Gene AcHAIR for regulating and controlling development of epidermal hair of kiwi fruit, recombinant vector, agrobacterium and application of gene AcHAIR

Abstract

The invention provides a gene AcHAIR for regulating and controlling development of epidermal hairs of kiwi fruits, the nucleotide sequence of the gene AcHAIR is shown as SEQ. ID. NO.1, and the coded amino acid sequence is shown as SEQ. ID. NO.2. The gene AcHAIR can be used for regulating and controlling development of epidermal hairs of kiwi fruits. The invention also provides a recombinant expression vector containing the AcHAIR gene, and an agrobacterium strain containing the AcHAIR gene. In addition, the invention also provides an application of the AcHAIR gene in cultivation of high-quality kiwi fruits. Overexpression of the AcHAIR gene can obviously promote growth of epidermal hair on leaves and stems in plants, and the AcHAIR gene is of great significance in exploring regulation and control mechanisms of development of the epidermal hair of kiwi fruits and cultivating high-quality kiwi fruit varieties.

Description

Technical Field

[0001] The invention relates to the field of plant molecular genetic engineering, and in particular to a gene AcHAIR for regulating kiwifruit epidermal hair development, a recombinant vector, Agrobacterium and application thereof. Background Art

[0002] Kiwi fruit (Actinidia chinensis Planch), also known as vine pear, sheep peach, wood seed, hairy wood fruit, kiwi fruit, and hemp vine fruit, is generally oval in shape, green-brown in appearance, and covered with epidermal hairs. It is a fruit with fresh quality, rich nutrition, and delicious flavor.

[0003] In addition to kiwifruit, epidermal hairs can be seen on the leaves, stems and flower organs of many terrestrial plants. These epidermal hairs originate from the proliferation of epidermal cells, which undergo cell division, differentiation and growth to produce tissues extending from the epidermal surface. The resulting hair-like protrusion structure is the first key barrier against biotic stress and environmental changes. It is involved in most processes of plant adaptation to external environmental changes (biotic and abiotic stresses), and is irreplaceable in physical protection and chemical defense. Plant epidermal hairs can reduce ultraviolet rays, low temperatures, excessive transpiration, and insect consumption and damage to plants, reduce the incidence of plant infection by pathogens, and contribute to plant growth and development.

[0004] Given that epidermal hairs can provide plants with a powerful physical and chemical defense system, studying the genes that regulate epidermal hairs and the mechanisms of epidermal hair development has important theoretical and practical significance. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a gene AcHAIR for regulating the development of kiwifruit epidermal hairs, and its recombinant vector, Agrobacterium and application. The overexpression of the gene AcHAIR can significantly promote the growth of epidermal hairs on leaves in the plant, which has important theoretical and practical significance for exploring the regulatory mechanism of kiwifruit epidermal hair development.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A gene AcHAIR for regulating the development of kiwifruit epidermal hairs, wherein the nucleotide sequence of the gene AcHAIR is shown in SEQ.ID.NO.1, and the encoded amino acid sequence is shown in SEQ.ID.NO.2.

[0008] As one of the preferred embodiments of the present invention, the gene AcHAIR is used to positively regulate the development of epidermal hairs in kiwifruit plants.

[0009] A recombinant expression vector contains the above-mentioned gene AcHAIR for regulating kiwifruit epidermal hair development.

[0010] As one of the preferred embodiments of the present invention, the recombinant expression vector is specifically pCAMBIA2300-AcHAIR-HA, and the construction method is as follows: inserting the nucleotide sequence of the gene AcHAIR into the eukaryotic expression vector pCAMBIA2300-HA.

[0011] A recombinant Agrobacterium containing the above-mentioned gene AcHAIR for regulating the development of kiwifruit epidermal hairs.

[0012] As one of the preferred embodiments of the present invention, the construction method of the recombinant Agrobacterium is as follows: the vector pCAMBIA2300-AcHAIR-HA constructed by the gene AcHAIR regulating kiwifruit epidermal hair development is introduced into the Agrobacterium GV3101 cell.

[0013] An application of the above-mentioned gene AcHAIR for regulating kiwifruit epidermal hair development in cultivating high-quality kiwifruit.

[0014] As one of the preferred embodiments of the present invention, by overexpressing the gene AcHAIR in kiwifruit plants, the growth of epidermal hairs on leaves of the plants is promoted, thereby enhancing the defense capability of the plants against environmental stress.

