Peach heat shock protein PpHSP20-32 and its use

Abstract

The invention discloses a peach heat shock protein PpHSP20‑32 and a use thereof. The invention separates the complete cDNA of the heat shock protein PpHSP20‑32 gene from peach, connects it to a plant expression vector, transforms the plant by Agrobacterium infection, and obtains a transgenic plant, wherein the plant becomes taller, and the width and length of the rosette leaves become larger. The gene is of great significance to plant growth and development, and can improve the heat resistance of the plant, so the gene can also be used in plant genetic improvement.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to peach heat shock protein PpHSP20-32 and application thereof. Background Art

[0002] As the global climate changes, temperature is the most important factor affecting plant growth and geographical distribution. Due to their sedentary nature, plants are subject to large temperature fluctuations caused by factors such as day and night alternation, seasonal changes, and weather changes, which has led to the evolution of plants with strong temperature adaptability. High or low temperature stress, as one of the important abiotic stress factors, seriously affects plant growth. Many mechanisms of plant response to temperature stress have been revealed, however, the perception and conduction of plants to non-stress temperature changes in the environment is still unknown.

[0003] A large number of studies have shown that heat shock proteins (HSP) play an important role in temperature response, including growth and development as well as stress response. According to the molecular weight of heat shock proteins, they are divided into five categories: HSP100, HSP90, HSP70, HSP60 and HSP20, among which HSP20 is the most common in plants. HSP20 is also called small heat shock protein (sHSP), with a molecular weight of 15-42kDa.

[0004] Through high temperature pretreatment, plants can acquire a certain degree of heat tolerance. As an important class of molecular chaperones, the accumulation of HSP20 plays a key role in this process. HSP20 is also involved in biological processes such as salt stress and drought stress. Most HSP20s genes are induced by heat, and the expression of some genes is enhanced by sodium chloride stress or drought stress. At present, the research on HSP20 mainly focuses on its heat tolerance. In pepper, overexpression of CaHSP16.4 can improve the stability of the active oxygen scavenging system and thus enhance tolerance to high temperature and drought stress. In addition, many HSP20s are related to plant development, such as seed maturation and germination, pollen development and hypocotyl elongation.

[0005] At present, the research on HSP20 function in different plants mainly focuses on its heat resistance, while there are few reports on its relationship with plant height and expression under non-stress temperature. Currently, only one study has shown that the AtHSP22 gene promotes hypocotyl elongation under high temperature conditions (29°C), but whether it causes changes in plant height has not been reported. Summary of the invention

[0006] One of the objectives of the present invention is to provide peach (Prunus persica L.) heat shock protein PpHSP20-32 and the protein encoded by it.

[0007] The second purpose of the present invention is to provide peach heat shock protein PpHSP20-32 and the protein and recombinant vector encoded thereby for use in plant growth and development.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides peach heat shock protein PpHSP20-32, the sequence of which is shown as SEQ ID NO.1.

[0010] The present invention also provides a protein encoded by peach heat shock protein PpHSP20-32, and its amino acid sequence is shown in SEQ ID NO.2.

[0011] The present invention also provides a recombinant vector comprising the peach heat shock protein PpHSP20-32. Preferably, the recombinant vector is an Agrobacterium expression vector.

[0012] The gene or protein is used to obtain a transgenic plant, and the plant includes but is not limited to Arabidopsis thaliana.

[0013] Specifically, the steps of obtaining the transgenic Arabidopsis thaliana include:

[0014] A: constructing a recombinant vector containing the peach heat shock protein PpHSP20-32;

[0015] B: Transform the constructed recombinant vector into Arabidopsis tissues or cells;

[0016] C: Transgenic Arabidopsis plants were obtained by cultivation and screening.

[0017] Among them, SEQ ID NO.1 in the sequence has a total of 462 bp, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2 in the sequence table, with a total of 153 amino acids.

[0018] The term "recombinant expression vector" in the present invention refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0019] The vector containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins. The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, etc.

[0020] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0021] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. Transformation of plants can be carried out using methods such as Agrobacterium transformation or gene gun transformation, such as spraying, leaf disc method, rice embryo transformation method, etc. Transformed plant cells, tissues or organs can be regenerated into plants using conventional methods.

[0022] The present invention also provides uses of peach heat shock protein PpHSP20-32, proteins encoded by peach heat shock protein PpHSP20-32 and recombinant vectors of peach heat shock protein PpHSP20-32 in plant growth and development, mainly in increasing plant height and / or rosette leaf width, length and / or heat resistance.

