Application of overexpression of StHsfA2 gene in improving potato yield and quality

Abstract

The application discloses application of overexpression of a StHsfA2 gene in improving potato yield and quality and belongs to the technical field of genetic engineering. The application finds that overexpression of the StHsfA2 gene can significantly improve photochemical efficiency and relative water content of potato leaves under high temperature, reduce relative electrical conductivity of the leaves, and significantly improve potato yield and quality under a high-temperature environment, specifically including the number of potato tubers, yield and starch content, and provides important gene resources, theoretical support and technical support for improvement of heat tolerance of potato and cultivation of heat-tolerant potato.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of overexpression of the StHsfA2 gene in improving potato yield and quality. Background Technology

[0002] As one of the world's most important food crops, the growth and yield of potatoes are affected by a variety of environmental factors, among which high-temperature stress is a significant contributor to yield reduction. High temperatures during the potato growing season can cause yield losses of up to 80% in severe cases. With the increasing severity of global warming, high-temperature weather is becoming more frequent in major potato-producing areas, posing a serious challenge to potato cultivation and production. Furthermore, high temperatures can delay tuber formation, leading to deformities, secondary growth, and loss of the tuber skin. Therefore, discovering and utilizing new genes to improve the heat resistance of potatoes is of great significance for their production.

[0003] The number of genes reported to contribute to heat tolerance in potatoes is very limited; for a long time, only a few genes, such as StHsfA3 and StSP6A, have been shown to aid in this development. Existing research indicates that heat shock transcription factors (HSFs) play a crucial role in plant responses to high-temperature stress. HsfA2, an important member of the HSF family, enhances the thermal stability of plant cells and thus improves heat tolerance by regulating the expression of heat shock proteins (HSPs) and other related genes. However, the application of HsfA2 in potatoes and its impact on potato heat tolerance remains largely unexplored and lacks in-depth, systematic research and practice. Summary of the Invention

[0004] The purpose of this invention is to provide the application of overexpressing the StHsfA2 gene in improving the heat resistance of potatoes, thereby addressing the problems existing in the prior art. This invention discovers that overexpressing the StHsfA2 gene can significantly improve the photochemical efficiency and relative water content of potatoes under high temperatures, reduce the relative electrical conductivity of leaves, and significantly improve potato yield and quality under high-temperature conditions, specifically including the number of potato tubers, yield, and starch content. This provides important genetic resources, theoretical support, and technical support for improving potato heat resistance traits and cultivating heat-resistant potatoes.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of overexpression of the StHsfA2 gene in improving potato yield and quality, wherein the potato is a potato cultivated under high temperature conditions.

[0007] The present invention also provides the application of recombinant vectors overexpressing the StHsfA2 gene in improving potato yield and quality, wherein the potatoes are potatoes cultivated under high temperature conditions.

[0008] The present invention also provides the application of recombinant microorganisms overexpressing the StHsfA2 gene in improving potato yield and quality, wherein the potatoes are potatoes cultivated under high temperature conditions.

[0009] Furthermore, the potato yield and quality include tuber quantity, tuber yield, and tuber starch content.

[0010] The present invention also provides a method for improving potato yield and quality in high-temperature environments, comprising the steps of using genetic transformation technology to stably overexpress the StHsfA2 gene in potato plants to obtain transgenic lines; and cultivating the transgenic lines in a high-temperature environment.

[0011] Optionally, the high-temperature environment refers to an environment where the daily average temperature reaches 25°C or higher and the nighttime average temperature reaches 20°C or higher.

[0012] The present invention also provides the application of the StHsfA2 gene in the breeding of high-yielding potato lines for planting in high-temperature regions.

[0013] The present invention also provides the application of recombinant vectors overexpressing the StHsfA2 gene in the breeding of high-yielding potato lines for planting in high-temperature regions.

[0014] The present invention also provides the application of recombinant microorganisms overexpressing the StHsfA2 gene in the cultivation of high-yielding potato lines for planting in high-temperature regions.

[0015] This invention also provides a method for cultivating high-yield potato lines, including the step of using genetic transformation technology to stably overexpress the StHsfA2 gene in potato plants to construct high-yield potato lines.

[0016] The present invention discloses the following technical effects:

[0017] This invention has found that overexpression of the StHsfA2 gene can significantly improve the heat resistance of potatoes, increase the photochemical efficiency and relative water content of leaves under high temperature, reduce the relative electrical conductivity of leaves, and significantly improve potato yield and quality under high temperature conditions, specifically including the number of potato tubers, yield and starch content.

