CN115449520A

CN115449520A - Insect-resistant gene and glyphosate-resistant gene expression vector and application thereof

Info

Publication number: CN115449520A
Application number: CN202110641143.0A
Authority: CN
Inventors: 王东芳; 张意红
Original assignee: Hangzhou Fangyun Biotechnology Co ltd
Current assignee: Hangzhou Fangyun Biotechnology Co ltd
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2022-12-09

Abstract

The invention discloses an insect-resistant gene and glyphosate-resistant gene expression vector and application thereof, wherein the expression vector contains an insect-resistant gene cry1Ab, an insect-resistant gene cry2Ae and a glyphosate-resistant gene cp4. According to the invention, a chloroplast signal peptide sequence is added in front of the Cry2Ae gene, so that on one hand, the expression quantity of Cry2Ae protein is increased, and the increase amplitude is 20% -100%; on the other hand, the interference of the Cry2Ae protein with high expression quantity on the growth and development of plants is reduced. Meanwhile, the carrier simultaneously expresses Cry1Ab, cry2Ae and Cp4 proteins, and transgenic crops obtained by transforming crops with the carrier can simultaneously resist main lepidoptera pests of crops such as Spodoptera frugiperda, cotton bollworm, beet armyworm, corn borer, chilo suppressalis and the like, thereby widening the insect-resistant range of the transgenic crops, effectively delaying the generation of pest resistance and simultaneously enabling the transgenic crops to have the capability of resisting glyphosate.

Description

Insect-resistant gene and glyphosate-resistant gene expression vector and application thereof

(I) the technical field

The invention belongs to the field of cultivation of new varieties of transgenic plants, and particularly relates to an expression vector for simultaneously and efficiently expressing two insect-resistant genes and a glyphosate-resistant gene and application thereof in cultivation of insect-resistant and glyphosate-resistant plant cells and plants.

(II) background of the invention

The rapid global population growth has driven a substantial increase in food demand. In fact, grain production has been significantly increased over the past few decades by conventional variety improvement and cultivation technology improvements, particularly the use of pesticides. The cellular genetics and the cellular biology technology which arose in the last 60 s of the century triggered a green revolution, which significantly improved the yield of grain crops and significantly enhanced the disease and insect resistance. However, the widespread adoption of these technologies also brings new problems to the environment and human health, such as increased soil erosion due to the use of pesticides and fertilizers. Meanwhile, after the technologies are used for 20 years, the contribution to grain yield increase is also obviously reduced. To overcome these problems, scientists from governments, universities, research institutions, and corporations are seeking new techniques and methods. With the rapid development of DNA technology, scientists have developed new modern breeding techniques and methods up to the last 80 th century, of which transgenic technology using agrobacterium-mediated transformation and biolistic transformation was the most successful.

With the development of biotechnology and the increase of investment of various countries in the field of biotechnology, global breeders and biotechnology research institutions can break through the traditional breeding framework and breeding years to cultivate new plant varieties. The approval of the commercial planting of transgenic insect-resistant cotton was approved in the united states in 1995, the promotion of transgenic insect-resistant cotton was started in 1996, and the introduction of transgenic insect-resistant cotton was started in 1996 in china. In 2019-2021, agricultural transgenic organism safety certificates of insect-resistant herbicide-tolerant corn and soybean are examined and approved for the first time in China, and the fact that transgenic organism breeding technology enters an accelerated stage is marked. In addition, spodoptera frugiperda has invaded China from Yunnan in 2018-2019 as a omnivorous pest, the pest is high in harmfulness and strong in drug resistance, and has serious harm to crops such as corn in China at present, and the pest such as Spodoptera frugiperda is urgently prevented and treated by a biological breeding method.

Bacillus thuringiensis (Bacillus thuringiensis) is a common gram-positive soil microorganism that produces an insecticidal protein with high selectivity for a particular organism. The safety test for Bt protein has been carried out for decades, and it is non-toxic to human and animals. The Cry1Ab gene is cloned from Bacillus thuringiensis HD-1 strain, and its expression product Cry1Ab protein has broad spectrum insecticidal activity in Lepidoptera pests (Hfte H and Whiteley H R. Microbiological Reviews,1989,53 (2): 242-255.; van Frankenhuyzen K. J Invertebr Pathol 2009,101 (1): 1-16). By 1 month 2012, a total of approximately 70 Cry2 proteins were identified, covering a9 subclass from Cry2Aa to Cry2Ai, and the majority of the Cry2Ae class, with Cry2Ae proteins being only toxic to lepidopteran insects, and Cry2Ae being more virulent than Cry2Aa to cotton bollworms of america (Zheng a et al. Although the molecular weight of the Cry2 protein is smaller than that of Cry1, the Cry2 has wider insecticidal range to lepidoptera compared with Cry1 toxin, and has asymmetric cross resistance with Cry1, so that the Cry2 protein has a greater application prospect in the aspect of agricultural pest control. In recent years, second generation transgenic plants based on cry2Ae monovalent gene or cry2Ae + cry1Ac bivalent gene are gradually replacing first generation transgenic plants based on cry1A type gene to alleviate the adaptation of target pests to insect-resistant plants and the risk of resistance, such as transgenic cotton Bollgard II (Tabashnik B E et al. Appl. Environ. Microbiol.2002,68 (8): 3790-3794.). The Cry1Ab has extremely low amino acid similarity with a Cry2Ae protein functional region, and is only 17.9 percent. Therefore, the two have greatly different insecticidal spectrums, are not easy to generate delivery resistance, and are very suitable for combined use.

Weeds are the second most important factor affecting crop production. It competes with crops for water resources, fertilizers, light sources, etc., causing a decrease in crop yield and also reducing the quality of agricultural products. The traditional manual weeding method is time-consuming and labor-consuming, has low efficiency and high mechanical weeding cost, and chemical weeding has high technical requirements and extremely high possibility of causing phytotoxicity to plants, influencing the growth and development of the crops, and simultaneously possibly influencing adjacent sensitive crops.

At present, transgenic approaches to glyphosate tolerance in plants are the most widely used weed solution. The glyphosate, the chemical name of which is N- (methyl phosphate) glycine, is an organic phosphine herbicide, is a systemic conduction type broad-spectrum biocidal herbicide, and has long-term safe use records of more than 40 years. The glyphosate-tolerant trait can be conferred on plants by expressing EPSPS genes such as CP4 (derived from Agrobacterium CP4 strain, encoding CP4EPSPS protein) (Patgette, SR et al 1996.Pages 53-84in Herbicide-Resistant Crops) and genes such as Gat (derived from glyphosate N-acetyltransferase of soil microorganism metagenome) which can express enzymes degrading glyphosate.

The method for simultaneously expressing multiple target proteins by plants mainly comprises a multiple expression frame molecule cloning and stacking method, a hybridization stacking method and a gene fusion method, wherein the multiple expression frame molecule cloning and stacking method is widely used due to the advantages of good stability, strong predictability, easy plant transformation in the later period and the like. The effect of the multiple expression cassette stacking strategy is influenced by many factors, including gene selection, coding sequence selection, selection of regulatory elements including promoters, terminators, etc., for each expression cassette, and the arrangement of reading frames. These factors all have different effects on the expression level and stability of the target gene among different generations of expression patterns, especially on the condition of high-generation gene silencing. Therefore, an expression vector capable of efficiently and stably expressing a plurality of target genes needs to be strictly explored and tested, and the vector plays a decisive role in obtaining stable and efficient transgenic crops.

Disclosure of the invention

The invention aims to provide a binary vector for stably and efficiently expressing insect-resistant genes (cry 1Ab and cry2 Ae) and glyphosate-resistant genes (cp 4 genes) at the same time, and application thereof in preparing insect-resistant and glyphosate-resistant plant cells and crops, so that corresponding transgenic crops have wider insecticidal spectrum, the generation resistance of insects to insecticidal proteins is delayed, and the insects are simultaneously glyphosate-resistant.

The technical scheme adopted by the invention is as follows:

the invention provides an expression vector for simultaneously stably and efficiently expressing an insect-resistant gene and a herbicide-tolerant glyphosate gene, which contains an insect-resistant gene cry1Ab, an insect-resistant gene cry2Ae and a glyphosate-tolerant gene cp4.

Furthermore, the amino acid sequence coded by the insect-resistant gene cry1Ab is shown in SEQ ID No:2, the nucleotide sequence is optimized by a codon, and the sequence is SEQ ID No:1 is shown in the specification; the amino acid sequence of the insect-resistant gene cry2Ae code is shown as SEQ ID No:4, the nucleotide sequence is optimized by a codon, and the sequence is SEQ ID No:3 is shown in the figure; the amino acid sequence coded by the glyphosate-tolerant gene cp4 is shown as SEQ ID No:6, the nucleotide sequence is optimized by a codon, and the sequence is SEQ ID No:5, respectively.

SEQ ID No:1cry1Ab Gene

atggacaacaacccgaacatcaacgagtgcatcccgtacaactgcctctccaacccggaggtggaggtgctcggcggcgagcgcatcgagaccggctacaccccgatcgacatctccctctccctcacccagttcctcctctccgagttcgtgccgggcgccggcttcgtgctcggcctcgtggacatcatctggggcatcttcggcccgtcccagtgggacgccttcctcgtgcagatcgagcagctcatcaaccagcgcatcgaggagttcgcccgcaaccaggccatctcccgcctggagggcctctccaacctctaccagatctacgccgagtccttccgcgagtgggaggccgacccgaccaacccggccctccgcgaggagatgcgcatccagttcaacgacatgaactccgccctcaccaccgccatcccgctcttcgccgtgcagaactaccaggtgccgctcctctccgtgtacgtgcaggccgccaacctccacctctccgtgctccgcgacgtgtccgtgttcggccagcgctggggcttcgacgccgccaccatcaactcccgctacaacgacctcacccgcctcatcggcaactacaccgaccacgccgtgcgctggtacaacaccggcctggagcgcgtgtggggcccggactcccgcgactggatcaggtacaaccagttccgccgcgagctcaccctcaccgtgctcgacatcgtgtccctcttcccgaactacgactcccgcacctacccgatccgcaccgtgtcccagctcacccgcgagatctacaccaacccggtgctggagaacttcgacggctccttccgcggctccgcccagggcatcgagggctccatccgctccccgcacctcatggacatcctcaactccatcaccatctacaccgacgcccaccgcggcgagtactactggtccggccaccagatcatggcctccccggtgggcttctccggcccggagttcaccttcccgctctacggcacgatgggcaacgccgccccgcagcagcgcatcgtggcccagctcggccagggcgtgtaccgcaccctctcctccaccctctaccgccgcccgttcaacatcggcatcaacaaccagcagctctccgtgctcgacggcaccgagttcgcctacggcacctcctccaacctcccgtccgccgtgtaccgcaagtccggcaccgtggactccctcgacgagatcccgccgcagaacaacaacgtgccgccgcgccagggcttctcccaccgcctctcccacgtgtccatgttccgctccggcttctccaactcctccgtgtccatcatccgcgccccgatgttctcctggattcaccgctccgccgagttcaacaacatcatcccgtcctcccagatcacccagatcccgctcaccaagtccaccaacctcggctccggcacctccgtggtgaagggcccgggcttcaccggcggcgacatcctccgccgcacctccccgggccagatctccaccctccgcgtgaacatcaccgccccgctctcccagcgctaccgcgtgcgcatccgctacgcctccaccaccaacctccagttccacacctccatcgacggccgcccgatcaaccagggcaacttctccgccaccatgtcctccggctccaacctccagtccggctccttccgcaccgtgggcttcaccaccccgttcaacttctccaacggctcctccgtgttcaccctctccgcccacgtgttcaactccggcaacgaggtgtacatcgaccgcatcgagttcgtgccggccgaggtgaccttcgaggccgagtacgacctggagcgcgcccagaaggccgtgaacgagctcttcacctcctccaaccagatcggcctcaagaccgacgtgaccgactaccacatcgaccaggtgtccaacctcgtggagtgcctctccgacgag。

