Trehalose transporter mutants, tret1 genes and uses thereof

Abstract

The application provides a trehalose transporter mutant, a Tret1 gene and application thereof, and relates to the technical field of biology.The amino acid sequence of the trehalose transporter mutant provided by the application is shown as SEQ ID NO.1.The inventors have found that, compared with a wild-type trehalose transporter, the trehalose transporter mutant provided by the application can significantly improve the transport capacity of insects for trehalose, reduce the supercooling point, and improve the cold resistance of insects.Therefore, the trehalose transporter mutant provided by the application can be used for regulating the trehalose transport efficiency and the cold resistance of insects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a trehalose transporter mutant, the Tret1 gene, and their applications. Background Technology

[0002] Insects are poikilothermic animals, and climate change has a significant impact on the seasonal fluctuations of their populations. Insects typically alter their structure and the content of substances within their bodies to cope with low temperatures, and their cold tolerance is a prerequisite for the existence and development of their populations. Through long-term adaptation to their environment, insects have developed a series of adaptive evolutionary mechanisms, which are currently a hot topic in insect ecology and evolutionary biology research. These mechanisms also provide a theoretical basis for pest forecasting and control, and can offer insights for the development of new pesticides. The cotton bollworm, belonging to the Noctuidae family of Lepidoptera, is widely distributed in major cotton-producing areas of my country, primarily coping with low temperatures through migration and diapause. Considering the geographical features of Xinjiang, with its plateaus, mountains, and deserts, the cotton bollworm in Xinjiang has formed a unique subpopulation. Xinjiang experiences relatively large temperature differences, with hot summers and cold winters, and the cotton bollworm, confined to the relatively closed Xinjiang basin, cannot migrate long distances to warmer regions. Therefore, the cotton bollworm in Xinjiang has evolved its own unique adaptive mechanisms to cope with the cold winters. Understanding the cold adaptation mechanisms of the cotton bollworm is helpful for the control of cold-resistant pests.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The primary objective of this invention is to provide a trehalose transporter mutant that enhances the transport efficiency of trehalose, thereby improving the insect's tolerance to low temperatures. This mutant can be used for pest control and can also be applied to natural enemies and economically important insects, expanding their suitable habitat range.

[0005] A second objective of the present invention is to provide the Tret1 gene to solve at least one of the above-mentioned problems.

[0006] A third objective of this invention is to provide the application of the above-mentioned trehalose transporter mutant or Tret1 gene in regulating insect cold resistance.

[0007] The fourth objective of this invention is to provide the application of the above-mentioned trehalose transporter mutant or the Tret1 gene in regulating the efficiency of trehalose transport in insects.

[0008] The fifth objective of this invention is to provide biological materials.

[0009] The sixth objective of this invention is to provide markers related to insect traits.

[0010] The seventh objective of this invention is to provide a kit for detecting insect traits.

[0011] The eighth objective of this invention is to provide a method for regulating insect traits.

[0012] In a first aspect, the present invention provides a trehalose transporter mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] In a second aspect, the present invention provides the Tret1 gene, which encodes the trehalose transporter mutant described above;

[0014] The nucleic acid sequence of the Tret1 gene is shown in SEQ ID NO.2.

[0015] Thirdly, the present invention provides the application of the above-mentioned trehalose transporter mutant or Tret1 gene in regulating insect cold resistance.

[0016] Fourthly, the present invention provides the application of the above-mentioned trehalose transporter mutant or Tret1 gene in regulating the efficiency of trehalose transport in insects.

[0017] Fifthly, the present invention provides biological materials selected from one or more of the following (n1) to (n3):

[0018] (n1) An expression cassette containing the Tret1 gene;

[0019] (n2) A vector containing the Tret1 gene, or a vector containing the expression cassette (n1);

[0020] (n3) A cell line containing the Tret1 gene, a cell line containing the expression cassette (n1), or a cell line containing the vector (n2).

[0021] In a sixth aspect, the present invention provides insect trait-related markers, said markers being selected from (a1): the Tret1 gene described above;

[0022] (a2) A fusion gene containing the Tret1 gene;

[0023] (a3): RNA transcribed from (a1) or (a2);

[0024] (a4): Protein expressed by any one of (a1) to (a3);

[0025] The traits include at least one of insect cold resistance and insect trehalose transport efficiency.

[0026] In a seventh aspect, the present invention provides a kit for detecting insect traits, the kit being used to detect the aforementioned markers;

[0027] Preferably, the kit includes a primer pair for detecting the Tret1 gene, the nucleic acid sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0028] Eighthly, the present invention provides a method for regulating insect traits, the method comprising: overexpressing or inhibiting the expression of the Tret1 gene;

[0029] The traits include at least one of insect cold resistance and insect trehalose transport efficiency.

