Nucleic acid molecule and application thereof in cultivation of insect-resistant plants

Abstract

The invention provides a novel nucleic acid molecule and application thereof in cultivating insect-resistant plants. The nucleic acid molecule provided by the invention can prevent soybean, corn and other plants from being damaged by lepidoptera insects, and can be used for cultivating novel insect-resistant plants.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology. Specifically, the present invention provides a new nucleic acid molecule and its application in cultivating insect-resistant plants. Background Art

[0002] Bt insecticidal crystal proteins from Bacillus thuringiensis have specific insecticidal activity against a variety of insects such as Lepidoptera, Diptera, Coleoptera, and Hymenoptera. Bt genes have become the most widely used and effective insect-resistant genes in plant genetic engineering and transgenic breeding. In 1981, Schnepft and Whiteley successfully cloned the first gene encoding Bt insecticidal protein, Cry1Aa. To date, more than 600 insecticidal protein genes have been isolated and cloned, and Cry2Ab insecticidal protein is one of them. Cry2Ab protein can be used to control many Lepidoptera pests, including cotton bollworm, fall armyworm, corn borer, and armyworm.

[0003] The original Bt gene from bacteria contains many elements that lead to unstable expression in eukaryotes, such as poly(A) signal sequences similar to those in plants, intron cleavage signal sites, AT-rich sequences, etc., which can lead to instability of the mRNA encoded by the gene; compared with plant genes, the GC content and codon usage frequency of the original Bt gene of bacteria are very different, and are not suitable for efficient expression in plants. After the original Bt gene was transformed into plants, the expression level of Bt insecticidal protein (ICP) in plants was generally low, and the insect resistance of plants was also poor. The content of toxic protein only accounted for 0.001% of soluble protein or was so low that it could not be detected, which was difficult to meet the requirements of production and application. After 1990, the research on the transformation of Bt genes gradually deepened. The expression level of modified Cry1Ab and Cry1Ac in transgenic soybeans was greatly improved, and the insecticidal protein content was 0.05-0.1% of the total soluble protein of the plant, achieving the purpose of insect resistance, thus making the application of transgenic Bt crops in agricultural production possible.

[0004] Codon optimization needs to consider a variety of factors to achieve good results, such as the host's preference for codon use, as well as mRNA secondary structure, restriction enzyme sites and GC content. It is not simply adjusting the synonymous codons of exogenous genes to codons with high expression abundance and high usage frequency in host cells to improve the translation efficiency of proteins. Sometimes the effect is often counterproductive. Therefore, codon optimization is not a simple experiment. In addition, plant nucleic acid sequence optimization is more difficult and more complicated. First, plants originated earlier than animals, and the genome size is much larger than that of microorganisms. The evolution of plant genomes is long and the evolution process is more complex, including high heterozygosity, highly repetitive sequences and complex polyploidy. Second, human research on plant genomes started late and the research is more difficult, so the existing data on plant genome structure and gene expression is not rich enough. Therefore, for a specific exogenous protein, it is not very certain what kind of coding method can achieve higher protein function efficiency.

[0005] In order to improve the functional efficiency of the insecticidal protein Cry2Ab in plants, the present invention obtains the nucleic acid molecule with the best insecticidal effect in plants through a large amount of biological information analysis and specific experimental tests. Summary of the invention

[0006] The present invention provides a nucleic acid molecule, characterized in that the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 2 or its reverse complementary sequence.

[0007] The present invention also provides an expression vector, characterized in that the expression vector contains the above-mentioned nucleic acid molecule.

[0008] The present invention also provides a host cell, characterized in that the host cell contains the above-mentioned nucleic acid molecule or the above-mentioned expression vector; the host cell is not an animal cell or a plant cell.

[0009] In some embodiments, the host cell is an Agrobacterium cell or an Escherichia coli cell.

[0010] The present invention also provides a method for producing transgenic plants, characterized in that the above-mentioned nucleic acid molecules, or expression vectors, or host cells are transformed into plants to obtain transformed plant cells, and then the transformed plant cells are cultured into transgenic plants.

[0011] The present invention also provides the use of the above nucleic acid molecules, expression vectors, host cells and methods in producing insect-resistant plants.

[0012] In some embodiments, the plant is soybean or corn.

[0013] The present invention also provides a method for protecting plants from insect attack, characterized in that it includes providing at least one transgenic plant cell in the diet of the target insect, wherein the transgenic plant cell contains the above-mentioned nucleic acid molecule or the above-mentioned expression vector in its genome; the target insect that ingests the transgenic plant cell is inhibited from further ingesting the plant.

[0014] In some embodiments, the plant is soybean or corn.