SEQ ID No:3cry2Ae Gene

atgaacaacgtgctcaacaacggccgcaccaccatctgcgacgcctacaacgtggtggcccacgacccgttctccttcgagcacaagtccctcgacaccatccgcaaggagtggatggagtggaagcgcaccgaccactccctctacgtggccccgatcgtgggcaccgtgtcctccttcctcctcaagaaggtgggctccctcatcggcaagcgcatcctctccgagctctggggcctcatcttcccgtccggctccaccaacctcatgcaggacatcctccgcgagaccgagcagttcctcaaccagcgcctcaacaccgacaccctcgcccgcgtgaacgccgagctggagggcctccaggccaacatccgcgagttcaaccagcaggtggacaacttcctcaacccgacccagaacccggtgccgctctccatcacctcctccgtgaacaccatgcagcagctcttcctcaaccgcctcccgcagttccgcgtgcagggctaccagctcctcctcctcccgctcttcgcccaggccgccaacatgcacctctccttcatccgcgacgtggtgctcaacgccgacgagtggggcatctccgccgccaccctccgcacctaccagaactacctcaagaactacaccaccgagtactccaactactgcatcaacacctaccagaccgccttccgcggcctcaacacccgcctccacgacatgctggagttccgcacctacatgttcctcaacgtgttcgagtacgtgtccatctggtccctcttcaagtaccagtccctcctcgtgtcctccggcgccaacctctacgcctccggctccggcccgcagcagacccagtccttcacctcccaggactggccgttcctctactccctcttccaggtgaactccaactacgtgctcaacggcttctccggcgcccgcctcacccagaccttcccgaacatcggcggcctcccgggcaccaccaccacccacgccctcctcgccgcccgcgtgaactactccggcggcgtgtcctccggcgacatcggcgccgtgttcaaccagaacttctcctgctccaccttcctcccgccgctcctcaccccgttcgtgcgctcctggctcgactccggctccgaccgcggcggcgtgaacaccgtgaccaactggcagaccgagtccttcgagtccaccctcggcctccgctgcggcgccttcaccgcccgcggcaactccaactacttcccggactacttcatccgcaacatctccggcgtgccgctcgtggtgcgcaacgaggacctccgccgcccgctccactacaacgagatccgcaacatcgagtccccgtccggcaccccgggcggcctccgcgcctacatggtgtccgtgcacaaccgcaagaacaacatctacgccgtgcacgagaacggcaccatgatccacctcgccccggaggactacaccggcttcaccatctccccgatccacgccacccaggtgaacaaccagacccgcaccttcatctccgagaagttcggcaaccagggcgactccctccgcttcgagcagtccaacaccaccgcccgctacaccctccgcggcaacggcaactcctacaacctctacctccgcgtgtcctccctcggcaactccaccatccgcgtgaccatcaacggccgcgtgtacaccgcctccaacgtgaacaccaccaccaacaacgacggcgtgaacgacaacggcgcccgcttcctcgacatcaacatgggcaacgtggtggcctccgacaacaccaacgtgccgctcgacatcaacgtgaccttcaactccggcacccagttcgagctcatgaacatcatgttcgtgccgaccaacctcccgccgatctac。

SEQ ID No:5cp4 gene

atgctacacggtgcaagcagccggccggcaaccgctcgcaaatcttccggcctttcgggaacggtcaggattccgggcgataagtccatatcccaccggtcgttcatgttcggcggtcttgccagcggtgagacgcgcatcacgggcctgcttgaaggtgaggacgtgatcaataccgggaaggccatgcaggctatgggagcgcgtatccgcaaggaaggtgacacatggatcattgacggcgttgggaatggcggtctgctcgcccctgaggcccctctcgacttcggcaatgcggcgacgggctgcaggctcactatgggactggtcggggtgtacgacttcgatagcacgttcatcggagacgcctcgctcacaaagcgcccaatgggccgcgttctgaacccgttgcgcgagatgggcgtacaggtcaaatccgaggatggtgaccgtttgcccgttacgctgcgcgggccgaagacgcctaccccgattacctaccgcgtgccaatggcatccgcccaggtcaagtcagccgtgctcctcgccggactgaacactccgggcatcaccacggtgatcgagcccatcatgaccagggatcataccgaaaagatgcttcaggggtttggcgccaacctgacggtcgagacggacgctgacggcgtcaggaccatccgccttgagggcaggggtaaactgactggccaagtcatcgatgttccgggagacccgtcgtccacggccttcccgttggttgcggcgctgctcgtgccggggagtgacgtgaccatcctgaacgtcctcatgaacccgaccaggaccggcctgatcctcacgcttcaggagatgggagccgacatcgaggtgatcaacccgcgcctggcaggcggtgaagacgttgcggatctgcgcgtgcgctcctctaccctgaagggcgtgacggtcccggaagatcgcgcgccgtccatgatagacgagtatcctattctggccgtcgccgctgcgttcgccgaaggggccacggtcatgaacggtcttgaggaactccgcgtgaaggaatcggatcgcctgtcggcggtggccaatggcctgaagctcaacggtgttgactgcgacgagggtgagacctcactcgtggtccgtggccggcctgatggcaagggcctcggcaacgccagtggagcggccgtcgccacgcacctcgatcatcgcatcgcgatgtccttcttggtgatgggtctcgtctcagagaacccggtgaccgtcgatgacgccacgatgatagcgacgagcttcccagagttcatggatctgatggcgggcctcggggccaagatcgaactgtctgacacgaaggccgcttga。

The expression vector of the invention constructs two insect-resistant protein coding genes cry1Ab and cry2Ae with very low homology and a glyphosate-resistant gene cp4 into the same plant expression vector, so that corresponding transgenic crops have wider insecticidal spectrum, the generation of resistance of insects to insecticidal proteins is delayed, and the glyphosate-resistant genes are simultaneously glyphosate-resistant. The carrier can transfer the three genes into a receptor plant at the same time, so that the plant has the characteristics of resisting the main lepidoptera pests such as spodoptera frugiperda, cotton bollworm, spodoptera exigua, corn borer and the like and resisting glyphosate herbicide. Cry1Ab has good killing effect on spodoptera frugiperda, ostrinia nubilalis and other lepidoptera pests, and can effectively solve the control problem of main lepidoptera pests of crops by combining the excellent killing effect of Cry2Ae on cotton bollworms and other lepidoptera pests. In particular, spodoptera frugiperda has invaded China from Yunnan in recent years, and the character of the vector endowed with plants is very critical to effectively controlling spodoptera frugiperda.

The glyphosate-tolerant gene comprises an EPSPS gene insensitive to glyphosate and an acetyl transferase gene with detoxification effect on glyphosate. The glyphosate-tolerant gene can be used as a selection marker and can also endow plants with glyphosate-tolerant characters, and optionally, other herbicide-tolerant genes can be overexpressed to realize the functions, such as a glufosinate-tolerant gene (bar/pat) (Wehrmann A et al nat Biotechnol.1996,14 (10): 1274-8), a nicosulfuron-tolerant gene (CYP 81A 9) (Liu X.et al. Theor Appl Gene t.2019,132 (5): 1351-1361), and the like.

The expression vector of the invention comprises promoters, which comprise a maize ubiquitin promoter, a composite promoter p35S-intron consisting of CaMV35S promoter and the second intron of maize actin2, and can also be other strong constitutive promoters, such as rice ubiquitin promoter (Wang J, oard J H. Plant Cell Reports, 2003.), cassava veinmosaicvirus (CsVMV) promoter, australianbannanacarea virus (BSV) promoter, mirabilismosis virus (MMV) promoter, and the like. In addition, the expression of a target gene can be enhanced by using elements such AS enhancers and introns, for example, MAR (Matrix attachment regions) sequence of tobacco (Dolgova AS, dolgov SV.Biotech.2019,9 (5): 176.), intron of rice actin gene (Oszvald M et al Biotechnology and Bioprocess Engineering,2007,12 (6): 676-683.), intron of maize HSP70 gene, etc. (Brown S M, santino C G.CA2100A1.1993). The nucleotide sequence of the ubiquitin promoter pZmUbi is shown as SEQ ID No:11, the nucleotide sequence of the composite promoter p35S-intron is shown as SEQ ID No:7, wherein the sequence of p35S is shown as SEQ ID No:7, the second intron sequence of the maize Actin2 gene is shown as SEQ ID No: indicated by 787bp-905bp in 7. The invention also provides another p35S-intron2 composite promoter which is sequentially a p35S promoter and a first intron sequence of a corn HSP70 gene from 5 'end to 3', wherein the nucleotide sequence of the p35S promoter is SEQ ID No:7, and the nucleotide sequence of the first intron of corn HSP70 is shown in SEQ ID No: shown at 12.

SEQ ID No：7p35S-intron

atggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgataacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtggcacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagaccttcctctatataaggaagttcatttcatttggagaggacacgctgaaatcaccagtctctctctacaaatctatctctctcgaggttggttctgctgtccggttgtcaatcctttagctaccatacatgtgtttcagttttcttttgcctgtttattcatttctgatttaataaactgatgggattttgatgccaaacacag

The expression vector of the invention also comprises a terminator, and the terminator can be a terminator derived from a plant, or a terminator derived from viruses and other organisms, or an artificially synthesized terminator. The terminator comprises a terminator Tnos (nucleotide sequence shown in SEQ ID No: 9) of the Agrobacterium tumefaciens nopaline synthase gene and a CaMV35S terminator T35S.

Furthermore, the expression vector also comprises a chloroplast signal peptide sequence used for enhancing the expression of the cry2Ae gene, wherein the signal peptide comprises a chloroplast signal peptide sequence (a nucleotide sequence is shown as SEQ ID No: 10) of a maize EPSPS gene or a chloroplast signal peptide sequence (a nucleotide sequence is shown as SEQ ID No: 8) of a rice EPSPS gene.

