Potato insect-resistant gene StSOBIR1, recombinant vector and application thereof

Abstract

The application discloses a potato insect-resistant gene StSOBIR1 as well as a recombinant vector and application thereof, and belongs to the technical field of biotechnology. A base sequence of the potato insect-resistant gene StSOBIR1 is shown in SEQ ID No: 1; or a gene with more than 95% homology with the base sequence of SEQ ID No: 1. An amino acid sequence coded by StSOBIR1 is shown in SEQ ID No: 2; and the recombinant vector comprises an inverse complementary sequence of nucleotides 930-1179 in SEQ ID No: 1. Experiments prove that the recombinant vector can be introduced into a receptor plant to prepare transgenic potatoes with silenced StSOBIR1 genes, so that the plant insect resistance is significantly improved when the plant is harmed by potato tuber moths. Therefore, the StSOBIR1 gene can be used for cultivating and identifying insect-resistant plant varieties required by agricultural and ecological environment management, and plays an important role in the field of agricultural pest insect-resistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a potato insect-resistant gene StSOBIR1, its recombinant vector, and its applications. Background Technology

[0002] China is a major potato producer globally, with an average annual output of over 80 million tons, ranking first in the world. In 2015, China launched its potato staple food strategy, making potatoes the fourth largest staple food after rice, wheat, and corn. In recent years, while my country's potato industry has developed rapidly, potato production also faces numerous challenges. Currently, the main method for controlling potato pests is the use of chemical pesticides, which play an indispensable role in effectively controlling yield losses caused by potato pests and ensuring food security. However, while controlling potato pests, chemical pesticides also have negative effects on beneficial organisms such as natural enemies, and their unscientific use can impact human health and the ecological environment. Therefore, the scientific and rational use of chemical pesticides, reducing their application, identifying insect-resistant genes, and utilizing plant resistance to control pests are particularly important for producing pollution-free potatoes and are among the needs of my country's agriculture.

[0003] Insect-resistant transgenic crops bred using plant insect-resistant genes can suppress pest populations, thereby enhancing plant resistance. They also offer advantages such as low cost, comprehensive protection, and high specificity, making them a current hot topic in agricultural bioengineering research. Conventional insect-resistant transgenic crops are mainly based on the insecticidal activity of insect-resistant proteins. However, plants themselves possess numerous insect-resistant regulatory genes that regulate the synthesis and accumulation of insect-resistant substances (such as insect-resistant compounds and proteins) within the plant, thereby improving plant resistance to pests. Currently, the application of these insect-resistant regulatory genes in plant insect-resistant breeding is relatively limited. Summary of the Invention

[0004] The purpose of this invention is to provide a potato insect-resistant gene StSOBIR1, its recombinant vector, and its applications.

[0005] A potato insect-resistant gene, StSOBIR1, has the function of negatively regulating potato insect resistance. The base sequence of the potato insect-resistant gene StSOBIR1 is shown in SEQ ID No:1 and consists of 2169 nucleotides.

[0006] Or, a gene with more than 95% homology to the base sequence shown in SEQ ID No:1.

[0007] A protein encoded by the potato insect-resistant gene StSOBIR1 has the function of negatively regulating potato insect resistance. The amino acid sequence of the protein encoded by the potato insect-resistant gene StSOBIR1 is shown in SEQ ID No:2. The lower the expression level and / or activity of this protein in plants, the stronger the insect resistance of the plants.

[0008] A recombinant vector comprising the reverse complementary sequence of nucleotides 930-1179 of SEQ ID No:1.

[0009] The recombinant vector was prepared by performing a two-step double enzyme digestion reaction at the two Xhol and Xbal sites of the pHELLSGATE8 vector, and inserting the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:1, as shown in SEQ ID No:3 and SEQ ID No:4, to obtain the recombinant vector.

[0010] The above-mentioned recombinant vector was used in the preparation of insect-resistant transgenic potatoes, and this recombinant vector can be used for plant genetic transformation.

