any one of SEQ ID NO:43 to SEQ ID NO:60.

19. The method of cultivating a plant for controlling Ostrinia furnacalis (Hubern) according to claim 17, wherein the ACh1 protein has an amino acid sequence shown in any one of SEQ ID NO:19 to SEQ ID NO: 36.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0088] FIG. 1 is a construction flowchart of a recombinant expression vector DBN01-P containing an ACe1 nucleotide sequence for the use of the insecticidal protein according to the present application.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0089] The technical schemes of the use of the insecticidal protein of the present application are further described below by specific embodiments.

Example 1: Acquisition and Synthesis of Gene

[0090] 1. Acquisition of the Nucleotide Sequence

[0091] An amino acid sequence of an ACe1 insecticidal protein is shown in Table 1; and an ACe1 nucleotide sequence encoding the amino acid sequence corresponding to the ACe1 insecticidal protein is shown in Table 1.

TABLE-US-00001 TABLE 1 ACe1 protein and corresponding amino acid and nucleotide sequence thereof Insecticidal protein Amino acid Bacterial nucleotide Plant nucleotide name SEQ ID NO. SEQ ID NO. SEQ ID NO. ACe1_3 SEQ ID NO: 1 SEQ ID NO: 19 SEQ ID NO: 43 ACe1_4 SEQ ID NO: 2 SEQ ID NO: 20 SEQ ID NO: 44 ACe1_5 SEQ ID NO: 3 SEQ ID NO: 21 SEQ ID NO: 45 ACe1_6 SEQ ID NO: 4 SEQ ID NO: 22 SEQ ID NO: 46 ACe1_8 SEQ ID NO: 5 SEQ ID NO: 23 SEQ ID NO: 47 ACe1_9 SEQ ID NO: 6 SEQ ID NO: 24 SEQ ID NO: 48 ACe1_10 SEQ ID NO: 7 SEQ ID NO: 25 SEQ ID NO: 49 ACe1_11 SEQ ID NO: 8 SEQ ID NO: 26 SEQ ID NO: 50 ACe1_12 SEQ ID NO: 9 SEQ ID NO: 27 SEQ ID NO: 51 ACe1_13 SEQ ID NO: 10 SEQ ID NO: 28 SEQ ID NO: 52 ACe1_14 SEQ ID NO: 11 SEQ ID NO: 29 SEQ ID NO: 53 ACe1_15 SEQ ID NO: 12 SEQ ID NO: 30 SEQ ID NO: 54 ACe1_16 SEQ ID NO: 13 SEQ ID NO: 31 SEQ ID NO: 55 ACe1_17 SEQ ID NO: 14 SEQ ID NO: 32 SEQ ID NO: 56 ACe1_18 SEQ ID NO: 15 SEQ ID NO: 33 SEQ ID NO: 57 ACe1_19 SEQ ID NO: 16 SEQ ID NO: 34 SEQ ID NO: 58 ACe1_20 SEQ ID NO: 17 SEQ ID NO: 35 SEQ ID NO: 59 ACe1_21 SEQ ID NO: 18 SEQ ID NO: 36 SEQ ID NO: 60

[0092] 2. Synthesis of Above Nucleotide Sequence

[0093] Nucleotide sequences (as shown in SEQ ID NO:19 to SEQ ID NO:36 in the sequence listing) of the above 18 ACe1 proteins are synthesized by Nanjing Genscript Biotechnology Co., Ltd.

Example 2: Construction of Recombinant Expression Vector and Transformation of Recombinant Expression Vector into Escherichia coli to Obtain an ACe1 Protein

[0094] 1. Construction of Recombinant Expression Vector Containing an ACe1 Gene

[0095] The nucleotide sequences of the ACe1 proteins (ACe1_3 to ACe1_6, ACe1_8 to ACe1_21) synthesized in the Example 1 are linked into a protein expression vector pET28a (Novagen, USA, CAT: 69864-3); operation steps are performed according to the specification of the product pET28a vector of Novagen, so as to obtain recombinant expression vectors DBN01-P to DBN18-P; and a construction flow is shown in FIG. 1 (where Kan represents a kanamycin resistance gene, f1 ori represents the origin of replication of phage f1, Lad is a Lad initiation codon, ACe1_3 is an ACe1_3 nucleotide sequence (SEQ ID NO:19), and MCS represents multiple cloning sites).

[0096] The ACe1 protein and names of the corresponding recombinant expression vectors thereof are shown in Table 2.