SEQ ID No:8 chloroplast signal peptide of rice EPSPS gene

atggcggcgaccatggcgtccaacgctgcggctgcggctgcggtgtccctggaccaggccgtggctgcgtcggcagcgttctcgtcgcggaagcagctgcggctgcctgccgcagcgcgcggagggatgcgggtgcgggtgcgggcgcggggtcggcgggaggcggtggtggtggcgtccgcgtcgtcgtcgtcggtggcagcgccggcggcgaaggctgag

SEQ ID No:9Tnos terminator

cccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc

SEQ ID No:10 maize EPSPS gene chloroplast signal peptide sequence

atggcggccatggcgaccaaggccgccgcgggcaccgtgtcgctggacctcgccgcgccgtcgcgccgccaccaccgcccgagctcggcgcgcccgcccgcccgccccgccgtccgcgggctgcgggcgcctgggcgccgcgtgatcgccgcgccgccggcggcggcagcggcggcggcggtgcaggcgggtgccgag

SEQ ID No:11 maize pUBI

ctacagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcag

Further, the expression vector comprises a maize ubiquitin promoter (ZmUbipromoter) pZmUbi which starts the expression of the insect-resistant gene cry1Ab, a terminator Tnos of the Agrobacterium tumefaciens nopaline synthase gene which terminates the expression of the gene; a maize ubiquitin promoter (ZmUbipromoter) pZmUbi for starting the expression of the insect-resistant gene cry2Ae, chloroplast signal peptide of a maize EPSPS gene, and a terminator Tnos for terminating the expression of the gene, namely, an Agrobacterium tumefaciens nopaline synthase gene; the composite promoter p35S-intron consisting of the CaMV35S promoter for starting the expression of the glyphosate-tolerant gene cp4 and the second intron of the maize actin2, the chloroplast signal peptide of the rice EPSPS gene and the CaMV35S terminator T35S for terminating the expression of the gene.

Furthermore, the expression vector also comprises a substrate attachment region sequence for improving the expression of the insect-resistant gene or the herbicide-resistant gene, wherein the substrate attachment region sequence is a tobacco MAR sequence, and the nucleotide sequence is SEQ ID No:13, the expression of the insect-resistant gene or the herbicide-resistant gene is further improved by adding a matrix attachment region sequence (MAR) to the 5 'end of the promoter or the 3' end of the terminator of the expression vector target gene.

The basic vector used to provide the backbone of the expression vector in the present invention may be a pCambia series of vectors (CAMBIA, canberra, aus trilia) or other vectors, preferably pCambia1300 vector.

The expression vector comprises a promoter-cry 2Ae gene-promoter-cry 1Ab gene-promoter-cp 4 gene; or MAR-promoter-cry 2Ae gene-promoter-cry 1Ab gene-promoter-cp 4 gene.

The invention also provides an application of the expression vector in preparing transgenic plant cells, wherein the application comprises the steps of electrically shocking and transforming the expression vector (1300-p 35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae or 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae) to the agrobacterium EHA105 to obtain an agrobacterium strain containing the expression vector, and infecting plants with the agrobacterium strain to obtain plant cells containing the expression vector.

The invention also provides a plant cell containing the T-DNA for stably and efficiently expressing the insect-resistant glyphosate-tolerant gene, and the glyphosate-tolerant gene cp4 is used as a screening marker in the culture of the plant transgenic cell.

The invention also provides a transgenic plant containing the T-DNA for stably and efficiently expressing the insect-resistant glyphosate-tolerant gene.

The invention further provides a method for obtaining plant cells or plants simultaneously stably and efficiently expressing the insect-resistant and glyphosate-resistant gene by using the vector, wherein the method can be an agrobacterium-mediated transformation method, a gene gun method, a protoplast infection method or other plant genetic transformation methods, and preferably the agrobacterium-mediated transformation method.

The expression vector constructed by the invention is suitable for expression of monocotyledons or dicotyledons, wherein the monocotyledons comprise: corn, rice, etc.; dicotyledonous plants include: soybean, rape, cotton, etc.

The transgenic plant has lepidoptera pest resistance, and comprises spodoptera frugiperda, cotton bollworm, spodoptera exigua, ostrinia nubilalis, chilo suppressalis and the like.

Compared with the prior art, the invention has the following beneficial effects: according to the invention, a chloroplast signal peptide sequence is added in front of the Cry2Ae gene, so that the expression quantity of Cry2Ae protein is increased by 20-100%; on the other hand, the interference of the Cry2Ae protein with high expression quantity on the growth and development of plants is reduced. Meanwhile, the carrier simultaneously expresses Cry1Ab, cry2Ae and Cp4 proteins, transgenic crops obtained by transforming crops with the carrier can simultaneously resist main lepidoptera pests of crops such as spodoptera frugiperda, cotton bollworm, spodoptera exigua, corn borer, chilo suppressalis and the like, the insect resistance range of the transgenic crops is widened, particularly, the transgenic crops have very good resistance to spodoptera frugiperda invading China, the generation of pest resistance is effectively delayed, the transgenic crops have the glyphosate resistance, and the transgenic crops with the composite character accord with the development direction of the current transgenic crops and can better meet the requirement of large-scale agricultural production.

Description of the drawings

FIG. 1: a structural schematic diagram of a vector T-DNA for simultaneously expressing cry1Ab, cry2Ae and cp4 genes.

pZmUbi represents the maize ubiquitin promoter; CTP1 and CTP2 represent different chloroplast signal peptides respectively; p35S-intron represents a composite promoter consisting of a CaMV35S promoter and a plant gene intron; cry2Ae represents the cry2Ae gene and terminator; cry1Ab denotes cry1Ab gene and terminator; cp4 denotes the cp4-EPSPS gene and the terminator.

FIG. 2 is a schematic diagram: schematic structural diagram of vector T-DNA for increasing MAR regulatory sequence and simultaneously expressing cry1Ab, cry2Ae and cp4 genes.

MARs represent sequences of Matrix attachment regions (Matrix attachment regions) of plants, having functions of stabilizing and enhancing gene expression; pZmUbi represents the maize ubiquitin promoter; CTP1 and CTP2 represent different chloroplast signal peptides respectively; p35S-intron represents a composite promoter consisting of a CaMV35S promoter and a plant gene intron; cry2Ae represents the cry2Ae gene and terminator; cry1Ab stands for cry1Ab gene and terminator; cp4 denotes the cp4-EPSPS gene and terminator.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto:

it is within the scope of the present invention to modify or replace the steps, methods or conditions of the present invention without departing from the spirit of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 selection of promoters

To obtain a better promoter than p35S, we increased expression efficiency by fusing the second intron of the maize Actin2 gene 3' to the p35S promoter as follows:

1. T-DNA vector construction:

(1) T-DNA vector construction containing composite promoter p35S-intron

Artificially synthesizing a nucleotide sequence OsCPT-cp4 which sequentially comprises a chloroplast signal peptide coding sequence (OsCPT) of the rice EPSPS gene and a glyphosate-tolerant gene cp4 (optimized by codons) from a 5 'end to a 3', wherein the chloroplast signal peptide sequence of the rice EPSPS gene is shown as SEQ ID No:8, the nucleotide sequence of the cp4 gene after codon optimization is shown as SEQ ID No:5, respectively.

Using corn genome (B73) as template, cloning second intron segment ZmActin2-intron of corn Actin2 gene by PCR (I-F: 5'-CTCTCTCGAGGTTGGTTCTGCTGTCCGGTTGTC; I-R:5' -CATGTGTGGCAGATCTGTGTTTGGCATCAAAAATC), and the nucleotide sequence is shown as SEQ ID No:7 as shown in 788bp-905 bp;

using artificially synthesized nucleotide sequence OsCPT-cp4 containing chloroplast signal peptide coding sequence (OsCTP) of rice EPSPS gene and cp4 gene from 5 'end to 3' end as template, and cloning OsCTP-cp4 (primer is cp4-F:5 '-GATGCCAAACAGATCTGCACCATGGCGAC; cp4-R:5' -GGAGCCTCGAGTTATCAGCGGCCTTCGTGTCAGAC) by PCR;

using the PCR products ZmActin2-intron and ZmCTP-cp4 as templates, cloning Actin2-intron-OsCT P-cp4 fragment (I-F: 5'-CTCTCTCGAGGTTGGTTCTGCTGTCCGGTTGTC; cp4-R:5' -GGAGCCTCGAGTTATCAGCGGCCTTCGTCGTCAGAC) by PCR, and setting an XhoI site at the 5 'end and the 3' end of the sequence respectively.

Carrying out enzyme digestion on the pCambia1300 vector by using restriction enzyme XhoI, and simultaneously carrying out dephosphorylation treatment on the vector by using FastAP to prevent self-ligation; the nucleotide fragment Actin2-intron-OsCTP-cp4 obtained by the PCR was digested with XhoI. Then, the vector 1300-p35S-intron-cp4 is obtained after the enzyme-cut vector and the fragment are connected. The promoter p35S is a self-contained fragment on a pCambia1300 vector, and the nucleotide sequence of the self-contained fragment is shown as SEQ ID No: the 1bp-787bp of 7 (i.e., 8714bp-7934bp on pCambia1300 vector (NCBI SEQ ID NO: AF 234296).

(2) Construction of T-DNA vector containing p35S promoter

As a comparison, the nucleotide fragment OsCPT-cp4 artificially synthesized and containing rice EPSPS gene chloroplast signal peptide coding sequence (OsCTP) and cp4 gene is used as a template, and the OsCTP-cp4 fragment (a primer is cp4-F2:5'-TCTCTCGAGACCATGGCGGCGACCATGGCGTCC; cp4-R:5' -GGAGCCTCGAGTTATCAAGCGGCCCTTCGTCGTCAGAC) is cloned by PCR, wherein a XhoI site is respectively arranged at the 5 'end and the 3' end of the sequence. Carrying out enzyme digestion on the pCambia1300 vector by using restriction enzyme XhoI, and simultaneously carrying out dephosphorylation treatment on the vector by using FastAP to prevent self-ligation; and (3) connecting the nucleotide fragment OsCTP-cp4 obtained by the PCR with XhoI and the OsCTP-cp4 fragment obtained by enzyme digestion with a pCambia1300 vector to obtain the vector 1300-p35S-cp4.

3. T-DNA vector for transforming agrobacterium

And (3) electrically shocking the T-DNA vectors (1300-p 35S-intron-cp4 and 1300-p35S-cp 4) obtained in the step (2) to transform the agrobacterium EHA105, screening by adopting kan to obtain an agrobacterium strain containing the T-DNA vectors, adding glycerol, and storing to a refrigerator of-80 ℃ for infection of transgenic crops.

4. Rice transformation:

the method for obtaining transgenic rice adopts the prior art (Luzhong, gong ancestor Xun. Life sciences 1998, 10. Molecular plant breeding 2003, 1. Mature and full 'Xishui-134' seeds are selected to be hulled, and callus is generated by induction and is used as a transformation material.

And (3) respectively drawing the agrobacterium containing the T-DNA vectors (1300-p 35S-intron-cp4 and 1300-p35S-cp 4) constructed in the step (3). Selecting a single colony, inoculating the single colony to a culture medium, and culturing at 28 ℃ until OD600 is 0.6 to obtain agrobacterium liquid serving as agrobacterium for transformation. The composition of the culture medium is as follows: 3g/L K ₂ HPO ₄ 、1g/LNaH ₂ PO ₄ 、1g/LNH ₄ Cl、0.3g/L MgSO ₄ ·7H ₂ O、0.15g/L KCl、0.01g/L CaCl ₂ 、0.0025g/L FeSO ₄ ·7H ₂ O, 5g/L sucrose, 20mg/L acetosyringone, solvent is water, pH =5.8.