[0030] As a further technical solution, overexpression of the Tret1 gene improves the cold resistance of insects;

[0031] Suppressing the expression of the Tret1 gene reduces the cold resistance of insects.

[0032] As a further technical solution, overexpression of the Tret1 gene improves the efficiency of trehalose transport in insects.

[0033] Suppressing the expression of the Tret1 gene reduces the efficiency of trehalose transport in insects.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The amino acid sequence of the trehalose transporter mutant provided by this invention is shown in SEQ ID NO.1. The inventors have found that, compared with the wild-type trehalose transporter, the trehalose transporter mutant provided by this invention can significantly improve the insect's ability to transport trehalose, lower the supercooling point, and enhance the insect's cold resistance. Therefore, the trehalose transporter mutant provided by this invention can be used to regulate the efficiency of trehalose transport and cold resistance in insects. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 Madhton plot for GWAS association analysis of temperature and genotype;

[0038] Figure 2 The haplotype distribution frequency of Tret1 in different geographical populations;

[0039] Figure 3Results on subcellular localization and trehalose transport efficiency of different haplotypes of the trehalose transporter Tret1;

[0040] Figure 4 This is a schematic diagram of the construction of segregating populations of different haplotypes Tret1 using nonsynonymous mutations (chr3:6626270, AA / GG) as molecular markers;

[0041] Figure 5 To determine the trehalose content in different haplotype populations of the Tret1 gene under low-temperature stress;

[0042] Figure 6 This study aims to determine the supercooling point of different haplotype populations of the Tret1 gene under low-temperature stress. Detailed Implementation

[0043] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0044] In a first aspect, the present invention provides a trehalose transporter mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0045] (SEQ ID NO.1).

[0046] The inventors have discovered that, compared to wild-type trehalose transporters, the trehalose transporter mutant provided by this invention significantly enhances insects' ability to transport trehalose, lowers their supercooling point, and improves their cold resistance. Therefore, the trehalose transporter mutant provided by this invention can be used to regulate trehalose transport efficiency and cold resistance in insects.

[0047] In a second aspect, the present invention provides the Tret1 gene, which encodes the trehalose transporter mutant described above;

[0048] The nucleic acid sequence of the Tret1 gene is shown in SEQ ID NO.2:

[0049]

[0050] Thirdly, the present invention provides the application of the above-mentioned trehalose transporter mutant or Tret1 gene in regulating insect cold resistance.

[0051] Fourthly, the present invention provides the application of the above-mentioned trehalose transporter mutant or Tret1 gene in regulating the efficiency of trehalose transport in insects.

[0052] In this invention, the insects include, but are not limited to, cotton bollworms, or other insects containing the Tret1 gene that are well known to those skilled in the art.

[0053] Fifthly, the present invention provides biological materials containing the aforementioned Tret1 gene. These biological materials include, but are not limited to, expression cassettes, vectors, and cell lines. The "expression cassette" refers to a nucleic acid construct containing the target gene and elements for regulating the target gene. The "vector" refers to a substance capable of replicating and / or expressing the target gene, and of introducing the target gene into prokaryotic or eukaryotic cells. The vector includes, but is not limited to, plasmids, bacteriophages, viral genomes, or viruses.

[0054] Specifically, it is selected from the following: (n1) an expression cassette containing the Tret1 gene; (n2) a vector containing the Tret1 gene, or a vector containing the expression cassette of (n1); (n3) a cell line containing the Tret1 gene, a cell line containing the expression cassette of (n1), or a cell line containing the vector of (n2).

[0055] In a sixth aspect, the present invention provides insect trait-related markers, said markers being selected from (a1): the Tret1 gene described above;

[0056] (a2) A fusion gene containing the Tret1 gene;

[0057] (a3): RNA transcribed from (a1) or (a2);

[0058] (a4): Protein expressed by any one of (a1) to (a3);

[0059] The traits include at least one of insect cold resistance and insect trehalose transport efficiency.

[0060] As described above, the Tret1 gene is related to insect cold resistance and trehalose transport efficiency. Therefore, the Tret1 gene can be used as a marker to characterize insect cold resistance and trehalose transport efficiency. The term "fusion gene containing the Tret1 gene" refers to a fusion gene that, in addition to the Tret1 gene, may contain other functional units, including but not limited to gene portions acting as markers, such as fluorescent protein genes or selection marker genes; gene portions encoding other trait markers; or elements regulating gene expression, such as promoters or enhancers. Correspondingly, the RNA or protein molecules transcribed from the Tret1 gene and its fusion gene can also serve as markers related to insect traits.

