CN100420751C - A kind of insecticidal gene and its use - Google Patents

Abstract

The invention discloses an insecticidal gene, the nucleotide sequence of which is SEQ ID NO:2. The present invention also discloses an insecticidal protein encoded by the above insecticidal gene, the amino acid sequence of which is SEQ ID NO:1. The novel insecticidal protein of the present invention has an amino acid sequence different from that of currently known insecticidal proteins, and it has high insecticidal activity against main agricultural pests, including armyworm, corn borer, beet armyworm and rice stem borer.

Description

A kind of insecticidal gene and its application

technical field

The invention relates to an insecticidal gene and its application.

Background technique

Insecticidal toxin is a protein with important application value. It can be used to modify insecticidal microbial pesticides and transgenic insect-resistant crops. At present, transgenic insect-resistant technology has been widely used commercially (James C.2004 ISAAA Briefs No.32.Ithaca, NY: ISAAA.43pp), which provides a low-cost new technology for pest control. At present, plant transgenic technology has matured, and the key to transgenic insect resistance is to obtain insecticidal proteins with excellent performance.

At present, there are many kinds of insecticidal proteins. More commonly used is Bt crystal protein, such as Cry1Ab, Cry1C, Cry2, Cry3 etc. (Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J. & Dean, D.H. 1998 Microbiol. Mol. Biol. Rev. 62, 807-813). The transgenic insect-resistant plants that have been commercialized now are all based on Bt crystal insecticidal protein. However, it has been found that a variety of insects can develop resistance to crystalline insecticidal proteins. The development of resistance affects the insect resistance properties and availability of these crystalline toxins (Ferre, J. & Van Rie, J. (2002) Annu. Rev. Entomol. 47, 501-533). The joint use or rotation of different insecticidal proteins without cross resistance can effectively slow down the occurrence of pest resistance (Zhao J-Z, Cao J, Li YX, Collins HL, Roush RT, Earle ED & SheltonAM.2003 Nature Biotechnol.21, 1493 -1497). Therefore, obtaining new insecticidal toxins has important application value for improving insecticidal ability and application.

Contents of the invention

Aiming at the deficiencies in the prior art, the invention provides a novel insecticidal gene and its application.

In order to achieve the above object, the present invention is achieved through such a technical scheme: a kind of insecticidal gene is provided, and the nucleotide sequence of the gene is SEQ ID NO:2.

The present invention also provides the insecticidal protein encoded by the above insecticidal gene, the amino acid sequence of the protein is SEQ ID NO:1.

The present invention also provides an insecticidal gene, which is synthesized using a partial fragment of SEQ ID NO:1.

The present invention also provides an insecticidal protein encoded by the above insecticidal gene. The amino acid sequence of the protein is: compared with SEQ ID NO: 1, it has more than 91% identity.

The present invention also provides the use of the above insecticidal gene.

The novel insecticidal protein of the present invention has an amino acid sequence different from that of currently known insecticidal proteins, and it has high insecticidal activity against main agricultural pests, including armyworm, corn borer, beet armyworm and rice stem borer.

Detailed ways

The methods of molecular biology used in the following examples of the present invention are all known techniques. Documents such as Current Protocols in Molecular Biology published by John Wiley and Sons, written by Ausubel, and Molecular Cloning: A Laboratory Manual, 3rd ED, published by Cold Spring Harbor Laboratory Press (2001) written by J. Sambrook, etc., have detailed descriptions.

Embodiment 1, the clone acquisition of gene and synthetic gene:

The steps of gene cloning are as follows:

1. Take 300 different Bt strains and culture them with TB culture solution at 28°C for 24 hours, then mix all the culture solutions, according to Reddy et al. (Reddy, A., Battisti, L. & Thorne, C.B. (1987) J. Bacteriol.169, 5263-5270) extract the plasmid of Bt. This plasmid is a mixture of plasmids from 300 different Bt strains.