Putting the callus to be transformed into agrobacterium liquid with OD600 of 0.6 for culturing, leading agrobacterium to be combined with the surface of the callus, then transferring the callus into a co-culture medium (MS +2 mg/L2, 4-dichlorophenoxyacetic acid (2, 4-D) +30g/L glucose +30g/L sucrose +3g/L agar (sigma 7921) +20mg/L acetosyringone), and co-culturing for 2-3 days at 28 ℃. The transformed callus was rinsed with sterile water, transferred to selection medium (MS +2 mg/L2, 4-D +30g/L sucrose +3g/L agar (Sigma 7921) +20mg/L acetosyringone +2mM glyphosate (Sigma)), and selected for two months at 28 ℃ (once with intermediate subculture). After screening, the calli with good growth activity are transferred to a pre-differentiation medium (MS +0.1g/L inositol +5mg/L abscisic acid (ABA) +1mg/L naphthylacetic acid (NAA) +5 mg/L6-benzyladenine (6-BA) +20g/L sorbitol) +30g/L sucrose +2.5g/L plant gel (gelrite)), and cultured for 20 days at 28 ℃, then the pre-differentiated calli are transferred to the differentiation medium, and differentiated germination is carried out for 14 hours every day. After 2-3 weeks, the resistant regenerated plants are transferred to rooting medium (1/2MS +0.2mg/L NAA +20g/L sucrose +2.5g/L gelrite) for strong seedlings to root, and finally the regenerated plants are washed off and the agar is transplanted to the greenhouse. Transgenic rice plants containing vectors 1300-p35S-intron-CP4 and 1300-p35S-CP4 were designated ICP and CP, respectively.

5. Analysis of the expression level of the target gene cp 4:

respectively selecting 20 plants of ICP and CP transgenic rice plant leaves with good growth vigor and identified as single copy through molecular biology for different generations, and analyzing the expression quantity of the CP 4in the obtained transgenic rice plant leaves through an ELISA method. ELISA assay was carried out using the detection kit of the Hippocampus Biotech Co.Ltd. (cat # AA 0841). The experimental method is carried out according to the instruction of the kit, and the specific steps are as follows:

(1) The CP4EPSPS standard sample concentration is diluted to 2.5ppb, 1.25ppb, 0.625ppb and 0.3125ppb;

(2) Adding 100 μ l of diluted standard sample into each hole of the enzyme-linked plate, sealing the hole of the enzyme-linked plate with Parafilm, incubating in a horizontal oscillator at room temperature in a slow shading manner for 45min, and preventing liquid in the hole from spilling or splashing on the sealing film;

(3) After incubation, the liquid in the wells was quickly poured out, each well was thoroughly washed 3 times with 250 μ l Washing Buffer (PBST Buffer), and the plate was spin-dried on absorbent paper;

(4) Adding 100 μ l of enzyme labeling solution into each well, shaking, sealing the enzyme linked plate with Parafilm membrane, and incubating in horizontal oscillator at room temperature in shade at slow speed for 45min;

(5) After incubation, the liquid in the wells was quickly poured out, each well was thoroughly washed 3 times with 250 μ l Washing Buffer (PBST Buffer), and the plate was spin-dried on absorbent paper;

(6) Adding 100 μ l of Substrate to the wells and shaking, sealing the enzyme-linked plate with Parafilm, incubating for 15-30min, during which the solution in the wells gradually changed from colorless to blue;

(7) Add 100. Mu.l of stop solution to the well and mix well, the solution turns from blue to yellow, OD450 was measured in microplate reader within 30 min. And drawing a standard curve graph by taking the absorbance value of the standard sample as a vertical coordinate and the concentration (ppb) of the CP4EPSPS standard sample as a horizontal coordinate according to the OD450 value of the standard sample. To exclude systematic errors between each measurement, a standard curve was made simultaneously for each measurement sample.The standard curve formula of the test is as follows: y =0.026x +0.114 (R) ² ＝0.9906)。

Diluting the crude protein sample (such as 200-500 times) as experimental sample, testing OD450 with standard sample detection method, and diluting the sample and detecting again if the sample OD450 value is out of the standard sample OD450 range; if the sample OD450 value is within the standard sample OD450 range, substituting the absorbance value of the sample into the standard curve, and reading the concentration corresponding to the sample from the standard curve, the concentration of the target protein in the sample can be calculated, and the result is shown in Table 1.

CP4 protein content (μ g/g) = sample concentration (ppb) × dilution factor × sample extract volume (μ L)/leaf mass (mg)/1000

Table 1: analysis of p35S-intron promoter-mediated target gene expression level

Remarking: ICP represents the transgenic plant of the vector 1300-p35S-intron-cp4, and cp represents the transgenic plant of the vector 1300-p35S-cp4. Data in the table represent the mean ± standard deviation of the expression levels of 20 independent transgenic material. * Indicates a significant difference from the control at a significant level of 0.1% by t.test.

Test results show that the expression level of the cp 4in the leaves of the transgenic rice plants obtained by the transformation vector 1300-p35S-intron-cp4 is obviously higher than that of the cp 4in the leaves of the transgenic rice plants obtained by the transformation vector 1300-p35S-cp4. Therefore, the promoter p35S-intron is considered to be capable of mediating high expression of a target gene in plants such as rice better than p 35S. We therefore chose to use the promoter p35S-intron to carry out subsequent experiments.

Example 2 cloning of chloroplast signal peptide sequence of maize EPSPS Gene mediating cry2Ae expression

1. T-DNA vector construction

Artificially synthesizing a chloroplast signal peptide coding sequence (ZmCTP) of a maize EPSPS gene, a nucleotide sequence of a cry2Ae gene and a nucleotide sequence ZmCTP-cry2Ae-Tnos of a Tnos terminator sequence in sequence from a 5 'end to a 3', wherein the nucleotide sequence of the cry2Ae gene is shown as SEQ ID No:3, the chloroplast signal peptide sequence of the EPSPS gene of the maize is shown as SEQ ID No:10, the nucleotide sequence of the Tnos terminator is shown as SEQ ID No: shown at 9.

The sequence ZmCTP-cry2Ae-Tnos (primer 2Ab-F:5 '-TTTAGGATCCATGCGGCCATGGCGACCAAG; 2Ab-R:5' -CCCGGCTGGTTACCGATAGACGACACC) is cloned by PCR with artificially synthesized ZmCTP-cry2Ae-Tnos fragment as a template, and a BamHI site and a KpnI site are respectively arranged at the 5 'end and the 3' end of the sequence.

The maize UBI promoter pZmUbi (primer UBI-F2:5 '-CAGTGCCAAGCTTCTACAGGCAGCGTGACCCCGGTC; primer UBI-R2:5' -ATGGTGGATCCCTGCAAGAAGTAACAAACAACAGGGT) is cloned by PCR by taking a maize genome (B73) as a template, and the nucleotide sequence is shown as SEQ ID No: shown at 11. A HindIII site and a BamHI site are respectively arranged at the 5 'end and the 3' end of the promoter. The nucleotide sequence fragment obtained by PCR cloning is denoted as pZmUbi2.

Carrying out double enzyme digestion on the pCambia1300 vector by using restriction enzymes HindIII and BamHI; carrying out double enzyme digestion on the maize UBI promoter pZmUbi2 cloned by PCR by using HindIII and BamHI; the ZmCTP-cry2Ae-Tnos fragment cloned by PCR was double digested with BamHI and KpnI. Recovering the carrier and the two fragments, and obtaining the final carrier 1300-pZmUbi2-ZmCTP-cry2Ae after three-section connection of the carrier and the fragments.

As a control, the sequence cry2Ae-Tnos (primer 2Ab-F:5 '-CTGCAGGGATCCACATAACAACGTGCTCAACAAC; 2Ab-R:5' -CCCGGCTGGTTACCGATAGACAC) was cloned by PCR using an artificially synthesized ZmCTP-cry2Ae-Tnos fragment as a template, and a BamHI site and a KpnI site were placed at the 5 'end and the 3' end of the sequence, respectively. The final vector 1300-pZmUbi2-cry2Ae was obtained by ligating cry2Ae-Tnos and pZmUbi2 cleavage products into pCambia1300 vector by the same method as above.

The obtained T-DNA vectors (1300-pZmUbi 2-ZmCTP-cry2Ae and 1300-pZmUbi2-cry2 Ae) are electrically shocked to transform the agrobacterium EHA105, kan screening is adopted to obtain agrobacterium strains containing the T-DNA vectors, glycerol is added, and the agrobacterium strains are stored in a refrigerator with the temperature of-80 ℃ for being used for infection of transgenic crops.

2. Transformation of rice

The transformation procedure was identical to that of example 1, except that the glyphosate in the selection medium was replaced with 50mg/L hygromycin (Hyg). Transgenic rice plants containing the vectors 1300-pZmUbi2-ZmCTP-cry2Ae and 1300-pZmUbi2-cry2Ae were obtained and named CTP2Ae and 2Ae, respectively.

3. Analysis of expression level of target Gene

Respectively selecting 20 plants with good growth vigor and identified as single-copy CTP2Ae and 2Ae transgenic rice plant leaves by molecular biology for different generations, and analyzing Cry2Ae expression quantity in the obtained transgenic rice by an ELISA method. ELISA was measured using a detection kit (ENVIROLOGIX, cat. No.: AP-005-CT NW V10). The experimental method is carried out according to the instruction of the kit, and the specific steps are as follows:

(1) The standard sample is diluted to 2.5ppb, 1.25ppb, 0.625ppb and 0.3125ppb to be used as a standard sample;

(2) Adding standard samples with different concentrations into each hole of the enzyme-linked plate, respectively adding 100 μ l of standard samples with different concentrations, sealing the holes of the enzyme-linked plate by using a Parafilm, and incubating for 45min in a horizontal oscillator in a slow shading manner at room temperature, wherein liquid in the holes is prevented from spilling or splashing on a sealing film;

(6) Add 100. Mu.l of Substrate to the wells and shake well, seal the plate with Parafilm, incubate for 15-30min, during which time the solution in the wells gradually changes from colorless to blue;

(7) Adding 100 μ l stop solution into the well, mixing, changing the solution from blue to yellow, and measuring OD450 value in a microplate reader within 30min. And drawing a standard curve graph by taking the absorbance value of the standard as a vertical coordinate and the concentration (ppb) of the Cry2Ae standard as a horizontal coordinate according to the OD450 value of the standard sample. To exclude systematic errors between each measurement, a standard curve was made simultaneously for each measurement sample. The standard curve formula of the test is as follows: y =0.701x +0.089 (R) ² ＝0.9926)。

Appropriately diluting (such as 200-500 times) the protein samples extracted from the leaves of the transgenic rice plants of different generations, and if the OD450 value of the sample is out of the range of the OD450 of the standard sample, diluting the sample again and detecting again; if the sample OD450 value is within the standard sample OD450 range, substituting the absorbance value of the sample into the standard curve, and reading the concentration corresponding to the sample from the standard curve, the concentration of the target protein in the sample can be calculated, and the result is shown in Table 2.

Cry2Ae protein content (μ g/g) = sample concentration (ppb) × dilution multiple × sample extract volume (μ L)/leaf mass (mg)/1000

Table 2: zmCTP mediated target gene expression level analysis

Remarking: CTP2Ae represents the transgenic plant of the vector 1300-pZmUbi-ZmCTP-cry2Ae, and 2Ae represents the transgenic plant of the vector 1300-pZmUbi2-cry2Ae. Data in the table represent the mean ± standard deviation of the expression levels of 20 independent transgenic material. * Indicates a significant difference from the control at a significant level of 0.1% by t.test.