[0011] A method to improve the insect resistance of potatoes: suppressing the expression of the potato insect resistance gene StSOBIR1 in recipient plants, thereby reducing the expression level and / or activity of the protein encoded by the potato insect resistance gene StSOBIR1 in the plants, and obtaining insect-resistant plants;

[0012] As an embodiment of the present invention, an RNAi interference vector is introduced into a recipient plant. The RNAi interference vector is a recombinant vector containing the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:1. The recombinant vector can inhibit the expression of the potato insect-resistant gene StSOBIR1 in the recipient plant.

[0013] Methods for introducing RNAi interference vectors into recipient plants include: transforming plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and then culturing the transformed plant tissues into plants.

[0014] A method for cloning the potato insect-resistant gene StSOBIR1 includes the following steps:

[0015] Construction of potato cDNA library: Total RNA was extracted from potato leaves, and cDNA was synthesized using total RNA as a template under the action of reverse transcriptase;

[0016] Using the above cDNA as a template, PCR amplification was performed using primers to obtain the potato insect-resistant gene StSOBIR1 fragment;

[0017] After the amplified fragment was recovered, it was ligated into a cloning vector, and after sequencing, the potato insect-resistant gene StSOBIR1 shown in SEQ ID No:1 was obtained.

[0018] The beneficial effects of this invention are as follows: This invention achieves potato resistance to chewing insects by inhibiting the expression level of the insect-resistant gene StSOBIR1 in potatoes. Experiments have shown that the size and / or weight of pests on transgenic potatoes with the StSOBIR1 gene silenced are smaller than those on wild-type potatoes, and the content of important insect-resistant secondary metabolites α-solanine and α-chaconine in transgenic potatoes with the StSOBIR1 gene silenced is significantly higher than that in wild-type potatoes. When transgenic potatoes with the StSOBIR1 gene silenced are attacked by the potato tuber moth, their resistance to the potato tuber moth is significantly enhanced. Therefore, the potato insect-resistant gene StSOBIR1 is associated with potato resistance to the potato tuber moth. Attached Figure Description

[0019] Figure 1 Phenotypic results of the aboveground parts of potato lines with StSOBIR1 gene silence.

[0020] Figure 2 The weight statistics of potato tuber moths after feeding the potato tuber moths with the StSOBIR1 gene silenced potato lines for 10 days.

[0021] Figure 3 The results show the content of solanine, an insect-resistant secondary metabolite, in potato lines with the StSOBIR1 gene silenced 48 hours after simulated insect pest treatment. A represents the content of α-solanine; B represents the content of α-chaconine. Specific implementation methods

[0022] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0024] The potatoes used in the following examples are of variety E3 (hereinafter also referred to as wild-type potatoes).

[0025] Example 1: Obtaining a recombinant vector that inhibits StSOBIR1 gene expression:

[0026] 1. Primer design and synthesis

[0027] The present invention designs the following primers: upstream primer StSOBIR1-QF1: 5'-TCTTACAATGCAGCCAACCA-3'; downstream primer StSOBIR1-QR1: 5'-TGCAGTAAACATACAAGATTTGACA-3'; upstream primer StSOBIR1-QF2: 5'-tttggagaggacacgctcgagTAACAAGAAGAAAAGGAAGTTAAGATCTTG-3'; downstream primer StSOBIR1-QR2: 5'-tggggtaccgaattcctcgagTCCACATCCACCTTTTCCAATC-3'; upstream primer StSOBIR1-QF3: 5'-gataagcttggatcctctagaTAACAAGAAGAAAAGGAAGTTAAGATCTTG-3'; downstream primer StSOBIR1-QR3: 5'-tcattaaagcaggactctagaTCCACATCCACCTTTTCCAATC-3'.

[0028] Both upstream and downstream primers 2 and 3 introduced Xhol and Xbal restriction sites at their respective ends. The primers were synthesized by a biotechnology company.