[0023] As an embodiment of the present invention, the polynucleotide is cloned into an appropriate vector by conventional methods, and the recombinant vector carrying the exogenous gene is introduced into plant cells to make the plant cells express the peach heat shock protein PpHSP20-32. The plant cells can be regenerated into plants to obtain plants that overexpress the polypeptide.

[0024] Specifically, a method for cultivating transgenic plants, the peach heat shock protein PpHSP20-32 gene is introduced into a target plant to obtain a transgenic plant, wherein the plant height, rosette leaf width or length and / or heat resistance of the transgenic plant are higher than that of the target plant.

[0025] The implementation method of "promoting the expression of PpHSP20-32 gene in target plants" can be as follows (1) or (2) or (3):

[0026] (1) introducing the PpHSP20-32 gene into target plants;

[0027] (2) introduction of strong promoters and / or enhancers;

[0028] (3) Other common methods in this field.

[0029] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants or forestry plants. The plants may be, for example (but not limited to): dicots, monocots or gymnosperms. More specifically, the plants include (but not limited to): wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries, beans, lentils, peas, soybeans, rapeseed, mustard, poppies, olea, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts, gourds, cucumbers, watermelons, cotton, flax, jute, citrus, lemons, grapefruits, spinach, lettuce, asparagus, cabbage, Chinese cabbage, Chinese cabbage, carrots, onions, potatoes, tomatoes, green peppers, avocados, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapevines, oyster grass, bananas, natural rubber trees and ornamental plants.

[0030] As a preferred embodiment, the "plants" include but are not limited to peach, Solanaceae, Cruciferae, Rosaceae, and Vitaceae. For example, the "plants" include but are not limited to: tobacco and tomato of Solanaceae; Arabidopsis of Cruciferae, apple and strawberry of Rosaceae; grape of Vitaceae, etc.

[0031] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and these different parts all have our target gene or nucleic acid. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0032] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method in this patent have the same characteristics.

[0033] The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.

[0034] The present invention has the following advantages:

[0035] 1. The present invention clones PpHSP20-32 in the peach heat shock protein PpHSP20 gene family, and finally uses an Agrobacterium-mediated method to transfer it into Arabidopsis to verify the function of the target gene, which is beneficial to clarify the molecular mechanism of peach dwarfing from a molecular mechanism perspective, and is of great significance for the use of molecular means to cultivate dwarf germplasm.

[0036] 2. The present invention can also isolate the complete cDNA of the heat shock protein PpHSP20-32 gene from peach, connect it to a plant expression vector, and transform the plant using the Agrobacterium infection method to obtain transgenic plants. The plants become taller and the width and length of the rosette leaves become larger, indicating that the gene is of great significance to plant growth and development, and can improve the heat resistance of plants. Therefore, the gene can also be used in plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis diagram of PCR amplification of peach heat shock protein PpHSP20-32;

[0038] The M of the left band in the figure is DL2000 marker, and the gene fragment size of PpHSP20-32 is 462bp.

[0039] Figure 2 This is the evolutionary analysis of peach PpHSP20-32 and Arabidopsis AtHSP17.6.

[0040] Figure 3 The expression of peach PpHSP20-32 in different tissues of 'Zhongyoutao No. 14'.

[0041] Figure 4 It is the positive plant identification of peach PpHSP20-32 transgenic Arabidopsis thaliana;

[0042] In the figure, A: transgenic plants; WT: wild-type Arabidopsis control; H2O: blank control; M: DL2000Marker; P: plasmid control.

[0043] Figure 5 This is the observation and variance analysis of rosette leaf phenotype of peach PpHSP20-32 transgenic Arabidopsis thaliana two weeks later.

[0044] Figure 6 This is the observation and variance analysis of plant height phenotype of peach PpHSP20-32 transgenic Arabidopsis thaliana three weeks after transplantation.

[0045] Figure 7 This is the change in germination rate of peach PpHSP20-32 transgenic Arabidopsis after high temperature treatment. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0047] As used throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The specification subsequently describes the preferred embodiments of the present invention, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the attached claims.