Test results show that the expression level of cry2Ae in the transgenic rice plant leaves obtained by the transformation vector 1300-pZmUbi2-ZmCTP-cry2Ae is obviously higher than the expression level of cry2Ae in the transgenic rice plant leaves obtained by the transformation vector 1300-pZmUbi2-cry2Ae. Therefore, the maize EPSPS chloroplast signal peptide ZmCTP is considered to be capable of effectively improving the high expression of the cry2Ae gene of the target protein in rice and other plants. We therefore increased ZmCTP in subsequent expression vector constructions.

Example 3 construction of expression vector containing insect-resistant protein coding genes cry1Ab, cry2Ae and Glyphosate-resistant gene cp4

In order to construct a vector for simultaneously and efficiently expressing cry1Ab, cry2Ae and cp4 genes, a nucleotide sequence OsCPT-cp4 which is a chloroplast signal peptide coding sequence (OsCPT) of a rice EPSPS gene and a glyphosate-resistant gene cp4 (optimized by codons) is artificially synthesized from a 5 'end to a 3' end; the artificially synthesized nucleotide sequence from 5 'end to 3' of chloroplast signal peptide coding sequence (ZmCTP) of EPSPS gene, nucleotide sequence of cry2Ae gene and nucleotide sequence ZmCTP-cry2Ae-Tnos of Tnos terminator sequence. In addition, a nucleotide fragment cry1Ab-Tnos which is composed of a cry1Ab gene nucleotide sequence and a Tnos terminator sequence in sequence from 5 'end to 3' is artificially synthesized, wherein the nucleotide sequence of the cry1Ab gene is shown as SEQ ID No:1, and the nucleotide sequence of the Tnos terminator is shown as SEQ ID No. 9.

1. Constructing a glyphosate-tolerant gene cp4 expression frame:

the same procedure as in example 1 was repeated except for using the T-DNA vector 1300-p35S-intron-cp4.

2. Constructing an expression frame of an insect-resistant gene cry1 Ab:

the nucleotide sequence was cloned by PCR using an artificially synthesized nucleotide fragment containing the cry1Ab gene and the Tnos terminator in sequence from 5 'end to 3' as a template (primer 1Ab-F:5 '-GCAGGATCCACCATGGACAACCGAGATCAC; 1Ab-R:5' -CCGCCGGAATTCGATCTAGTAACATAGATACACACAC), and a BamHI site and an EcoRI site were provided at 5 'end and 3' end of the sequence, respectively.

The maize UBI promoter pZmUbi (primer UBI-F1:5 '-CTAGATCGGTACCCCTACAGTGCAGCGTGACCCCGGTC; primer UBI-R1:5' -ATGGTGGATCCTGCAGAAGTAACACAAACAACAGGGT) is cloned by PCR by taking a maize genome (B73) as a template, and the nucleotide sequence is shown as SEQ ID No: as shown in fig. 11. A KpnI site and a BamHI site are respectively arranged at the 5 'end and the 3' end of the promoter. The nucleotide sequence fragment obtained by PCR cloning is denoted as pZmUbi1 (the sequence is the same as pZmUbi2, and the restriction enzyme cutting site is different).

3. Constructing an expression frame of an insect-resistant gene cry2 Ae:

the same procedure as in example 2 was repeated except for using the T-DNA vector 1300-pZmUbi2-ZmCTP-cry2Ae.

4. Construction of 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae vector:

the vector 1300-p35S-intron-cp4 constructed in example 1 was double-digested with KpnI and EcoRI, the nucleotide fragment containing the cry1Ab gene and the Tnos terminator cloned in the above PCR was double-digested with BamHI and EcoRI, pZmUbi1 cloned in the above PCR was double-digested with KpnI and BamHI, and the vector recovered by the above digestion was ligated with the two nucleotide fragments in three pieces to obtain a transit vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab. Carrying out double enzyme digestion on the transition vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab by using HindIII and KpnI, and recovering the enzyme digested vector; the vector 1300-pZmUbi2-ZmCTP-cry2Ae constructed in example 2 was digested with HindIII and KpnI to recover the pZmUbi2-ZmCTP-cry2Ae fragment. The recovered vector and fragment are connected to obtain the final vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae.

5. Construction of 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2Ae vector:

a MAR fragment (primer is MARF:5 '-GTGCCAAGCTTCGATTAAATCCCAATTATTT; MARR:5' -CTGTAGAAGCTTACTATTTTCAGAAGAAGTTCCCAAT) is cloned by PCR by taking a tobacco genome (Nicotiana benthamiana) as a template, and the sequence nucleotide sequence is shown as SEQ ID No:13, a HindIII site is respectively arranged at the 5 'end and the 3' end of the sequence. Respectively carrying out enzyme digestion on the vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae and the PCR product MAR by using restriction endonuclease HindIII, carrying out dephosphorylation treatment on the vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae by using FastAP enzyme, recovering the enzyme-digested 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae vector and MAR fragment, and cloning after connection to obtain the final vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2Ae.

The obtained T-DNA vectors (1300-p 35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae and 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae) are respectively subjected to electric shock to transform the agrobacterium EHA105 to obtain an agrobacterium strain containing the T-DNA vector, and the agrobacterium strain is stored in a refrigerator with the temperature of-80 ℃ after being added with glycerol for being used for infection of transgenic crops.

Example 4 obtaining transgenic insect-resistant herbicide-tolerant maize

The transformation technology of corn is mature. References such as Vladimir Sidorov&<xnotran> David Duncan (in M.Pa ul Scott (ed.), methods in Molecular Biology: transgenic Maize, vol:526;Yuji Ishida,Yukoh Hiei a nd Toshihiko Komari.Nature Protocols.2007,2:1614-1622. : 8-10 Hi-II , ( 1.0-1.5 mm). 3 T-DNA (1300-p 35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae) (MS +2mg/L (2,4- ) 2,4-D +30g/L +3g/L (sigma 7921) +40mg/L ) 2-3 (22 ℃). (MS +2 mg/L2,4-D +30g/L +2.5g/L (gelrite) +5mg/L AgNO </xnotran> ₃ +200mg/L acetosyringone), dark culture at 28 ℃ for 10-14 days. All calli were transferred to selection medium (same as callus induction medium) with 2mM glyphosate and incubated in the dark at 28 ℃ for 2-3 weeks. All tissues were transferred to fresh 2mM glyphosate in selection medium and incubated at 28 ℃ for 2-3 weeks in the dark. Then, all the screened viable embryonic tissues were transferred to a regeneration medium (MS +30g/L sucrose +0.5 mg/L6-furfurylaminopurine (kinetin) +2.5g/L gelrite +200mg/L acetosyringone), and cultured in the dark at 28 ℃ for 10-14 days, one strain per dish. Transferring the embryonic tissue to a fresh regeneration medium, and culturing for 10-14 days at 26 ℃ by illumination. All fully developed plants are transferred to rooting medium (1/2MS +20g/L sucrose +2.5g/L gelrite +200mg/L acetosyringone), and cultured under light at 26 ℃ until roots are fully developed. Transgenic maize plants containing the T-DNA vectors 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae and 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2Ae, respectively, were obtained and designated C1A2A and C1AM2A, respectively.

Example 5 obtaining of transgenic insect-resistant herbicide-tolerant Rice

In this example, transgenic rice plants of the vectors 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae and 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2Ae prepared in example 3 were obtained in the same manner as in example 1.

Example 6 acquisition of transgenic insect-resistant herbicide-tolerant soybeans

The procedure used here to obtain transgenic soybeans is from the established art (Deng et al plant Physiology communications.1998,34, 381-387, ma et al science Agricuicura Sinica.2008, 41-668, zhou et al journal of northern Agricultural university.2001, 32. Healthy, full and mature soybeans of 'Tianlong No. 1' are selected, sterilized by 80% ethanol for 2 minutes, washed by sterile water and then placed in a dryer filled with chlorine (generated by the reaction of 50ml of NaClO and 2ml of concentrated HCl) for sterilization for 4-6 hours. Spreading sterilized semen glycines in B5 culture medium in clean bench, culturing at 25 deg.C for 5 days with optical density of 90-150 μmol photon/m ² S level. When the cotyledon turns green and breaks the seed coat, the aseptic bean sprouts grow. The bean sprouts with the hypocotyl removed were cut into five-five pieces in length so that two pieces of explants had cotyledons and epicotyls. The explants are cut at about 7-8 of the node of the cotyledon and epicotyl and can be used as the target tissue to be infected.

Monoclonal agrobacteria containing the vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae prepared in example 3 and 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2Ae were separately cultured for use. The prepared explants were immersed in the Agrobacterium suspension and co-incubated for 30 minutes at 28 ℃. Then, the excess cell suspension on the infected tissue is absorbed and cleaned by absorbent paper, and then transferred to 1/10B5 co-culture medium for dark culture at 25 ℃ for 3-5 days.

The co-cultured plant tissue was washed with B5 liquid medium to remove excess Agrobacterium, and then placed in B5 solid medium for 5 days at 25 ℃ until it germinated. The induced germ tissue was transferred to B5 selection medium containing 0.5mM glyphosate and incubated at 25 ℃ with light for 4 weeks, during which the medium was changed every two weeks. Transferring the selected embryo tissue to a solid culture medium, culturing at 25 deg.C, and growing into plantlet. Subsequently, transgenic plants were transferred to 1/2B5 medium for rooting induction. Finally, the grown plantlets are washed to remove agar and planted in a greenhouse.

Example 7 determination of insect resistance of transgenic maize

1. Test maize lines: the transgenic corn of the test is an insect-resistant glyphosate-tolerant transgenic strain C1A2A and C1AM2A which is introduced with cry1Ab, cry2Ae and cp4 genes. The control was non-transgenic parent material PH4CV.

2. Spodoptera frugiperda resistance assay:

(1) Indoor live test:

after the transgenic corn of different generations and the control conventional corn germinate in a greenhouse and are sprayed with glyphosate 20 days later to determine that the transgenic corn is a transgenic plant, 10 plants are respectively taken, each plant is inoculated with 10 spodoptera frugiperda of 1 year, and the death rate is observed after 6 days. Spodoptera frugiperda is from the subject group, and egg masses produced by breeding Spodoptera frugiperda female insects are collected in the field. The egg mass is placed at the temperature of 28 +/-1 ℃, the RH is 70 +/-5%, and the egg mass is incubated under the condition of 18h (L: D), and the larvae incubated within 12 hours are selected for bioassay experiments. The test results are shown in table 3.

TABLE 3 Spodoptera frugiperda indoor bioactivity assay results ^#

^# The data in the table are mean ± standard deviation of mortality, n = 10. C1A2A represents 1300-p35S-intron-cp4-pUbi1-cry1Ab-pUbi2-cry2Ae vector transgenic maize plants; c1AM2A represents a transgenic maize plant of vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae; PH4CV indicates non-transgenic control plants.

(2) Field life test:

and carrying out artificial inoculation on the Spodoptera frugiperda for initial larva incubation when the corn plants grow to the middle period of heart leaves (6-8 leaves are completely unfolded). The blackhead egg mass (about 40-60 larvae) was placed in a 1.5ml centrifuge tube and placed in the heart plexus after the larvae had hatched. 10 insects were inoculated per treatment. Leaf feeding grade was investigated 20d after midcardiac inoculation.