[0029] 2. Obtaining the StSOBIR1 gene fragment using PCR method

[0030] Based on the protein sequence ID provided in the protein phosphorylation database, the nucleic acid sequence of this gene (ID LOC102583521) was found on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Primer 1 was designed using primer-blast, and the primer contains the full-length CDS region of this gene. In this invention, high-quality gDNA double strands were used as templates to amplify the StSOBIR1 sequence using the upstream primer StSOBIR1-QF1 and the downstream primer StSOBIR1-QR1. The PCR conditions were: ① 94℃, 10 min; ② 94℃, 30 s; 55℃, 30 s; 72℃, 100 s; 35 cycles; ③ 72℃, 10 min.

[0031] 3. Cloning identification and sequencing

[0032] The amplified fragment was analyzed by 1% agarose gel electrophoresis at 120V for 30 min. The product was then recovered using a commercial gel extraction kit, and the concentration of the recovered product was determined using a micro spectrophotometer. DH5α competent cells were ligated and transformed, and the bacterial culture was verified by PCR using M13 primers. The product was again detected by 1% agarose gel electrophoresis at 120V for 30 min. The bacterial culture containing the target band was sent to a sequencing company for sequencing. Finally, the results were compared with those from NCBI using Geneious software. After verification, the plasmid was stored. This invention, through nucleotide sequence determination and analysis, ultimately obtained the potato insect-resistant gene StSOBIR1, whose nucleotide sequence is shown in SEQ ID No:1, and its amino acid sequence is shown in SEQ ID No:2.

[0033] 4. Carrier Construction

[0034] Using the above PCR products as templates, the StSOBIR1 sequence fragment was amplified by PCR using StSOBIR1-QF2, StSOBIR1-QR2 and StSOBIR1-QF3, StSOBIR1-QR3 respectively, under the same conditions, resulting in two 250bp reverse complementary PCR fragments, as shown in SEQ ID No:3 and SEQ ID No:4.

[0035] First, the two XHo I sites on the pHellsgate8 vector were double-digested with the restriction endonuclease XHo I. The digested product was then ligated with the PCR product SEQ ID No:3 using a commercial ligation transformation kit, and the recombination ligation effect was verified by sequencing. Next, the two XBa I sites on the recombinant product were double-digested with the restriction endonuclease XBa I. The digested product was then ligated with the PCR product SEQ ID No:4 using a commercial ligation transformation kit, and the recombination ligation effect was verified by sequencing, thus obtaining the plant recombinant vector.

[0036] The StSOBIR1 gene and its recombinant vector described in this invention can be used for plant genetic transformation and applied in the cultivation of plants with enhanced stress resistance.

[0037] Example 2: Construction and functional identification of StSOBIR1 gene-silenced potato lines

[0038] I. Construction of StSOBIR1 gene-silenced potato lines

[0039] Potato tubers were sterilized and sown in 1 / 2 MS medium for dark culture until germination. Then, they were transferred to light conditions for 6-8 days of culture. Sterile seedling cotyledons were collected and pre-cultured for two days. The plant recombinant vector obtained in Example 1 was transformed into Agrobacterium using the freeze-thaw method to obtain recombinant Agrobacterium. Positive transformants (containing the StSOBIR1 gene shown in SEQ ID NO:1) were identified by PCR and used to infect the cotyledons of sterile potato seedlings. In this example, Agrobacterium activated on a medium containing 50 mg / L kanamycin was used to infect the cotyledons of sterile seedlings. After dark culture for 2 days, the seedlings were transferred to selection medium and subcultured every 2 weeks until green buds appeared. The resistant buds were then transferred to rooting medium and cultured under light. After 2-3 weeks of rooting, leaves from the regenerated plants were harvested and tested by PCR. PCR conditions were: ① 94℃, 3 min; ② 94℃, 30 s; 58℃, 30 s; 72℃, 30 s; 30 cycles; ③ 72℃, 10 min; ④ 25℃, 1 min.