[0061] In a seventh aspect, the present invention provides a kit for detecting insect traits, the kit being used to detect the aforementioned markers;

[0062] Preferably, the kit includes a primer pair for detecting the Tret1 gene, the nucleic acid sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8:

[0063] SNP-tret1-F: CCCTGAAGATGCTCGTAAA (SEQ ID NO.7);

[0064] SNP-tret1-R: TGATAGCGGTGGCAATAAA (SEQ ID NO. 8).

[0065] Eighthly, the present invention provides a method for regulating insect traits, the method comprising: overexpressing or inhibiting the expression of the Tret1 gene;

[0066] The traits include at least one of insect cold resistance and insect trehalose transport efficiency.

[0067] In some preferred embodiments, overexpression of the Tret1 gene enhances the insect's cold resistance;

[0068] Suppressing the expression of the Tret1 gene reduces the cold resistance of insects.

[0069] In some preferred embodiments, overexpression of the Tret1 gene improves the efficiency of trehalose transport in insects;

[0070] Suppressing the expression of the Tret1 gene reduces the efficiency of trehalose transport in insects.

[0071] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0072] It should be noted that the amino acid sequence of the wild-type (Tret1-GG) trehalose transporter is as follows:

[0073] (SEQ ID NO.3).

[0074] The nucleic acid sequence of the wild-type (Tret1-GG) trehalose transporter gene is as follows:

[0075]

[0076] Example 1: Association analysis of Tret1 gene and low temperature

[0077] Experimental methods:

[0078] GWAS analysis: Sequencing data were aligned to the *Helicoverpa armigera* reference genome using the default parameters of BWA software, and the data were filtered using SAMtools. SNP loci were then identified using GATK software with the following parameters: QD ≥ 2.0, MQ ≥ 40.0, FS ≤ 60.0, SOR < 3.0, MQRankSum ≥ -12.5, and ReadPosRankSum ≥ -8.0. Historical average data from sampling sites in Xinjiang, North China, and South China were downloaded from the WorldClim website. A linear model (LMM) was used to perform association analysis between genotype and temperature data using GEMMA software. Significance thresholds were determined using Bonfeeoni correction (corrected P-value = 0.05 / N).

[0079] Experimental results:

[0080] GWAS analysis was used to correlate cotton bollworm resequencing data with sampling point temperature information. A significant association signal was identified on chromosome 3. Local analysis revealed a gene, Tret1, within this region, containing a nonsynonymous mutation (chr3:6626270). This nonsynonymous mutation was significantly associated with low temperature (P = 5.02 × 10⁻⁶). -9 )(like Figure 1 (As shown). The accuracy of this locus was confirmed using Sanger sequencing. Analysis of the haplotype distribution of this nonsynonymous mutation revealed that haplotype A (TAT, Ile, isoleucine) is mainly distributed in the cooler Xinjiang region, while haplotype G (TGT, Thre, threonine) is mainly distributed in the warmer South China region (e.g., as shown). Figure 2 (As shown). Subsequent analysis of the spatiotemporal expression pattern of Tret1 revealed that the gene is highly expressed in the pupal stage and fat body, both of which are critical instars and tissues for insects to resist low temperatures.

[0081] Example 2: Intracellular detection of the trehalose transport capacity of different haplotypes of Tret1

[0082] Experimental methods:

[0083] CDS cloning of different haplotypes of the Tret1 gene, construction of eukaryotic expression vectors, and determination of intracellular trehalose content: RNA was extracted from cotton bollworm samples using Trizol reagent, and first-strand cDNA synthesis was performed using the SuperScript™ III First-Stand Synthesis System reverse transcription kit. Primers for amplifying the full-length Tret1 gene were designed.

[0084] Tret1-CDS-F:

[0085] 5'-CCCAAGCTTGCCACCATGAGTTTCAATAAAAACAACC-3' (SEQ ID NO. 5);

[0086] Tret1-CDS-R:

[0087] 5'-TCCCCGCGGGCCACCCCCTCCGCCTCCACAGCCATTTTGTGGTTGCTTACT-3' (SEQ ID NO. 6).

[0088] The full-length haplotypes of different types were amplified using primers Tret1-CDS-F and Tret1-CDS-R. After obtaining the target fragment, it was ligated to the p-EGFP-N1 plasmid by enzyme digestion. The accuracy of the constructed plasmid was verified by PCR sequencing before cell experiments were conducted.

[0089] Subcellular localization assay: Cells were seeded in 24-well plates with glass slides. After about 70% cell coverage, transfection was performed. After 24-36 hours, the cells were prepared for observation. After fixation with 4% paraformaldehyde for 20 minutes, the cells were washed with PBS, and Hoechst was added to stain the nuclei for 5 minutes in the dark. The localization was observed under a fluorescence microscope.