Insect resistance grading: using a grade 9 standard (Marcon et al, 1999): grade 1-3: the wormhole needle is needle-punched (level 1: rare, dispersed; level 2: medium amount; level 3: large amount). 4-6 grade: the size of the head of the wormhole match (4 grade: rare and scattered; 5 grade: medium quantity; 6 grade: large quantity). 7-9 level: the wormholes are larger than the match heads (7 grade: rare dispersion; 8 grade: medium amount; 9 grade: large amount). Resistance grade classification: grade 1-2 (high resistance), grade 3-4 (insect resistance), grade 5-6 (insect feeling), grade 7-9 (high feeling).

Planting and managing test corns: no insecticidal pesticide was used during the entire planting process. And (3) fertilizer application: the compound fertilizer is 15 kg per mu before sowing, and 20 kg per mu is added in the 6-7 leaf period (about 40 days after sowing).

The results of the field resistance test for Spodoptera frugiperda are shown in Table 4.

TABLE 4 Spodoptera frugiperda field test results

Maize line	Generation T2	Resistance to insect conditions
			C1A2A-5	100％ ^# Small amount of food intake spot	1
C1A2A-26	Food spot of 100% in small amount	1
			C1A2A-39	Food spot of 100% in small amount	1
C1A2A-67	Eating 78% of the raw materials in small amount	3
			C1A2A-73	Food spot of 100% in small amount	1
C1AM2A-14	Food spot of 100% in small amount	1
			C1AM2A-18	Eating in small amount of 90%	2
C1AM2A-31	Food spot of 100% in small amount	1
			C1AM2A-39	Food spot of 100% in small amount	1
C1AM2A-55	Small amount of 100% food intake spot	1
			PH4CV	Large amount of food is taken, and the leaves have large amount of holes	8.5

^# Representing the mortality rate of corn borer. C1A2A tableTransgenic maize plants showing the 1300-p35S-intron-cp4-pUbi1-cry1Ab-pUbi2-cry2Ae vector; c1AM2A represents a transgenic maize plant of vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae; PH4CV represents non-transgenic control plants. The numbers following the line name represent the number of different transformants.

Indoor bioassay results show that most of transformants have 100% killing effect on young spodoptera frugiperda within 72 hours, and field test results show that most of transformants have high resistance to spodoptera frugiperda.

2. And (3) identifying the resistance of the cotton bollworms:

(1) Indoor bioassay

After transgenic corns of different generations and control conventional corns germinate in a greenhouse and are sprayed with glyphosate 20 days later to determine that the transgenic corns are transgenic, 10 plants are taken respectively, each plant is inoculated with 10 cotton bollworms of 1 year old, and the death rate is observed after 6 days. The bollworm egg blocks are purchased from Henan family cloud biopesticide (Henan Jiyuan white cloud industry Co., ltd.). The egg mass is placed at the temperature of 28 +/-1 ℃, the RH is 70 +/-5%, and the egg mass is incubated under the condition of 18h (L: D), and the larvae incubated within 12 hours are selected for bioassay experiments. The test results are shown in table 5.

TABLE 5 measurement of the indoor biological Activity of Helicoverpa armigera

^# The data in the table are mean ± standard deviation of mortality, n = 10. C1A2A represents 1300-p35S-intron-cp4-pUbi1-cry1Ab-pUbi2-cry2Ae vector transgenic maize plants; c1AM2A represents a transgenic maize plant of vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae; PH4CV represents non-transgenic control plants.

(2) Field life test:

when the corn plant grows to the middle stage of heart leaf (6-8 leaves are completely unfolded), the cotton bollworm is artificially inoculated to the larva which is just hatched. The blackhead egg mass (about 40-60 larvae) was placed in a 1.5ml centrifuge tube and placed in the heart plexus after the larvae had hatched. 10 insects were inoculated per treatment. Leaf feeding grade was investigated 20d after midcardiac inoculation.

Insect resistance grading: using a grade 9 standard (Marcon et al, 1999): grade 1-3: the wormhole is needle-punched (level 1: sparse, dispersed; level 2: medium amount; level 3: large amount). 4-6 grade: the size of the head of the wormhole match (4 grade: rare and scattered; 5 grade: medium quantity; 6 grade: large quantity). 7-9 grade: the wormholes are larger than the match heads (7: rare dispersion; 8: medium number; 9: large number). Resistance grade classification: grade 1-2 (high resistance), grade 3-4 (insect resistance), grade 5-6 (insect feeling), grade 7-9 (high feeling).

The results of the bollworm field resistance test are shown in Table 6.

TABLE 6 Cotton bollworm field test results

Maize line	Generation T2	Resistance to insect conditions
			C1A2A-5	100％ ^# Small amount of food intake spot	1
C1A2A-26	Food spot of 100% in small amount	1
			C1A2A-39	Small amount of 100% food intake spot	1
C1A2A-67	Taking 85% of the raw materials in small amount	3
			C1A2A-73	Food spot of 100% in small amount	1
C1AM2A-14	Food spot of 100% in small amount	1
			C1AM2A-18	Food spot of 100% in small amount	1
C1AM2A-31	Small amount of 100% food intake spot	1
			C1AM2A-39	Eating in 900% small amount	2
C1AM2A-55	Food spot of 100% in small amount	1
			PH4CV	Large amount of food can be taken, and the leaf has large amount of cavity	8.5

^# Representing the mortality rate of corn borer. C1A2A represents 1300-p35S-intron-cp4-pUbi1-cry1Ab-pUbi2-cry2Ae vector transgenic maize plants; c1AM2A represents a transgenic maize plant of vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae; PH4CV represents non-transgenic control plants. The numbers following the line name represent the numbers of the different transformants.

Indoor bioassay results show that most of transformants have 100% killing effect on cotton bollworm low-age larvae within 72 hours, and field test results show that most of transformants show high resistance to cotton bollworms.

3. Resistance identification of corn borers

(1) Indoor bioassay

After the transgenic corn of different generations and the control conventional corn germinate in a greenhouse and are sprayed with glyphosate 20 days later to determine that the transgenic corn is transgenic, 10 plants are respectively taken, each plant is inoculated with 10 corn borers of 1 year old, and the death rate is observed after 6 days. The corn borer egg masses are all purchased from Henan family cloud biological pesticide (Henan Jiyuan Baiyun industry Co., ltd.). And (3) placing the egg masses at the temperature of 28 +/-1 ℃, RH 70 +/-5% and 16h (L: D) for incubation, and selecting larvae which are incubated within 12 hours for bioassay experiments. The test results are shown in table 7.

TABLE 7 indoor bioactivity assay results for corn borer

(2) Field life test:

and (3) carrying out artificial inoculation on the larvae of the corn borers in the early hatching period when the corn plants grow to the middle stage of heart leaves (6-8 leaves are completely unfolded). The blackhead eggs (about 40-60 larvae) were placed in 1.5ml centrifuge tubes and placed in the cardiac plexus after the larvae hatched. 10 insects were inoculated per treatment. Leaf feeding grade was investigated 20d after midcardiac inoculation.

Insect-resistant grading: using a grade 9 standard (Marcon et al, 1999): grade 1-3: the wormhole needle is needle-punched (level 1: rare, dispersed; level 2: medium amount; level 3: large amount). 4-6 grade: the size of the match head with wormholes (4 grade: rare and scattered; 5 grade: medium quantity; 6 grade: large quantity). 7-9 level: the wormholes are larger than the match heads (7: rare dispersion; 8: medium number; 9: large number). Resistance grade classification: grade 1-2 (high resistance), grade 3-4 (insect resistance), grade 5-6 (insect feeling), grade 7-9 (high feeling).

The results of the resistance effect test for corn borer in field are shown in table 8.

TABLE 8 test results for corn borer in field

Corn strain	Generation T2	Resistance to insect conditions
			C1A2A-5	100％ ^# Small amount of food intake spot	1
C1A2A-26	Small amount of 100% food intake spot	1
			C1A2A-39	100％Small amount of food intake spot	1
C1A2A-67	Eating in small amount of 100%	1
			C1A2A-73	Food spot of 100% in small amount	1
C1AM2A-14	Food spot of 100% in small amount	1
			C1AM2A-18	Food spot of 100% in small amount	1
C1AM2A-31	Food spot of 100% in small amount	1
			C1AM2A-39	Eating in small amount of 100%	1
C1AM2A-55	Food spot of 100% in small amount	1
			PH4CV	Large amount of food is taken, and the leaves have large amount of holes	8.0

^# Representing the mortality rate of corn borer. C1A2A represents 1300-p35S-intron-cp4-pUbi1-cry1Ab-pUbi2-cry2Ae vector transgenic maize plants; c1AM2A represents a transgenic maize plant of vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2 Ae; PH4CV indicates non-transgenic control plants. The numbers following the line name represent the number of different transformants.

Indoor bioassay results show that most of transformants have 100% killing effect on young larvae of ostrinia nubilalis within 72 hours, and field test results show that most of transformants have high resistance to ostrinia nubilalis.

Example 8 measurement of the glyphosate resistance of transgenic maize

Test maize lines: the transgenic corn of the test is insect-resistant and glyphosate-tolerant transgenic strains C1A2A and C1AM2A which are introduced with cry1Ab, cry2Ae and cp4 genes. The control was non-transgenic parent material PH4CV.

And (3) determining the glyphosate resistance of the field: transgenic corn and conventional corn seeds of the T2 generation were sprayed 20 days after germination, at 4-6 leaf stage, with 41% glyphosate isopropylamine salt (monsanto) diluted in tap water at a volume ratio of 1.

The glyphosate resistance of the transgenic corn of different generations is determined by spraying glyphosate in the field during the period of 4-6 leaves. The results are shown in Table 9.

TABLE 9 transgenic insect-resistant herbicide-resistant corn Glyphosate-resistant Capacity test ^#

^# Spraying 40L of 1:100, respectively; 1:200 or 1 times diluted noda (41% glyphosate, montelukast product). C1A2A represents 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-pZmUbi2-cry2Ae vector transgenic maize plants; c1AM2A transgenic maize expressing vector 1300-p35S-intron-cp4-pZmUbi1-cry1Ab-MAR-pZmUbi2-cry2AePlant growing; PH4CV indicates non-transgenic control plants. The numbers following the line name represent the number of different transformants.

The results show that most transformants can tolerate higher concentrations of glyphosate. The resistance levels of C1A2A-5, C1A2A-26, C1A2A-67, C1AM2A-14, C1AM2A-18, C1AM2A-31 and C1AM2A-39 are higher, and 40L of 1: there was no observable adverse effect on transgenic corn under 50-diluted noda (41% glyphosate isopropylamine salt, montmorindo).

It is also noted that the above-mentioned lists merely illustrate several embodiments of the invention. The invention is not limited to the above embodiments but may be extended and expanded in many ways. All extensions that can be derived or suggested by a person of ordinary skill in the art from the present disclosure should be considered within the scope of the present invention.