[0048] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0049] The 'Zhongyoutao No. 14' in the present invention is a new variety of semi-dwarf nectarine bred by Wang Zhiqiang and others from the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences using hybrid breeding methods. This variety has been launched on the market and can be purchased on the market. The experimental materials used in the present invention are provided by the National Peach and Grape Improvement Center of the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences.

[0050] Embodiment 1

[0051] 1. Isolation of gene PpHSP20-32

[0052] Total RNA from leaves of 'Zhongyoutao No. 14' was extracted using a column-type plant total RNA extraction and purification kit (Shanghai Biotech), and reverse transcription was performed using the FastQuant RT Kit (with gDNase) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain single-stranded cDNA. PCR amplification was performed using the single-stranded cDNA as a template and the following sequences as primers to obtain the full-length sequence of the gene PpHSP20-32. The PCR amplification electrophoresis is shown in the figure below. Figure 1 The full-length sequence of gene PpHSP20-32 is shown in SEQ ID NO.1 in the sequence list, with a total of 462 bp, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2 in the sequence list, with a total of 153 amino acids. The amino acid sequence alignment results of peach PpHSP20-32 and Arabidopsis thaliana AtHSP17.6 (NP_172220) are shown in Figure 2 . Figure 2 The results showed that the amino acid sequence similarity between PpHSP20-32 in peach, ie, PpHSP20-32 and AtHSP17.6, was 49.07% (the homology was not high).

[0053] The primer sequences are:

[0054] Forward PpHSP20-32-F: 5′-ATGGCATTAATTCCTAGTACCATTT-3′;

[0055] Reverse PpHSP20-32-R: 5′-TTAGTGCCCAGAAATTTGAACG-3′.

[0056] PCR amplification conditions were as follows: 98°C for 10 s, 55°C for 15 s, 72°C for 40 s, 35 cycles; and stored at 4°C.

[0057] 2. Expression analysis of gene PpHSP20-32

[0058] Fluorescence quantitative PCR operation steps: The total RNA from the stem tip, flower, embryo, young leaf, mature leaf and fruit of 'Zhongyoutao No. 14' was extracted using a column-type plant total RNA extraction and purification kit (Shanghai Shenggong), and reverse transcription was performed using the FastQuant RT Kit (with gDNase) reverse transcription kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain single-stranded cDNA, and amplification reaction was performed using the fluorescence quantitative kit SYBR Select Master Mix (Applied Biosystems, Mardrid, CA, USA), and qRT-PCR detection was performed using the ABI PRISM 7500 FAST Sequence Detection System (Applied Biosystems, Mardrid, CA, USA) fluorescence quantitative instrument.

[0059] The fluorescence quantitative PCR reaction system was 20 μl, including 200 ng cDNA (1 μL), SYBR Select Master Mix (10 μL), 0.5 μmol·L -1 Upstream and downstream primers (1 μL each) and RNase-free water (7 μL).

[0060] Reaction program: 95℃ pre-denaturation, 2min; 95℃ denaturation 30s, 60℃ annealing 30s, 72℃ extension 30s, 42 cycles in total. Each sample was repeated 3 times. Quantitative primers were designed using Primer Premier 5.0 software.

[0061] The quantitative primer sequences are:

[0062] Forward Q PpHSP20-32-F: 5′-AAGCAACCTCCTTCACCAACTCCA-3′;

[0063] Reverse Q PpHSP20-32-R: 5′-GGCATTCTCAGGCAAACTCAAACG-3′.

[0064] qRT-PCR was used to detect the expression of PpHSP20-32 genes in different tissues of 'Zhongyoutao No. 14'. Figure 3 It can be seen that PpHSP20-32 is expressed in the stem tips, young leaves, mature leaves, flowers, embryos, and fruits of the one-year-old branches of 'Zhongyoutao No. 14'. The expression level in mature leaves is the highest, and there is almost no expression in other parts.

[0065] 3. Functional identification test of PpHSP20-32 gene

[0066] To study the function of the PpHSP20-32 gene, its function was analyzed and identified using transgenic Arabidopsis thaliana.

[0067] 3.1 Construction of recombinant vector

[0068] The PpHSP20-32 gene amplified from the leaves of 'Zhongyoutao No. 14' was ligated to the overexpression vector pSAK-277 using a one-step cloning kit (Vazyme, Nanjing Novi Biotechnology Co., Ltd.) using T4 ligase from NEB at 16°C overnight. The primers are as follows:

[0069] G-PpHSP20-32-F:

[0070] 5′-CCCAAGCTTATGGCATTAATTCCTAGTACCATTT-3′;

[0071] G-PpHSP20-32-R:

[0072] 5′-TGCTCTAGATTAGTGCCCAGAAATTTGAACG-3′.