[0040] II. Effects of pHEllSGATE8-StSOBIR1-RNAi transgenic potatoes on insect resistance

[0041] Before insect feeding, wild-type potatoes and transgenic potatoes of pHEllSGATE8-StSOBIR1-RNAi were cultured under standard conditions: 16 hours of light, 8 hours of darkness, relative humidity 55±10%, and temperature 22℃. When the plants reached 3 weeks of age, newly hatched potato tuber moth larvae were collected with a brush and transferred to the plants, allowing them to grow freely. Five to six plants from each strain were used for the experiment, with approximately 10 newly hatched potato tuber moth larvae per plant. On the 10th day of growth, the plants were photographed for record-keeping, and the growth status and weight of the potato tuber moths were also photographed and recorded.

[0042] Experimental results showed that, compared with wild-type potato E3, the potato tuber moths feeding on transgenic potatoes had lighter body weights and significantly higher levels of α-solanine and α-chaconine, indicating that the transgenic potatoes exhibited significantly enhanced resistance to potatoes. Specifically:

[0043] (1) The transgenic potato plants of pHEllSGATE8-StSOBIR1-RNAi were photographed and recorded after 21 days of growth.

[0044] The results are as follows Figure 1 As shown, the transgenic potatoes of pHEllSGATE8-StSOBIR1-RNAi exhibited the same characteristics as the control wild-type potato E3.

[0045] (2) On the 10th day after feeding the potato tuber moth, the weight of the potato tuber moths fed with the corresponding plants was counted.

[0046] The results are as follows Figure 2 As shown, after newly hatched larvae were introduced into the corresponding plants and fed for 10 days, the weight of tuber moths that fed on transgenic potatoes was significantly lower than that of tuber moths that fed on wild-type potato E3.

[0047] (3) After the transgenic potato plants of pHEllSGATE8-StSOBIR1-RNAi grew to 21 days, two wounds were made on each side of the leaves parallel to the main vein using a sterile roller. 20 μL of the extracted tuber moth oral secretion was drawn up with a pipette tip and evenly applied to the wounds. Only the first three leaves of the pinnate compound leaf were treated each time. The leaves were collected after 48 hours and the contents of α-solanine and α-chaconine were detected.

[0048] The results are as follows Figure 3 As shown, the transgenic potatoes of pHEllSGATE8-StSOBIR1-RNAi had significantly higher contents of α-solanine and α-chaconine than wild-type potatoes.

[0049] The above results demonstrate that the potato insect-resistant gene StSOBIR1 is associated with potato resistance to the potato tuber moth. The protein encoded by the potato insect-resistant gene StSOBIR1 and its encoding gene can be used in research and industrial production to improve crop insect resistance using transgenic technology.

[0050] 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 it. Although the present invention has been described with reference to preferred embodiments, various changes in form and detail may be made thereto without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. The use of a recombinant vector based on potato insect-resistant gene StSOBIR1 in the preparation of transgenic potato resistant to potato tuber moth, characterized in that, The base sequence of the potato insect-resistant gene StSOBIR1 is shown in SEQ ID No:1, and the recombinant vector contains the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:

1.

2. Use according to claim 1, characterized in that, The recombinant vector was prepared by performing a double enzyme digestion reaction twice at the two Xhol and Xbal sites of the pHELLSGATE8 vector, and inserting the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:1, as shown in SEQ ID No:3 and SEQ ID No:4, to obtain the recombinant vector.

3. A method of increasing the resistance of potato tubers to potato tuber moths, characterized in that, An RNAi interference vector, which is a recombinant vector containing the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:1, is introduced into the recipient plant to inhibit the expression of the potato insect-resistant gene StSOBIR1 in the recipient plant.

4. The method of claim 3, wherein, The recombinant vector was prepared by performing a double enzyme digestion reaction twice at the two Xhol and Xbal sites of the pHELLSGATE8 vector, and inserting the reverse complementary sequence of nucleotides 930-1179 in SEQ ID No:1, as shown in SEQ ID No:3 and SEQ ID No:4, to obtain the recombinant vector.

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