Sequence listing

<110> Hangzhou rhyme Biotechnology Limited

<120> insect-resistant gene and glyphosate-resistant gene expression vector and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1974

<212> DNA

<213> Unknown (Unknown)

<400> 1

atggacaaca acccgaacat caacgagtgc atcccgtaca actgcctctc caacccggag 60

gtggaggtgc tcggcggcga gcgcatcgag accggctaca ccccgatcga catctccctc 120

tccctcaccc agttcctcct ctccgagttc gtgccgggcg ccggcttcgt gctcggcctc 180

gtggacatca tctggggcat cttcggcccg tcccagtggg acgccttcct cgtgcagatc 240

gagcagctca tcaaccagcg catcgaggag ttcgcccgca accaggccat ctcccgcctg 300

gagggcctct ccaacctcta ccagatctac gccgagtcct tccgcgagtg ggaggccgac 360

ccgaccaacc cggccctccg cgaggagatg cgcatccagt tcaacgacat gaactccgcc 420

ctcaccaccg ccatcccgct cttcgccgtg cagaactacc aggtgccgct cctctccgtg 480

tacgtgcagg ccgccaacct ccacctctcc gtgctccgcg acgtgtccgt gttcggccag 540

cgctggggct tcgacgccgc caccatcaac tcccgctaca acgacctcac ccgcctcatc 600

ggcaactaca ccgaccacgc cgtgcgctgg tacaacaccg gcctggagcg cgtgtggggc 660

ccggactccc gcgactggat caggtacaac cagttccgcc gcgagctcac cctcaccgtg 720

ctcgacatcg tgtccctctt cccgaactac gactcccgca cctacccgat ccgcaccgtg 780

tcccagctca cccgcgagat ctacaccaac ccggtgctgg agaacttcga cggctccttc 840

cgcggctccg cccagggcat cgagggctcc atccgctccc cgcacctcat ggacatcctc 900

aactccatca ccatctacac cgacgcccac cgcggcgagt actactggtc cggccaccag 960

atcatggcct ccccggtggg cttctccggc ccggagttca ccttcccgct ctacggcacg 1020

atgggcaacg ccgccccgca gcagcgcatc gtggcccagc tcggccaggg cgtgtaccgc 1080

accctctcct ccaccctcta ccgccgcccg ttcaacatcg gcatcaacaa ccagcagctc 1140

tccgtgctcg acggcaccga gttcgcctac ggcacctcct ccaacctccc gtccgccgtg 1200

taccgcaagt ccggcaccgt ggactccctc gacgagatcc cgccgcagaa caacaacgtg 1260

ccgccgcgcc agggcttctc ccaccgcctc tcccacgtgt ccatgttccg ctccggcttc 1320

tccaactcct ccgtgtccat catccgcgcc ccgatgttct cctggattca ccgctccgcc 1380

gagttcaaca acatcatccc gtcctcccag atcacccaga tcccgctcac caagtccacc 1440

aacctcggct ccggcacctc cgtggtgaag ggcccgggct tcaccggcgg cgacatcctc 1500

cgccgcacct ccccgggcca gatctccacc ctccgcgtga acatcaccgc cccgctctcc 1560

cagcgctacc gcgtgcgcat ccgctacgcc tccaccacca acctccagtt ccacacctcc 1620

atcgacggcc gcccgatcaa ccagggcaac ttctccgcca ccatgtcctc cggctccaac 1680

ctccagtccg gctccttccg caccgtgggc ttcaccaccc cgttcaactt ctccaacggc 1740

tcctccgtgt tcaccctctc cgcccacgtg ttcaactccg gcaacgaggt gtacatcgac 1800

cgcatcgagt tcgtgccggc cgaggtgacc ttcgaggccg agtacgacct ggagcgcgcc 1860

cagaaggccg tgaacgagct cttcacctcc tccaaccaga tcggcctcaa gaccgacgtg 1920

accgactacc acatcgacca ggtgtccaac ctcgtggagt gcctctccga cgag 1974

<210> 2

<211> 658

<212> PRT

<213> Unknown (Unknown)

<400> 2

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly

20 25 30

Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser

35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile

50 55 60

Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile

65 70 75 80

Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala

85 90 95

Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala

130 135 140

Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val

145 150 155 160

Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser

165 170 175

Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg

180 185 190

Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val

195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

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Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val

225 230 235 240

Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro

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

260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu

275 280 285

Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr

290 295 300

Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln

305 310 315 320

Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro

325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala

340 345 350

Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg

355 360 365

Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp

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Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val

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

405 410 415

Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His

420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile

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Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn

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

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

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Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg

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Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn

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Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn

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Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn

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Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu

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Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val

610 615 620

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

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Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser

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Asp Glu

<210> 3

<211> 1896

<212> DNA

<213> Unknown (Unknown)

<400> 3

atgaacaacg tgctcaacaa cggccgcacc accatctgcg acgcctacaa cgtggtggcc 60

cacgacccgt tctccttcga gcacaagtcc ctcgacacca tccgcaagga gtggatggag 120

tggaagcgca ccgaccactc cctctacgtg gccccgatcg tgggcaccgt gtcctccttc 180

ctcctcaaga aggtgggctc cctcatcggc aagcgcatcc tctccgagct ctggggcctc 240

atcttcccgt ccggctccac caacctcatg caggacatcc tccgcgagac cgagcagttc 300

ctcaaccagc gcctcaacac cgacaccctc gcccgcgtga acgccgagct ggagggcctc 360

caggccaaca tccgcgagtt caaccagcag gtggacaact tcctcaaccc gacccagaac 420

ccggtgccgc tctccatcac ctcctccgtg aacaccatgc agcagctctt cctcaaccgc 480

ctcccgcagt tccgcgtgca gggctaccag ctcctcctcc tcccgctctt cgcccaggcc 540

gccaacatgc acctctcctt catccgcgac gtggtgctca acgccgacga gtggggcatc 600

tccgccgcca ccctccgcac ctaccagaac tacctcaaga actacaccac cgagtactcc 660

aactactgca tcaacaccta ccagaccgcc ttccgcggcc tcaacacccg cctccacgac 720

atgctggagt tccgcaccta catgttcctc aacgtgttcg agtacgtgtc catctggtcc 780

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ggcccgcagc agacccagtc cttcacctcc caggactggc cgttcctcta ctccctcttc 900

caggtgaact ccaactacgt gctcaacggc ttctccggcg cccgcctcac ccagaccttc 960

ccgaacatcg gcggcctccc gggcaccacc accacccacg ccctcctcgc cgcccgcgtg 1020

aactactccg gcggcgtgtc ctccggcgac atcggcgccg tgttcaacca gaacttctcc 1080

tgctccacct tcctcccgcc gctcctcacc ccgttcgtgc gctcctggct cgactccggc 1140

tccgaccgcg gcggcgtgaa caccgtgacc aactggcaga ccgagtcctt cgagtccacc 1200

ctcggcctcc gctgcggcgc cttcaccgcc cgcggcaact ccaactactt cccggactac 1260

ttcatccgca acatctccgg cgtgccgctc gtggtgcgca acgaggacct ccgccgcccg 1320

ctccactaca acgagatccg caacatcgag tccccgtccg gcaccccggg cggcctccgc 1380

gcctacatgg tgtccgtgca caaccgcaag aacaacatct acgccgtgca cgagaacggc 1440

accatgatcc acctcgcccc ggaggactac accggcttca ccatctcccc gatccacgcc 1500

acccaggtga acaaccagac ccgcaccttc atctccgaga agttcggcaa ccagggcgac 1560

tccctccgct tcgagcagtc caacaccacc gcccgctaca ccctccgcgg caacggcaac 1620

tcctacaacc tctacctccg cgtgtcctcc ctcggcaact ccaccatccg cgtgaccatc 1680

aacggccgcg tgtacaccgc ctccaacgtg aacaccacca ccaacaacga cggcgtgaac 1740

gacaacggcg cccgcttcct cgacatcaac atgggcaacg tggtggcctc cgacaacacc 1800

aacgtgccgc tcgacatcaa cgtgaccttc aactccggca cccagttcga gctcatgaac 1860

atcatgttcg tgccgaccaa cctcccgccg atctac 1896

<210> 4

<211> 632

<212> PRT

<213> Unknown (Unknown)

<400> 4

Met Asn Asn Val Leu Asn Asn Gly Arg Thr Thr Ile Cys Asp Ala Tyr

1 5 10 15

Asn Val Val Ala His Asp Pro Phe Ser Phe Glu His Lys Ser Leu Asp

20 25 30

Thr Ile Arg Lys Glu Trp Met Glu Trp Lys Arg Thr Asp His Ser Leu

35 40 45

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

50 55 60

Val Gly Ser Leu Ile Gly Lys Arg Ile Leu Ser Glu Leu Trp Gly Leu

65 70 75 80

Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu

85 90 95

Thr Glu Gln Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg

100 105 110

Val Asn Ala Glu Leu Glu Gly Leu Gln Ala Asn Ile Arg Glu Phe Asn

115 120 125

Gln Gln Val Asp Asn Phe Leu Asn Pro Thr Gln Asn Pro Val Pro Leu

130 135 140

Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg

145 150 155 160

Leu Pro Gln Phe Arg Val Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu

165 170 175

Phe Ala Gln Ala Ala Asn Met His Leu Ser Phe Ile Arg Asp Val Val

180 185 190

Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr

195 200 205

Gln Asn Tyr Leu Lys Asn Tyr Thr Thr Glu Tyr Ser Asn Tyr Cys Ile

210 215 220

Asn Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp

225 230 235 240

Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val

245 250 255

Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly

260 265 270

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

275 280 285

Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser

290 295 300

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

305 310 315 320

Pro Asn Ile Gly Gly Leu Pro Gly Thr Thr Thr Thr His Ala Leu Leu

325 330 335

Ala Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Asp Ile Gly

340 345 350

Ala Val Phe Asn Gln Asn Phe Ser Cys Ser Thr Phe Leu Pro Pro Leu

355 360 365

Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg Gly

370 375 380

Gly Val Asn Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Ser Thr

385 390 395 400

Leu Gly Leu Arg Cys Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn Tyr

405 410 415

Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val Val

420 425 430

Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile Arg Asn

435 440 445

Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Leu Arg Ala Tyr Met Val

450 455 460

Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Val His Glu Asn Gly

465 470 475 480

Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile Ser

485 490 495

Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile Ser

500 505 510

Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Ser Asn

515 520 525

Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn Leu

530 535 540

Tyr Leu Arg Val Ser Ser Leu Gly Asn Ser Thr Ile Arg Val Thr Ile

545 550 555 560

Asn Gly Arg Val Tyr Thr Ala Ser Asn Val Asn Thr Thr Thr Asn Asn

565 570 575

Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Leu Asp Ile Asn Met Gly

580 585 590

Asn Val Val Ala Ser Asp Asn Thr Asn Val Pro Leu Asp Ile Asn Val

595 600 605

Thr Phe Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile Met Phe Val

610 615 620

Pro Thr Asn Leu Pro Pro Ile Tyr

625 630

<210> 5

<211> 1368

<212> DNA

<213> Unknown (Unknown)