[0015] The advantages of the present invention compared to the prior art are:

[0016] (1) The present invention screened and obtained a regulatory gene AcHAIR that affects the development of epidermal hairs by analyzing differentially expressed genes between kiwifruits with different epidermal hairs; AcHAIR belongs to the zinc finger gene family, in which HAIR is a positive regulator of epidermal hair biosynthesis; in order to further explore whether AcHAIR affects the function of epidermal hair development, the present invention further constructed a corresponding overexpression recombinant expression vector, obtained a recombinant plasmid, and revealed the function of AcHAIR in promoting the development of kiwifruit epidermal hairs through biochemical and molecular biological methods and transgenic biotechnology (overexpression of pCAMBIA2300-AcHAIR-HA stably transformed MicroTom tomatoes, the number of epidermal hairs on tomato leaves was significantly increased; at the same time, after overexpression of pCAMBIA2300-AcHAIR-HA stably transformed Arabidopsis, the epidermal hairs on Arabidopsis leaves also increased significantly, and even five-branched trichomes appeared);

[0017] (2) The present invention analyzes a new molecular regulatory mechanism that promotes the accumulation of kiwifruit epidermal hairs and reveals a new functional gene that promotes the development of kiwifruit epidermal hairs, providing a basis for enhancing the plant's defense ability against environmental stress and cultivating high-quality kiwifruit varieties;

[0018] (3) The present invention innovatively proposes a new index for quantifying the development of epidermal hairs, namely the branching index (the percentage of hairy branches on the first pair of true leaves of WT, transgenic lines and Arabidopsis; the average value of three pairs of leaves is taken);

[0019] (4) The AcHAIR gene and the corresponding transgenic plants of the present invention are obtained by conventional methods, the steps are simple, and the required materials are easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the electrophoresis diagram of the AcHAIR gene clone in Example 2 (in the figure, lane "M" is a 5K marker, and lane "1" is the target gene band AcHAIR, with a size of 678 bp);

[0021] Figure 2 This is the double enzyme digestion verification electrophoresis diagram of the overexpression vector pCAMBIA2300-AcHAIR-HA in Example 3 (in the figure, lane "M" is 2K marker, lane "1" is vector digestion, and the digestion sites are Sac1 and Spe1), and lane "2" is the target gene band AcHAIR);

[0022] Figure 3 It is the result diagram of the stable transformation of AcHAIR in tomato in Experimental Example 1 to promote the development of epidermal hairs in seedling stems and leaves (in the figure, Figure a shows the appearance of wild-type and 35S::AcHAIR transformed plants grown for 3 weeks, the left figure shows the appearance of WT wild-type plants, and the right figure shows the appearance of three 35S::AcHAIR transformed plants, with a scale of 1 mm; Figure b shows the results of quantitative detection of gene expression in transgenic plants; Figure c shows the results of semi-quantitative detection of gene expression in transgenic plants; wherein UBQ13 is an internal reference gene, 35S::AcHAIR-1, 35S::AcHAIR-2, and 35S::AcHAIR-5 represent three independent 35S::AcHAIR overexpressing transgenic tomato lines; and wherein, "**" indicates P<0.01);

[0023] Figure 4It is a result diagram of the promotion of epidermal hair development by stable transformation of 35S::AcHAIR in Arabidopsis thaliana in Experimental Example 2 (in the figure, Figure a shows the appearance of the wild type and three 35S::AcHAIR overexpressing plants and magnified leaves grown for 15 days, and the left figure shows the appearance of the WT wild-type plant, and the right figure shows the appearance of the three 35S::AcHAIR transformed plants, with a scale of 1 mm; Figure b shows the number of epidermal hairs on the first pair of true leaves of WT and transgenic Arabidopsis thaliana, and the average value of 15 pairs of leaves; Figure c shows semi-quantitative detection of gene expression in transgenic plants; wherein Actin is an internal reference gene, WT represents wild-type plants, 35S::AcHAIR-1, 35S::AcHAIR-3, and 35S::AcHAIR-4 represent three independent 35S::AcHAIR overexpressing transgenic Arabidopsis lines; and wherein, "**" indicates P<0.01);

[0024] Figure 5 These are the microscopic and statistical graphs of the wild-type and 35S::AcHAIR-transformed Arabidopsis in Experimental Example 3 (in the graph, Figure a is a microscope observation of the epidermal hairs on the cross-section of leaves of the wild-type and 35S::AcHAIR-transformed plants grown for 15 days, and the left figure is the appearance of the WT wild-type plant, and the right figure is the appearance of three 35S::AcHAIR-transformed plants, with a scale of 1 mm; Figure b is the percentage of hairy branches on the first pair of true leaves of WT and transgenic Arabidopsis, the average value of three pairs of leaves; wherein WT represents the wild-type plant, 35S::AcHAIR-1, 35S::AcHAIR-3, and 35S::AcHAIR-4 represent three independent 35S::AcHAIR-overexpressing transgenic Arabidopsis strains; and wherein, "br" represents the number of branches). DETAILED DESCRIPTION

[0025] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0026] Meanwhile, the reagents and culture media used in the present invention are conventional reagents and culture media in the art unless otherwise specified, and will not be described in detail. The experimental conditions and experimental methods used in the present invention are conventional conditions and methods in the art unless otherwise specified, such as the conditions and methods described in Sambrook, Russell's Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions and methods recommended by the manufacturer, and will not be described in detail.