[0018] This invention confirms that the StHsfA2 gene plays a regulatory role in potato heat tolerance and potato yield under high temperature, providing important gene resources, theoretical support, and technical support for the improvement of potato heat tolerance traits and the breeding of heat-resistant potatoes. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Western blot results of proteins in wild-type plant WT and transgenic plants StHsfA2-eGFP-#1, StHsfA2-eGFP-#4 and StHsfA2-eGFP-#6;

[0021] Figure 2 The images show the leaves of wild-type plant WT and transgenic plants StHsfA2-eGFP-#1, StHsfA2-eGFP-#4 and StHsfA2-eGFP-#6 under a fluorescence microscope. The scale bar is 20 μm.

[0022] Figure 3 The growth status of potato seedlings in an artificial climate chamber;

[0023] Figure 4 Photochemical efficiency of wild-type WT plants and transgenic plants OE-1, OE-4 and OE-6 under different treatments;

[0024] Figure 5 The relative water content of leaves of wild-type WT plants and transgenic plants OE-1, OE-4 and OE-6 under different treatments;

[0025] Figure 6 The relative electrical conductivity of leaves of wild-type WT plants and transgenic plants OE-1, OE-4 and OE-6 under different treatments;

[0026] Figure 7 The growth status of wild-type plants WT and transgenic plants OE-1, OE-4 and OE-6 under different treatments is shown. The scale bar of the large plant image is 2cm, and the scale bar of the small tuber image is 2cm.

[0027] Figure 8 The number of tubers in wild-type plants (WT) and transgenic plants (OE-1, OE-4, and OE-6) under different treatments;

[0028] Figure 9 The tuber yields of wild-type WT plants and transgenic plants OE-1, OE-4 and OE-6 under different treatments;

[0029] Figure 10 The starch content of tubers from wild-type plants (WT) and transgenic plants (OE-1, OE-4, and OE-6) under different treatments was calculated. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0036] Example 1: Construction and identification of heat shock transcription factor StHsfA2 overexpression vector and transgenic potato plants

[0037] The coding sequence of the heat shock transcription factor StHsfA2 (Soltu.DM.08G013140) was obtained from the potato genome information provided by the Solanum tuberosum v6.1 potato database in the Phytozome v13 website (https: / / phytozome-next.jgi.doe.gov / ). Upstream primer F-PCR and downstream primer R-PCR were designed using SnapGene software.

[0038] F-PCR (SEQ ID NO.1):

[0039] 5'-GAACACGGGGGACTCTAGAATGGAGGAAGTAGTGAAAGTGAAGGT-3';

[0040] R-PCR (SEQ ID NO.2):

[0041] 5'-CTTGCTCACCATGGTACCACCCCATTCAGGTGTTTTTGCAACA-3'.

[0042] RNA was extracted from potato (Desiree variety) leaves and cDNA was obtained by reverse transcription. Using the cDNA as a template sequence, the potato StHsfA2 gene was amplified using upstream primer F-PCR and downstream primer R-PCR. After removing the stop codon, the full-length gene was 1020 bp. The amplified product was digested with enzymes, transformed, and ligated into the pBI121-eGFP vector, which carries an eGFP tag. After successful construction of the recombinant plasmid, it was transformed into Agrobacterium tumefaciens strain GV3101 using a heat shock method. The transformed Agrobacterium was used to infect potato leaves, and tissue culture was performed. StHsfA2 transgenic plants were obtained through resistance selection. Protein was extracted from young leaves and verified by Western blot using a GFP-specific recognition antibody. The results are shown in the figure below. Figure 1 The transgenic lines with the target band were obtained and named StHsfA2-eGFP-#1, StHsfA2-eGFP-#4 and StHsfA2-eGFP-#6, respectively.

[0043] Young leaves from the three transgenic lines were taken and the green fluorescence of the StHsfA2-eGFP fusion protein was observed under a laser microscope. The results are shown in the figure. Figure 2 All three strains successfully expressed the corresponding fusion proteins, further verifying the reliability of the positive plants.

[0044] Example 2: Identification of traits in transgenic potato plants

[0045] Wild-type potato (Desiree variety) was designated WT. Transgenic plants StHsfA2-eGFP-#1, StHsfA2-eGFP-#4, and StHsfA2-eGFP-#6 (hereinafter referred to as OE-1, OE-4, and OE-6, respectively) were cultured in the same suitable tissue culture environment, with the temperature controlled at approximately 20℃. Single stem nodes of the tissue culture seedlings were cut and axillary buds were induced to grow in the culture medium. After approximately 2-3 weeks, uniformly growing axillary buds were cut and cultured in the medium for another 2 weeks. Subsequently, uniformly growing plants were selected and transplanted into soil and placed in an artificial climate chamber for further cultivation. Figure 3 ).