[0015] The beneficial effect of the present invention is that the present invention targets Cry2Ab insecticidal protein, obtains a nucleic acid molecule with the best insecticidal effect in plants through a large amount of biological information analysis and specific experimental tests. The nucleic acid molecule can be used to control lepidopteran pests and cultivate new insect-resistant plants.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Physical map of expression vector pCAMBIA3300-Cry2Ab-bar, taking pAb1 as an example.

[0018] Figure 2 Insect resistance of pAb1-transfected soybean and corn. A: pAb1-transfected corn; B: corn receptor control B104; C: pAb1-transfected soybean; D: soybean receptor control Williams 82. DETAILED DESCRIPTION

[0019] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public publications cited herein are incorporated herein by reference in their entirety.

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of this application. Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0021] Example 1 Optimization of Cry2Ab protein coding sequence

[0022] There is codon preference in the translation process of organisms. The most basic principle of codon optimization is to replace the codons in the exogenous mRNA sequence with synonymous codons that are frequently used in host cells to ensure that the codons in the exogenous mRNA sequence and the codon usage bias of the host cell are more consistent, thereby improving the expression level of the protein. The degree of consistency is often expressed by the Codon Adaptation Index (CAI), which reflects the degree to which the synonymous codons in the coding region are consistent with the optimal use of codons in a specific species. The value range is between 0-1 and is species-specific. In theory, the closer this value is to 1, the higher the protein expression of the exogenous mRNA in the host cell. In addition, the secondary structure of mRNA has a certain influence on the translation efficiency, and the restriction enzyme cutting site will also affect the operation of constructing the expression vector. The GC content is related to the stability of DNA. If it is higher than 70%, it may indirectly affect the expression regulation of the gene.

[0023] In order to obtain a nucleic acid molecule encoding Cry2Ab protein (amino acid sequence as shown in SEQ ID NO.1) with better insecticidal effect, the present invention counted the codon usage between different genes of plants such as soybean and corn, and on the basis of focusing on the CAI (codon adaptability index) values ​​of codons of highly expressed genes in different species, paid attention to the parameters of codon usage frequency statistics such as relative usage of synonymous codons (RSCU, which refers to the relative probability of a specific codon in the synonymous codons encoding the corresponding amino acid), the number of effective codons (ENC, between 20-61, reflecting the preference for the non-balanced use of synonymous codons in the codon family), and more than a dozen key parameters related to the transcription and translation process, including the secondary structure of mRNA, species repeat sequence structure, rare codons, hidden editing sites, GC content and enzyme cutting sites, and developed a new algorithm model.

[0024] The algorithm model uses a random sampling strategy, and each calculation returns a local optimal result, so the results of each optimization will be different. Through multiple calculations, the model initially screened hundreds of nucleotide sequences, and then selected 7 sequences with a CAI value of not less than 0.6, a GC content of less than 70%, and an ENC value of not more than 30 from the top 20 sequences in the model score. They were named Ab1-Ab7, and the sequence-related parameter information is shown in Table 1.

[0025] Table 1 Cry2Ab nucleic acid molecule sequence list

[0026]

[0027] Example 2 Obtaining transgenic plants and identifying their insect resistance

[0028] The seven candidate nucleic acid molecules Ab1-Ab7 in Example 1 and the three previously disclosed

[0029] The nucleic acid molecules C1 (SEQ ID NO: 1 of patent number CA2795377C), C2 (SEQ ID NO: 2 of patent number CN104286014B) and C3 (SEQ ID NO: 26 of patent number EP3008187B1) of Cry2Ab gene are operably connected to the plastid transit peptide (PTP) gene (SEQ ID NO: 9 of CA2795377C) and NOS promoter at the 5' end, and connected to the NOS terminator at the 3' end. The obtained Cry2Ab expression cassette is then connected to the bar gene expression cassette to construct 10 plant expression vectors pCAMBIA3300-Cry2Ab-bar (bar gene expression cassette is used as a selection marker), which are numbered pAb1-pAb7, pC1-pC3, and the expression cassette element information of the vector T-DNA region is shown in Table 2, and the physical map is shown in Figure 1 (Take pAb1 vector as an example).

[0030] After the above expression vectors were transformed into Escherichia coli DH5α, the plasmids were extracted and then transferred into Agrobacterium EHA105 or LBA4404. The above 10 vectors were transformed into dicotyledonous representative species soybean and monocotyledonous representative species corn respectively through Agrobacterium-mediated method. Williams 82 was selected for soybean and inbred line B104 was selected for corn. Glufosinate-ammonium was used as a screening agent, and finally 89 soybean and 86 corn positive transgenic plants were obtained (vector construction and plant genetic transformation adopted the common methods in the art), and the insect resistance of these transgenic plants was identified.