[0073] Detect positive clones. Then send the positive clones to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0074] 3.2 Screening of positive transgenic Arabidopsis strains

[0075] Since an efficient and mature genetic transformation system has not yet been established for peach, the model plant Arabidopsis thaliana was used to verify the function of the PpHSP20-32 gene. The sequenced pSAK-277-PpHSP20-32 plasmid was extracted and transferred into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method. The concentration of Agrobacterium culture solution was adjusted to OD 600To 0.8, wild-type Arabidopsis plants were infected by the flower dipping method, and after harvesting T0 generation seeds, resistant plants were screened on MS medium containing kanamycin. After growing into seedlings, DNA was extracted, and positive transgenic plants were obtained by PCR detection, and T1 generation seeds were collected. After the T1 generation seeds were sown, the differences between the T2 generation transgenic Arabidopsis and the wild type were observed. The following experiments all used T2 generation and subsequent homozygous lines.

[0076] PCR molecular identification of positive plant primers:

[0077] Forward PpHSP20-32-F: 5′-ATGGCATTAATTCCTAGTACCATTT-3′;

[0078] Reverse PpHSP20-32-R: 5′-TTAGTGCCCAGAAATTTGAACG-3′.

[0079] Used to confirm fragment insertion.

[0080] like Figure 4 In the transgenic plants, a target band of the same size as the plasmid DNA could be amplified, while the wild-type Arabidopsis plant DNA and the blank control did not amplify the corresponding band, proving that PpHSP20-32 has been integrated into the wild-type Arabidopsis genome.

[0081] Example 2 Use of gene PpHSP20-32 in transgenic Arabidopsis

[0082] Effect of 1 gene PpHSP20-32 on the phenotype of transgenic Arabidopsis plants

[0083] T2 transgenic plants (three transgenic lines L1, L2 and L3) and wild-type Arabidopsis were sown on MS medium for growth. After growing two cotyledons, they were transplanted into the matrix for observation of plant phenotypes. When they grew for two weeks, phenotypic observation and variance analysis were performed. Compared with wild-type Arabidopsis, the T2 transgenic lines of PpHSP20-32 showed an increase in the length and width of the rosette leaves. Figure 5 When the plants were grown for three weeks, the plant morphology was observed and the plant heights of the three transgenic lines were all higher than those of the WT. The average plant height of L1 was 34.4 cm, which was significantly higher than that of the WT (29.3 cm). There was no significant difference in the internode length between the transgenic lines and the WT, but the number of nodes was significantly higher than that of the WT, indicating that the increase in plant height of the transgenic lines may be due to the increase in the number of nodes, which indicates that overexpression of PpHSP20-32 leads to an increase in plant height, such as Figure 6 .

[0084] 2. Response of gene PpHSP20-32 to temperature

[0085] The T2 transgenic and wild-type Arabidopsis seeds were treated at 46°C for 30 minutes and then transferred to 25°C for heat resistance testing. No seeds germinated within 48 hours after high temperature treatment. The seed germination rate of the PpHSP20-32 transgenic line was significantly improved 60 hours after high temperature treatment, and the germination rates of the L1, L2 and L3 lines reached 82%, 73% and 80%, respectively; while the WT seed germination rate was only 3%, significantly lower than that of the transgenic line seeds. At 84 hours, the seed germination rates of the transgenic lines reached 95%, 94% and 92%, respectively; the WT seed germination rate was significantly increased to 62%, such as Figure 7 This indicates that transgenic Arabidopsis plants overexpressing PpHSP20-32 exhibit high temperature tolerance.

[0086] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Peach heat shock protein PpHSP20-32 Use for increasing plant height and / or rosette leaf width and length, characterized in that: Peach heat shock protein PpHSP20-32 The nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the plant is peach or Arabidopsis thaliana.

2. A method for cultivating transgenic plants with increased plant height and / or rosette leaf width and length, characterized in that: The nucleotide sequence fragment as shown in SEQ ID NO.1 in claim 1 is introduced into a target plant to obtain a transgenic plant, wherein the plant height and / or rosette leaf width and length of the transgenic plant are higher than those of the target plant, and the target plant is peach or Arabidopsis thaliana.

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