[0046] After one week of growth, the potato plants were randomly divided into a control group and a high-temperature treatment group. The control group was kept at 22°C day / 16°C night, while the high-temperature treatment group was kept at 35°C day / 29°C night. The daytime duration was 8 hours, the relative humidity was 60%, and the light intensity was 260 μmol / m². -2 s -1 After a week of high-temperature treatment, potato leaves from the same location were selected for photochemical efficiency, relative water content, and relative conductivity measurements. The results are as follows: Figures 4-6 As shown, under normal conditions, there were no significant differences in photochemical efficiency, relative water content, and relative electrical conductivity between wild-type and transgenic plants OE-1, OE-4, and OE-6. However, under high-temperature conditions, transgenic plants OE-1, OE-4, and OE-6 exhibited higher photochemical efficiency and relative water content, and lower relative electrical conductivity. This indicates that overexpression of StHsfA2 can significantly enhance the growth of potatoes under high-temperature conditions.

[0047] All plants from the high-temperature treatment group and the control group were transferred to an artificial climate chamber with the same ambient temperature environment (same as the control group) to resume growth. After 6 weeks of growth, their growth and tuber yield were recorded. Results are shown below. Figures 7-10 It is evident that while wild-type plants recovered from high-temperature treatment, their growth was still significantly lower than that of the control group, indicating irreversible damage. Their tuber quantity, tuber yield, and tuber starch content were also significantly lower than those of the control group and the transgenic plants treated with high temperature. In contrast, transgenic plants OE-1, OE-4, and OE-6 recovered from high-temperature treatment, showing no significant difference in growth compared to the control group (wild-type and transgenic plants), and exhibited significant advantages in tuber quantity, tuber yield, and tuber starch content.

[0048] The above results indicate that overexpression of the heat shock transcription factor StHsfA2 gene can significantly improve the growth status, yield, and quality of potatoes cultivated under high-temperature conditions, without affecting their growth under suitable conditions. Furthermore, potatoes overexpressing the StHsfA2 gene exhibit excellent environmental adaptability under high-temperature stress, possessing both heat tolerance and high yield, making them highly valuable for application.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of overexpression of StHsfA2 gene for increasing yield and quality of potato, characterized in that, The potatoes mentioned are potatoes cultivated in a high-temperature environment; The potato yield and quality include tuber quantity, tuber yield, and tuber starch content; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature environment refers to an environment where the daily average temperature reaches 25°C or higher and the nighttime average temperature reaches 20°C or higher.

2. Use of a recombinant vector overexpressing StHsfA2 gene for increasing potato yield and quality, characterized in that, The potatoes mentioned are potatoes cultivated in a high-temperature environment; The potato yield and quality include tuber quantity, tuber yield, and tuber starch content; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature environment refers to an environment where the daily average temperature reaches 25°C or higher and the nighttime average temperature reaches 20°C or higher.

3. Use of a recombinant microorganism overexpressing the StHsfA2 gene for increasing potato yield and quality, characterized in that, The potatoes mentioned are potatoes cultivated in a high-temperature environment; the potato yield and quality include the number of tubers, the yield of tubers, and the starch content of tubers; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature environment refers to an environment where the daily average temperature reaches 25°C or higher and the nighttime average temperature reaches 20°C or higher.

4. A method of improving yield and quality of potatoes in a high temperature environment, characterized by, The process includes the steps of using genetic transformation technology to stably overexpress the StHsfA2 gene in potato plants to obtain transgenic lines; and cultivating the transgenic lines in a high-temperature environment. The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature environment refers to an environment where the average daily temperature reaches 25°C or higher and the average nighttime temperature reaches 20°C or higher. The potato yield and quality include tuber quantity, tuber yield, and tuber starch content.

5. Use of the StHsfA2 gene for breeding high-yielding potato lines, characterized in that, The high-yielding strains are intended for cultivation in high-temperature regions; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature region refers to an environment where the average daily temperature is above 25°C and the average nighttime temperature is above 20°C.

6. Use of a recombinant vector overexpressing the StHsfA2 gene for breeding high-yield potato lines, characterized in that, The high-yielding strains are intended for cultivation in high-temperature regions; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solnum tuberosum v6.1 on the phytozome v13 website. The high-temperature region refers to an environment where the average daily temperature is above 25°C and the average nighttime temperature is above 20°C.

7. Use of recombinant microorganism overexpressing StHsfA2 gene for breeding high-yielding potato lines, characterized in that, The high-yielding strains are intended for cultivation in high-temperature regions; The StHsfA2 gene is numbered Soltu.DM.08G013140 in the potato database Solanum tuberosum v6.1 in the phytozome v13 website; The environment of the high-temperature region is an environment in which the daily average temperature reaches 25°C or higher, and the average temperature at night reaches 20°C or higher.

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