[0090] Cellular trehalose content determination: Cells were transfected with different haplotypes of Tret1 plasmid (the experimental procedure was exactly the same except for the different genes carried by the plasmids), cultured for 36 hours, and the trehalose content was determined using a trehalose ELISA Kit. The experiment was repeated 3 times.

[0091] Experimental results:

[0092] Two haplotypes of the Tret1 gene, the full-length CDS (Tret-A and Tret-G), were cloned. After constructing eukaryotic expression plasmids, they were transfected into insect Hi5 cells. Subcellular localization showed that the Thr / Ile point mutation did not affect the localization of the Tret1 gene, but it significantly affected its ability to transport trehalose (e.g., Figure 3 As shown, Figure 3In this context, A represents the subcellular localization result of Tret-A type; Figure 3 B in the figure represents the subcellular localization result of Tret-G type; Figure 3 (C represents the trehalose transport efficiency of different haplotypes of the trehalose transporter Tret1). Haplotype A, containing Ile, showed significantly higher trehalose transport capacity than haplotype G, containing Thr. This also indicates that haplotype A, mainly distributed in the low-temperature Xinjiang region, has stronger low-temperature adaptability.

[0093] Example 3: Determination of differences in cold resistance among different haplotypes of Tret1 in insects

[0094] Experimental methods:

[0095] Separation and construction of different haplotype populations of the Tret1 gene, determination of trehalose content induced by low temperature, and determination of supercooling point: Design of primers for the detection of non-synonymous mutation sites of trehalose:

[0096] SNP-tret1-F: CCCTGAAGATGCTCGTAAA (SEQ ID NO.7);

[0097] SNP-tret1-R: TGATAGCGGTGGCAATAAA (SEQ ID NO. 8).

[0098] Using this nonsynonymous mutation site as a molecular marker, different haplotypes of the Tret1 gene were isolated. After three generations of population segregation, homozygous cotton bollworm populations containing different Tret1 haplotypes were obtained. Trehalose content in different haplotype populations under low-temperature induction was determined using a trehalose assay kit.

[0099] Supercooling point determination: Supercooling point was determined during the pupal stage using a SUN-V insect supercooling point analyzer (Beijing Pengcheng Company). A single pupa was placed in a 1.5ml centrifuge tube, with the tube in contact with the metal probe. The gap between the pupa and the centrifuge tube was filled with cotton. The tube was then placed at -20°C for supercooling point determination. The experiment was repeated three times, with 18 pupae per replicate.

[0100] Experimental results:

[0101] Segregating populations of Tret1 haplotypes A and G were constructed as follows: The 96S strain, reared for multiple generations indoors, was segregated using a nonsynonymous mutation (TAT / TGT, chr3:6626270) as a marker to ensure consistent genetic background among the segregating populations of different haplotypes. Through three generations of genetic segregation, a cotton bollworm population Tret1-AA carrying homozygous haplotype A and a population Tret1-GG carrying homozygous haplotype G were obtained (e.g., ...). Figure 4(As shown). Subsequently, the trehalose content of the two populations was measured under low-temperature stress, and it was found that the trehalose content of the population carrying haplotype A was significantly higher than that of the population carrying haplotype G under low-temperature stress (as shown). Figure 5 (As shown). Supercooling point is an important indicator of insect cold tolerance. Supercooling point measurements were performed on two haplotypes of Tret1, and it was found that the supercooling point of Tret1-AA was significantly lower than that of Tret1-GG, indicating that the cotton bollworm carrying haplotype A has stronger cold tolerance (e.g., as shown). Figure 6 (As shown).

[0102] summary:

[0103] Association analysis of omics data from field-collected samples with natural environmental factors identified an amino acid variation in Tret1 associated with low-temperature tolerance in Xinjiang. The results showed that this amino acid variation represents an adaptive evolutionary result of insects adapting to low temperatures under natural conditions. In vitro cell experiments and in vivo insect experiments confirmed that this variation significantly enhances insect tolerance to low temperatures. These experimental results deepen our understanding of insect adaptive evolution, provide potential targets for controlling cotton bollworm, and offer important insights for exploring the suitable habitats of economically important insects and natural enemies, as well as developing low-temperature tolerant insects.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of trehalose transporter mutants or the Tret1 gene in improving trehalose transport efficiency in cotton bollworm; The amino acid sequence of the trehalose transporter mutant is shown in SEQ ID NO.1; The nucleic acid sequence of the Tret1 gene is shown in SEQ ID NO.2.

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