[0031] Table 2 Element information of T-DNA regions of 10 expression vectors

[0032]

[0033] Williams 82 was used as a control for soybeans and B104 was used as a control for corn. Transgenic plants with good insect resistance were screened by leaf bioassay in the laboratory. The detached leaves were fed to the newly hatched larvae of cotton bollworm to screen the insect resistance of the materials. Two replicates were set for each material. When the mortality rate of the negative control did not exceed 20%, the average mortality rate of the transgenic plant material exceeded 40%, which was preliminarily identified as insect resistance.

[0034] The results of indoor bioassays of soybean and corn transgenic plants transferred into 10 different vectors are shown in Tables 3 and 4. The results showed that 31 transgenic soybean plants and 34 transgenic corn plants were resistant to cotton bollworms. Among them, the transgenic soybean plants and transgenic corn plants transferred into pAb1 had the highest proportion of resistant plants, reaching 72.7% and 71.4%, respectively. The CAI value and model score of the cry2Ab nucleic acid molecule sequence Ab1 in the pAb1 vector were not the highest in soybean and corn, with CAI values ​​of 0.79 and 0.80, and model scores of 80 and 77, respectively. However, unexpectedly, the Ab1 sequence had the highest proportion of resistant transgenic soybeans and corn.

[0035] Table 3 Indoor bioassay results of different soybean transgenic plants

[0036]

[0037] Table 4 Indoor bioassay results of different transgenic maize plants

[0038]

[0039] The resistance levels of the 31 transgenic soybean plants and 34 transgenic corn plants were further evaluated. The soybeans were numbered S1-S31, with Williams 82 as the control, and the corns were numbered M1-M34, with B104 as the control. Transgenic plants with better insect resistance were screened by leaf indoor bioassay. The detached leaves were fed to the newly hatched larvae of cotton bollworm to evaluate the insect resistance of the materials. The mortality rate and corrected mortality rate of the test insects were calculated according to the following formulas 1 and 2, respectively.

[0040]

[0041]

[0042] The results are shown in Table 5: the mortality of cotton bollworms feeding on the 31 transgenic soybean plants was significantly higher than that of the control, among which the 8 transgenic soybean plants S9-S16 (transfected with pAb1 vector) had high resistance to cotton bollworm, and the transgenic plants of the other 9 vectors were moderately resistant or resistant; the mortality of cotton bollworms feeding on the leaves of the 34 transgenic corn plants was significantly higher than that of the control, among which the 10 transgenic corn plants M9-M18 (transfected with pAb1 vector) had high resistance to cotton bollworm, and the transgenic plants of the other 9 vectors were moderately resistant or resistant.

[0043] The test results show that the nucleic acid molecule sequences of the above 18 transgenic plants with high resistance to cotton bollworm are all Ab1, and their insecticidal effects in corn and soybean are better than those of the three publicly known nucleic acid molecules C1-C3 of the Cry2Ab gene.

[0044] Table 5 Indoor bioassay results of soybean and corn transgenic plants resistant to cotton bollworm

[0045]

[0046]

[0047] The values ​​are expressed as the mean ± standard deviation of three biological replicates, and the significance of the differences between the data in the same column was analyzed using the LSD method (α = 0.05).

[0048] In summary, the present invention optimizes the coding sequence of the insecticidal protein Cry2Ab to obtain a nucleic acid molecule with optimal insecticidal effect in plants. It has been identified that transgenic corn and soybeans produced using this nucleic acid molecule have the characteristics of high resistance to Lepidoptera insects such as cotton bollworm.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 2 or its reverse complementary sequence.

2. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 1.

3. A host cell, characterized in that The host cell contains the nucleic acid molecule of claim 1 or the expression vector of claim 2; The host cell is neither an animal cell nor a plant cell.

4. The host cell according to claim 3, characterized in that The host cell is an Agrobacterium cell or an Escherichia coli cell.

5. A method for producing a transgenic plant, characterized in that The nucleic acid molecule according to claim 1, or the expression vector according to claim 2, or the host cell according to any one of claims 3 to 4 is transformed into a plant to obtain a transformed plant cell, and then the transformed plant cell is cultured into a transgenic plant.

6. Use of the nucleic acid molecule according to claim 1, the expression vector according to claim 2, the host cell according to claims 3-4, and the method according to claim 5 in producing insect-resistant plants.

7. The use according to claim 6, characterized in that: The plant is soybean or corn.

8. A method for protecting plants from insect infestation, characterized in that: The method comprises providing at least one transgenic plant cell in the diet of a target insect, wherein the transgenic plant cell comprises the nucleic acid molecule of claim 1 or the expression vector of claim 2 in its genome; the target insect that ingests the transgenic plant cell is inhibited from further ingesting the plant.

9. The method according to claim 8, characterized in that The plant is soybean or corn.

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