2. Using the above-mentioned Bt plasmid as template DNA, PCR amplification was performed with primers StartN 5'atg aac aag aat aat act aaatta agc aca aga and StopC 5'cat gtt act cga gaa ttc tcg ag tta. A total of 30 cycles of PCR amplification were performed. The first five cycles are 95°C, 30 seconds; 50°C, 60 seconds; 72°C, 2 minutes 30 seconds, followed by 25 cycles: 95°C, 30 seconds; 60°C, 60 seconds; 72°C, 2 minutes 30 seconds Second.

3. The PCR product was analyzed by electrophoresis, and the DNA of about 2.4 kb was recovered and cloned into the T-vector produced by Shanghai Sangon. The obtained clone was sequenced, and the amino acid sequence of the encoded protein is shown in Sequence 1 (SEQ ID NO: 1).

According to the codon usage frequency of maize genes, the above amino acid sequence was reverse-translated into an artificial gene with high GC content, which was artificially synthesized by Shanghai Shenggong Company (as described in SEQ ID NO: 2) and cloned into BamH1 and Sac of the expression vector PET28a Between sites (plasmid pET28a-LG01).

Embodiment 2, the preparation of albumen:

The expression vector pET28a-LG01 containing the above genes was transformed into Escherichia coli BL21star by a general standard method, and a colony containing the plasmid pET28a-LG01 was obtained. A single colony was inoculated into 100 ml of LB bacterial culture medium, cultured with shaking at 37°C until OD ₆₀₀ =0.6, added IPTG to 0.5 mM, and continued to culture under the same conditions for 4 hours. The culture solution was centrifuged at 5000 g for 10 minutes to pellet E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 ml of 20mM Tris-HCl buffer solution to the precipitate and ultrasonically pulverize it for the determination of insecticidal activity.

Example 3, the protein expressed in Escherichia coli has killing effect on various Lepidoptera insects:

The protein obtained in Example 2 was coated on the surface of the artificial diet for insects, and after standing for 2 hours, the newborn first-instar larvae were connected to measure the insecticidal activity. The preparation method of the negative control was the same as in Example 2, but the plasmid was the pET28a vector itself without any inserted DNA. Under the condition of 25°C, record the number of insects killed after 7 days. The insecticidal activity results are as follows:

Armyworm corn borer beet armyworm this invention 100% 80% 100% negative control 0% 0% 0%

Embodiment 4, the protein expressed in rice callus has insecticidal activity:

Expression was obtained in rice callus through the following steps, and it was shown to have insecticidal activity:

1. The gene of the present invention was excised from pET28a-LGO1 (Example 1) using BamHI and SacI. The maize Ubiqutin promoter was obtained from the maize genome (with a HindIII site at the 5' and a BamHI site at the 3') by PCR, and treated with HindIII and BamHI. Vector pCAM1300 was treated with HindIII and SacI. The above three DNA fragments were subjected to ligase to obtain the pCAMUbi-LG01 plant transgenic plasmid.

2. Inject pCAMUbi-LG01 into rice callus by gene gun, and carry out screening and culture under 50mg/L hygromycin. After 20 days, the rice callus obtained from the screening culture was ultrasonically crushed in 20 mM Tris-HCl. After shaking and mixing, the insecticidal activity was measured.

3. The mixed sample is coated on the surface of the artificial diet for insects, and the newborn first-instar larvae are inoculated on the artificial diet to measure the insecticidal activity. The negative control is the rice callus without introducing the foreign gene. The activity measurement was carried out at 125°C for 7 days, and then the insect mortality was counted.

The result shows that the insecticidal efficiency of the transgenic rice callus to rice leaf roller, rice stem borer, armyworm and corn borer is 100%.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

SEQ ID NO: 1

Met Asn Lys Asn Asn Ser Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile Asp Tyr Phe

Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met Asn Met Ile Phe Lys Thr

Asp Thr Gly Gly Asn Val Thr Leu Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Glu

Ile Ser Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Glu Leu Ile Ala Gln Val Asn

Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ser Asn Glu Gln Asn Gln Val Leu

Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met Leu His Ile Tyr Leu Pro Lys

Ile Thr Ser Met Leu Ser Asp Val Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu

Tyr Leu Ser Lys Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val

Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr Val Asn

Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Thr Leu Lys Val Lys Lys Asp Ser

Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr

Lys Asn Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly

Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Ala Lys Glu Asn

Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn Phe Leu Ile Val Leu Thr Ala

Leu Gln Ala Lys Ala Phe Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Gly

Ile Asp Tyr Thr Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val

Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys Val Lys Gly

Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro Gly His Ala Leu Val Gly Phe

Glu Met Ser Asn Asp Ser Ile Thr Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn

Tyr Gln Val Asp Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Thr Asp Lys Leu Phe

Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe Pro Asn Glu Tyr

Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys Thr Leu Arg Tyr Glu Val Thr Ala

Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser

Glu Ala Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val

Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp Glu Asn Ser

Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu

Ser Asn Lys Glu Thr Lys Leu Ile Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu

Asn Gly Ala Ile Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr

Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys Asp Gly Gly

Phe Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys Thr Glu Tyr Val Ile Gln Tyr Thr

Val Lys Gly Lys Ala Ser Ile Tyr Leu Lys Asp Glu Lys Asn Asn Glu Gly Ile Tyr Glu

Glu Ile Asn Asn Asp Leu Glu Asp Phe Gln Thr Val Thr Lys Arg Phe Ile Thr Gly Thr

Asp Ser Ser Gly Val His Leu Ile Phe Thr Ser Gln Asn Gly Asp Glu Ala Phe Gly Gly

Asn Phe Ile Ile Ser Glu Ile Arg Ser Ser Glu Glu Leu Leu Ser Pro Glu Leu Ile Lys

Ser Asp Ala Trp Val Gly Ser Gln Gly Thr Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn

Ser Asn Ala Asn Gly Thr Phe Arg Gln Asn Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser

Met Asn Phe Asn Val Asn Gly Phe Ala Lys Val Thr Val Arg Asn Ser Arg Glu Val Leu

Phe Glu Lys Asn Phe Ser Gln Leu Ser Pro Lys Asp Tyr Ser Glu Lys Phe Thr Thr Ala

Ala Asn Asn Thr Gly Phe Tyr Val Glu Leu Ser Arg Gly Thr Gln Gly Gly Asn Ile Thr

Phe Arg Asp Phe Ser Ile Lys

SEQ ID NO: 2

1 ATGAACAAGA ACAACAGCAA GCTGAGCACC AGGGCCCTGC CGAGCTTCAT CGACTACTTC

61 AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC

121 GACACCGGCG GCAACGTGAC CCTGGACGAG ATCCTGAAGA ACCAGCAGCT GCTGAACGAG

181 ATCAGCGGCA AGCTGGACGG CGTGAACGGC AGCCTGAACG AGCTGATCGC CCAGGTGAAC

241 CTGAACACCG AGCTGAGCAA GGAGATCCTG AAGATCAGCA ACGAGCAGAA CCAGGTGCTG

301 AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGCACATCTA CCTGCCGAAG

361 ATCACCAGCA TGCTGAGCGA CGTGATGAAG CAGAACTACG CCCTGAGCCT GCAGATCGAG

421 TACCTGAGCA AGCAGCTGCA GGAGATCAGC GACAAGCTGG ACATCATCAA CGTGAACGTG

481 CTGATCAACA GCACCCTGAC CGAGATCACC CCGGCCTACC AGAGGATCAA GTACGTGAAC

541 GAGAAGTTCG AGGAGCTGAC CTTCGCCACC GAGACCACCC TGAAGGTGAA GAAGGACAGC

601 AGCCCGGCCG ACATCCTGGA CGAGCTGACC GAGCTGACCG AGCTGGCCAA GAGCGTGACC

661 AAGAACGACG TGGACGGCTT CGAGTTCTAC CTGAACACCT TCCACGACGT GATGGTGGGC

721 AACAACCTGT TCGGCAGGAG CGCCCTGAAG ACCGCCAGCG AGCTGATCGC CAAGGAGAAC

781 GTGAAGACCA GCGGCAGCGA GGTGGGCAAC GTGTACAACT TCCTGATCGT GCTGACCGCC

841 CTGCAGGCCA AGGCCTTCCT GACCCTGACC ACCTGCAGGA AGCTGCTGGG CCTGGCCGGC

901 ATCGACTACA CCAGCATCAT GAACGAGCAC CTGAACAAGG AGAAGGAGGA GTTCAGGGTG

961 AACATCCTGC CGACCCTGAG CAACACCTTC AGCAACCCGA ACTACGCCAA GGTGAAGGGC

1021 AGCGACGAGG ACGCCAAGAT GATCGTGGAG GCCAAGCCGG GCCACGCCCT GGTGGGCTTC

1081 GAGATGAGCA ACGACAGCAT CACCGTGCTG AAGGTGTACG AGGCCAAGCT GAAGCAGAAC

1141 TACCAGGTGG ACAAGGACAG CCTGAGCGAG GTGATCTACG GCGACACCGA CAAGCTGTTC

1201 TGCCCGGACC AGAGCGAGCA GATCTACTAC ACCAACAACA TCGTGTTCCC GAACGAGTAC

1261 GTGATCACCA AGATCGACTT CACCAAGAAG ATGAAGACCC TGAGGTACGA GGTGACCGCC

1321 AACTTCTACG ACAGCAGCAC CGGCGAGATC GACCTGAACA AGAAGAAGGT GGAGAGCAGC

1381 GAGGCCGAGT ACAGGACCCT GAGCGCCAAC GACGACGGCG TGTACATGCC GCTGGGCGTG

1441 ATCAGCGAGA CCTTCCTGAC CCCGATCAAC GGCTTCGGCC TGCAGGCCGA CGAGAACAGC

1501 AGGCTGATCA CCCTGACCTG CAAGAGCTAC CTGAGGGAGC TGCTGCTGGC CACCGACCTG

1561 AGCAACAAGG AGACCAAGCT GATCGTGCCG CCGAGCGGCT TCATCAGCAA CATCGTGGAG

1621 AACGGCGCCA TCGAGGAGGA CAACCTGGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC

1681 GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTGT ACGTGCACAA GGACGGCGGC

1741 TTCAGCCAGT TCATCGGCGA CAAGCTGAAG CCGAAGACCG AGTACGTGAT CCAGTACACC

1801 GTGAAGGGCA AGGCCAGCAT CTACCTGAAG GACGAGAAGA ACAACGAGGG CATCTACGAG

1861 GAGATCAACA ACGACCTGGA GGACTTCCAG ACCGTGACCA AGAGGTTCAT CACCGGCACC

1921 GACAGCAGCG GCGTGCACCT GATTCTTCACC AGCCAGAACG GCGACGAGGC CTTCGGCGGC

1981 AACTTCATCA TCAGCGAGAT CAGGAGCAGC GAGGAGCTGC TGAGCCCGGA GCTGATCAAG

2041 AGCGACGCCT GGGTGGGCAG CCAGGGCACC TGGATCAGCG GCAACAGCT GACCATCAAC

2101 AGCAACGCCA ACGGCACCTT CAGGCAGAAC CTGCCGCTGG AGAGCTACAG CACCTACAGC

2161 ATGAACTTCA ACGTGAACGG CTTCGCCAAG GTGACCGTGA GGAACAGCAG GGAGGTGCTG

2221 TTCGAGAAGA ACTTCAGCCA GCTGAGCCCG AAGGACTACA GCGAGAAGTT CACCACCGCC

2281 GCCAACAACA CCGGCTTCTA CGTGGAGCTG AGCAGGGGCA CCCAGGGCGG CAACATCACC

2341 TTCAGGGACT TCAGCATCAA GTAA

Claims (3)

1. An insecticidal gene is characterized in that: the nucleotide sequence of the gene is SEQ ID NO:2. 2. The insecticidal protein encoded by the insecticidal gene according to claim 1, characterized in that: the amino acid sequence of the protein is SEQ ID NO:1. 3. the purposes of insecticidal gene as claimed in claim 1, it is characterized in that: be used for killing armyworm, corn borer, beet armyworm or Chiloborer.