TABLE-US-00002 TABLE 2 ACe1 protein and names of corresponding recombinant expression vectors thereof Insecticidal protein name Recombinant expression vector ACe1_3 DBN01-P ACe1_4 DBN02-P ACe1_5 DBN03-P ACe1_6 DBN04-P ACe1_8 DBN05-P ACe1_9 DBN06-P ACe1_10 DBN07-P ACe1_11 DBN08-P ACe1_12 DBN09-P ACe1_13 DBN10-P ACe1_14 DBN11-P ACe1_15 DBN12-P ACe1_16 DBN13-P ACe1_17 DBN14-P ACe1_18 DBN15-P ACe1_19 DBN16-P ACe1_20 DBN17-P ACe1_21 DBN18-P

[0097] 2. Transformation of Recombinant Expression Vector into Escherichia coli to Obtain ACe1 Protein

[0098] Then, the recombinant expression vectors DBNO1 to DBN18-P are transformed into Escherichia coli BL21(DE3) competent cells (Transgen, China, CAT: CD501) by a heat shock method; positive colonies are picked and placed in an LB liquid medium (10 g/L of a tryptone, 5 g/L of a yeast extract, 10 g/L of NaCl, 100 mg/L of an ampicillin, and pH is adjusted to 7.5 with NaOH); and cultured for 16 h at 37° C. and at 200 r/min. The culture solution is then transferred to an YT culture medium according to the proportion of 1:10; and culture is performed at 37° C. and at 200 r/min. When an OD=600 value of the culture solution reaches 0.6-0.8, IPTG is added until a final concentration is 0.5 mM, so as to perform inducible expression for 6h, and the culture solution is centrifuged to collect the cells; the supernatant is discarded, resuspending is performed after PBS is added, and then ultrasonic disruption is performed; and the expression protein is detected by SDS-PAGE, the protein concentration is estimated, and preservation is performed at −20° C. for later use.

Example 3, Identification of Inhibitory Activity Against Ostrinia furnacalis (Hubern) by Feeding the ACe1 Protein

[0099] Inhibitory activity against the Ostrinia furnacalis (Hubern), the Agrotis ipsilon (Rottemberg) and the Mythimna separate (Walker) is detected with the series of ACe1 proteins (ACe1_3 to ACe1_6, ACe1_8 to ACe1_21) obtained in part 2 of Example 2. A total of 18 treatments are designed for each pest, which respectively are ACe1_3 to ACe1_6, ACe1_8 to ACe1_21; and 1 negative control treatment is designed, which is GFP. Protein liquid of ACe1_3 to ACe1_6, ACe1_8 to ACe1_21, and GFP are respectively mixed in feed, and a final concentration is 50 μg/g. Each group of treatments is repeated for 3 times.

TABLE-US-00003 TABLE 3 Inhibitory activity results of Ostrinia furnacalis (Hubern), Agrotis ypsilon (Rottemberg) and Mythimna separate (Walker) that are fed with the ACe1 protein Serial Test insect Mythimna number Ostrinia furnacalis Agrotis ypsilon separate of proteins (Hubern) (Rottemberg) (Walker) ACe1_3 + S S ACe1_4 + S S ACe1_5 + S S ACe1_6 + S S ACe1_8 + S S ACe1_9 + S S ACe1_10 + S S ACe1_11 + S S ACe1_12 − − − ACe1_13 + S S ACe1_14 + S S ACe1_15 − − − ACe1_16 + S S ACe1_17 + S S ACe1_18 + S S ACe1_19 − − − ACe1_20 − − − ACe1_21 − − − GFP − − − “+” represents that there is a lethal effect; “−” represents that there is no inhibitory activity; ″NT″ stands for not tested; and ″S″ represents developmental inhibition

[0100] Results in Table 3 show that, ACe1_3 to ACe1_6, ACe1_8 to ACe1_11, ACe1_13, ACe1_14, ACe1_16 to ACe1_18 proteins show desirable inhibitory activity against the Ostrinia furnacalis (Hubern), and only show the effect of developmental inhibition on the Agrotis ipsilon (Rottemberg) and the Mythimna separate (Walker).

[0101] Therefore, it indicates that the ACe1 proteins (ACe1_3 to ACe1_6, ACe1_8 to ACe1_11, ACe1_13, ACe1_14, ACe1_16 to ACe1_18) show resistance activity against the Ostrinia furnacalis (Hubern), and this activity is sufficient to have adverse effects on the growth of the Ostrinia furnacalis (Hubern), so that the Ostrinia furnacalis (Hubern) can be controlled in the fields. In addition, it is also possible to reduce the occurrence of diseases on the transgenic ACe1 plants by controlling the damage of the Ostrinia furnacalis (Hubern), thereby greatly improving the yield and quality of the transgenic ACe1 plants.

[0102] In conclusion, through the use of the insecticidal protein of the present application, ACe1 protein that can kill the Ostrinia furnacalis (Hubern) is produced in a plant body to control the Ostrinia furnacalis (Hubern). Compared with an agricultural control method, a chemical control method, a physical control method and a biological control method used in the prior art, the present application achieves the protection of whole growth period and whole plant on the plants so as to control the infestation of the Ostrinia furnacalis (Hubern), and is pollution-free, residue-free, stable in effect, thorough, simple, convenient and economical.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical schemes of the present application and not to limit them. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical schemes of the present application may be modified or equivalently replaced without departing from the spirit and scope of the technical schemes of the present application.