<400> 5

atgctacacg gtgcaagcag ccggccggca accgctcgca aatcttccgg cctttcggga 60

acggtcagga ttccgggcga taagtccata tcccaccggt cgttcatgtt cggcggtctt 120

gccagcggtg agacgcgcat cacgggcctg cttgaaggtg aggacgtgat caataccggg 180

aaggccatgc aggctatggg agcgcgtatc cgcaaggaag gtgacacatg gatcattgac 240

ggcgttggga atggcggtct gctcgcccct gaggcccctc tcgacttcgg caatgcggcg 300

acgggctgca ggctcactat gggactggtc ggggtgtacg acttcgatag cacgttcatc 360

ggagacgcct cgctcacaaa gcgcccaatg ggccgcgttc tgaacccgtt gcgcgagatg 420

ggcgtacagg tcaaatccga ggatggtgac cgtttgcccg ttacgctgcg cgggccgaag 480

acgcctaccc cgattaccta ccgcgtgcca atggcatccg cccaggtcaa gtcagccgtg 540

ctcctcgccg gactgaacac tccgggcatc accacggtga tcgagcccat catgaccagg 600

gatcataccg aaaagatgct tcaggggttt ggcgccaacc tgacggtcga gacggacgct 660

gacggcgtca ggaccatccg ccttgagggc aggggtaaac tgactggcca agtcatcgat 720

gttccgggag acccgtcgtc cacggccttc ccgttggttg cggcgctgct cgtgccgggg 780

agtgacgtga ccatcctgaa cgtcctcatg aacccgacca ggaccggcct gatcctcacg 840

cttcaggaga tgggagccga catcgaggtg atcaacccgc gcctggcagg cggtgaagac 900

gttgcggatc tgcgcgtgcg ctcctctacc ctgaagggcg tgacggtccc ggaagatcgc 960

gcgccgtcca tgatagacga gtatcctatt ctggccgtcg ccgctgcgtt cgccgaaggg 1020

gccacggtca tgaacggtct tgaggaactc cgcgtgaagg aatcggatcg cctgtcggcg 1080

gtggccaatg gcctgaagct caacggtgtt gactgcgacg agggtgagac ctcactcgtg 1140

gtccgtggcc ggcctgatgg caagggcctc ggcaacgcca gtggagcggc cgtcgccacg 1200

cacctcgatc atcgcatcgc gatgtccttc ttggtgatgg gtctcgtctc agagaacccg 1260

gtgaccgtcg atgacgccac gatgatagcg acgagcttcc cagagttcat ggatctgatg 1320

gcgggcctcg gggccaagat cgaactgtct gacacgaagg ccgcttga 1368

<210> 6

<211> 455

<212> PRT

<213> Unknown (Unknown)

<400> 6

Met Leu His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser

1 5 10 15

Gly Leu Ser Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His

20 25 30

Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr

35 40 45

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

50 55 60

Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp

65 70 75 80

Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe

85 90 95

Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly Val

100 105 110

Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu Thr Lys Arg

115 120 125

Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val

130 135 140

Lys Ser Glu Asp Gly Asp Arg Leu Pro Val Thr Leu Arg Gly Pro Lys

145 150 155 160

Thr Pro Thr Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val

165 170 175

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

180 185 190

Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln

195 200 205

Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg

210 215 220

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

225 230 235 240

Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu

245 250 255

Leu Val Pro Gly Ser Asp Val Thr Ile Leu Asn Val Leu Met Asn Pro

260 265 270

Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile

275 280 285

Glu Val Ile Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu

290 295 300

Arg Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg

305 310 315 320

Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala

325 330 335

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

340 345 350

Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly Leu Lys Leu Asn

355 360 365

Gly Val Asp Cys Asp Glu Gly Glu Thr Ser Leu Val Val Arg Gly Arg

370 375 380

Pro Asp Gly Lys Gly Leu Gly Asn Ala Ser Gly Ala Ala Val Ala Thr

385 390 395 400

His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val

405 410 415

Ser Glu Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser

420 425 430

Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu

435 440 445

Leu Ser Asp Thr Lys Ala Ala

450 455

<210> 7

<211> 905

<212> DNA

<213> Unknown (Unknown)

<400> 7

atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac 60

caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat 120

tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa 180

tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc 240

aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 300

tcaaagcaag tggattgatg tgataacatg gtggagcacg acactctcgt ctactccaag 360

aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 420

atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca 480

gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt 540

caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 600

gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact 660

gacgtaaggg atgacgcaca atcccactat ccttcgcaag accttcctct atataaggaa 720

gttcatttca tttggagagg acacgctgaa atcaccagtc tctctctaca aatctatctc 780

tctcgaggtt ggttctgctg tccggttgtc aatcctttag ctaccataca tgtgtttcag 840

ttttcttttg cctgtttatt catttctgat ttaataaact gatgggattt tgatgccaaa 900

cacag 905

<210> 8

<211> 222

<212> DNA

<213> Unknown (Unknown)

<400> 8

atggcggcga ccatggcgtc caacgctgcg gctgcggctg cggtgtccct ggaccaggcc 60

gtggctgcgt cggcagcgtt ctcgtcgcgg aagcagctgc ggctgcctgc cgcagcgcgc 120

ggagggatgc gggtgcgggt gcgggcgcgg ggtcggcggg aggcggtggt ggtggcgtcc 180

gcgtcgtcgt cgtcggtggc agcgccggcg gcgaaggctg ag 222

<210> 9

<211> 256

<212> DNA

<213> Unknown (Unknown)

<400> 9

cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 60

gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 120

tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 180

tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 240

tctatgttac tagatc 256

<210> 10

<211> 198

<212> DNA

<213> Unknown (Unknown)

<400> 10

atggcggcca tggcgaccaa ggccgccgcg ggcaccgtgt cgctggacct cgccgcgccg 60

tcgcgccgcc accaccgccc gagctcggcg cgcccgcccg cccgccccgc cgtccgcggg 120

ctgcgggcgc ctgggcgccg cgtgatcgcc gcgccgccgg cggcggcagc ggcggcggcg 180

gtgcaggcgg gtgccgag 198

<210> 11

<211> 1992

<212> DNA

<213> Unknown (Unknown)

<400> 11

ctacagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380

agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440

gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500

ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560

gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620

aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680

catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740

ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800

ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860

gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920

atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980

ttacttctgc ag 1992

<210> 12

<211> 784

<212> DNA

<213> Unknown (Unknown)

<400> 12

gtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa tgctctcttg 60

tgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa atcgtgttct 120

gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat ggtagtacga 180

aacgaagata gaacctacac agcaatacga gaaatgtgta atttggtgct tagcggtatt 240

tatttaagca catgttggtg ttatagggca cttggattca gaagtttgct gttaatttag 300

gcacaggctt catactacat gggtcaatag tatagggatt catattatag gcgatactat 360

aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc tattctgttt 420

ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg acgaaatttt 480

gatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca cttgttttaa 540

atattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc tgcttgtttg 600

ttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac ctcctgatgg 660

tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat cttgctcgat 720

gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt cattgtaatg 780

caga 784

<210> 13

<211> 1168

<212> DNA

<213> Unknown (Unknown)

<400> 13

tcgattaaaa atcccaatta tatttggtct aatttagttt ggtattgagt aaaacaaatt 60

cgaaccaaac caaaatataa atatatagtt tttatatata tgcctttaag actttttata 120

gaattttctt taaaaaatat ctagaaatat ttgcgactct tctggcatgt aatatttcgt 180

taaatatgaa gtgctccatt tttattaact ttaaataatt ggttgtacga tcactttctt 240

atcaagtgtt actaaaatgc gtcaatctct ttgttcttcc atattcatat gtcaaaatct 300

atcaaaattc ttatatatct ttttcgaatt tgaagtgaaa tttcgataat ttaaaattaa 360

atagaacata tcattattta ggtatcatat tgatttttat acttaattac taaatttggt 420

taactttgaa agtgtacatc aacgaaaaat tagtcaaacg actaaaataa ataaatatca 480

tgtgttatta agaaaattct cctataagaa tattttaata gatcatatgt ttgtaaaaaa 540

aattaatttt tactaacaca tatatttact tatcaaaaat ttgacaaagt aagattaaaa 600

taatattcat ctaacaaaaa aaaaaccaga aaatgctgaa aacccggcaa aaccgaacca 660

atccaaaccg atatagttgg tttggtttga ttttgatata aaccgaacca actcggtcca 720

tttgcacccc taatcataat agctttaata tttcaagata ttattaagtt aacgttgtca 780

atatcctgga aattttgcaa aatgaatcaa gcctatatgg ctgtaatatg aatttaaaag 840

cagctcgatg tggtggtaat atgtaattta cttgattcta aaaaaatatc ccaagtatta 900

ataatttctg ctaggaagaa ggttagctac gatttacagc aaagccagaa tacaaagaac 960

cataaagtga ttgaagctcg aaatatacga aggaacaaat atttttaaaa aaatacgcaa 1020

tgacttggaa caaaagaaag tgatatattt tttgttctta aacaagcatc ccctctaaag 1080

aatggcagtt ttcctttgca tgtaactatt atgctccctt cgttacaaaa attttggact 1140

actattggga acttcttctg aaaatagt 1168

Claims

1. An expression vector of an insect-resistant gene and a glyphosate-tolerant gene is characterized in that the expression vector contains the insect-resistant gene cry1Ab, the insect-resistant gene cry2Ae and the glyphosate-tolerant gene cp4.

2. The expression vector of claim 1, wherein the nucleotide sequence of cry1Ab is SEQ ID No:1 is shown in the specification; the nucleotide sequence of the insect-resistant gene cry2Ae is SEQ ID No:3 is shown in the specification; the nucleotide sequence of the glyphosate-tolerant gene cp4 is SEQ ID No:5, respectively.

3. The expression vector of claim 1, characterized in that the expression vector comprises a maize ubiquitin promoter pZmUbi or a composite promoter; the composite promoter consists of a CaMV35S promoter and a second intron of a maize actin 2.

4. The expression vector of claim 3, wherein the nucleotide sequence of the maize ubiquitin promoter pZmUbi is as set forth in SEQ ID No:11 is shown in the figure; the nucleotide sequence of the composite promoter is shown as SEQ ID No: shown at 7.

5. The expression vector according to claim 1, wherein the expression vector comprises the terminator Tnos or the CaMV35S terminator T35S of the agrobacterium tumefaciens nopaline synthase gene.

6. The expression vector of claim 1, wherein said expression vector comprises a chloroplast signal peptide of a maize EPSPS gene or a rice EPSPS gene; the chloroplast signal peptide nucleotide sequence of the corn EPSPS gene is shown as SEQ ID No:10 is shown in the figure; the nucleotide sequence of chloroplast signal peptide of the rice EPSPS gene is shown as SEQ ID No: shown in fig. 8.

7. The expression vector of claim 1, wherein the expression vector comprises a tobacco MAR sequence having a nucleotide sequence of SEQ ID No: shown at 13.

8. The expression vector of claim 1, wherein the expression vector comprises a maize ubiquitin promoter pZmUbi for promoting the expression of the insect-resistant gene cry1Ab, a terminator Tnos for the Agrobacterium tumefaciens nopaline synthase gene for terminating the expression of the gene; the corn ubiquitin promoter pZmUbi for starting the expression of the insect-resistant gene cry2Ae, the chloroplast signal peptide of the corn EPSPS gene, and the terminator Tnos of the agrobacterium tumefaciens nopaline synthetase gene for terminating the expression of the gene; the composite promoter p35S-intron consisting of the CaMV35S promoter p35S for starting the expression of the glyphosate-tolerant gene cp4 and the gene intron, the chloroplast signal peptide of the rice EPSPS gene and the CaMV35S terminator T35S for terminating the expression of the gene.

9. Use of the expression vector of claim 1 in the preparation of a transgenic plant cell.

10. Use according to claim 9, characterized in that said plants comprise: corn, rice, soybean, canola or cotton.