[0027] Example 1

[0028] Screening and identification of the gene AcHAIR that regulates epidermal hair development:

[0029] The present invention analyzes differentially expressed genes between kiwifruits with different epidermal hairs, and identifies a gene that regulates epidermal hair development. At the same time, the gene belongs to the C2H2-GATA gene family after analysis, and positively regulates the biosynthesis of epidermal hairs, so the gene is involved in regulating epidermal hair development. It is named AcHAIR, the nucleotide sequence is shown in SEQ.ID.NO.1, and the encoded amino acid sequence is shown in SEQ.ID.NO.2.

[0030] Example 2

[0031] Cloning of AcHAIR gene:

[0032] According to the cDNA sequence of 'Hongyang' kiwifruit in the Kiwifruit Genome Database (Gene ID: Actinidia32415), PCR specific primers (AcHAIR F and AcHAIR R, sequences are shown in SEQ.ID.NO.3 and SEQ.ID.NO.4, respectively) were designed using primer design software Primer Premier 5.0; total RNA was extracted from Hongyang kiwifruit (using an RNA extraction kit for extraction), and the gene sequence of AcHAIR was isolated from Hongyang kiwifruit using reverse transcription PCR (RT-PCR) technology, as shown in SEQ ID NO: 1.

[0033] 1% agarose gel electrophoresis was used to detect the target fragment. The results were as follows Figure 1 As shown, the target band size is 678 bp.

[0034] Example 3

[0035] Construction of plant overexpression vector pCAMBIA2300-AcHAIR-HA:

[0036] (1) Extract the plasmid DNA containing the "target gene AcHAIR" and the expression vector pCAMBIA2300-HA plasmid at the same time, use the endonucleases Sac1 and Spe1 to double-digest pCAMBIA2300-HA, and purify it after digestion to obtain the linearized pCAMBIA2300-HA vector.

[0037] (2) Using homologous recombinase, the linearized pCAMBIA2300-HA vector and AcHAIR were connected and transformed respectively, and finally the recombinant plasmid pCAMBIA2300-AcHAIR-HA was obtained.

[0038] (3) After PCR detection and double enzyme digestion verification, sequencing is performed. The enzyme digestion verification results are as follows Figure 2.

[0039] Example 4

[0040] Recombinant vector was used to transform Agrobacterium tumefaciens GV3101:

[0041] (1) Thaw 100 μL Agrobacterium GV3101 competent cells on ice.

[0042] (2) Add 10 μL of recombinant plasmid pCAMBIA2300-AcHAIR-HA, mix gently, and place on ice for 30 min.

[0043] (3) Quickly freeze in liquid nitrogen for 5 min, incubate in a 37°C water bath for 5 min, then add 1 mL of YEP liquid culture medium without antibiotics and incubate at 28°C in a shaking incubator for 4 h.

[0044] (4) Centrifuge at 4000 rpm for 5 min to remove excess culture medium, apply an appropriate amount of bacterial solution on a YEP solid plate medium containing Kan&Rif, and culture at 28°C for 36 h.

[0045] Experimental Example 1

[0046] AcHAIR stably transformed tomato (MicroTom):

[0047] 1. Experimental Materials

[0048] Wild-type tomato seedlings with a growth period of 7 days.

[0049] 2. Experimental Methods

[0050] (1) Seed disinfection: Wash tomato seeds with 75% ethanol for 30 seconds, soak in 10% NaClO solution for 8 minutes, and then wash with sterile water for 7 to 8 times. Spread the washed seeds evenly on 1 / 2MS solid culture medium.

[0051] (2) Vernalization and light culture: The seeds were placed in the dark at 4°C for 12 h for vernalization, followed by light culture at 22°C for 16 h and 8 h in the dark for 7 days.

[0052] (3) Pre-culture: When the seedlings have only two cotyledons, cut the cotyledons into pieces and the stems into nodes, place them on a pre-culture medium (500 mL MS + 500 μL 1 mg / mL 6-BA + 20 μL 1 mg / mL IAA) and culture for 2 to 3 days.

[0053] (4) Co-cultivation: Activate "35S::AcHAIR (recombinant bacteria constructed in Example 4) with OD = 0.6", and soak the pre-cultured cotyledons and stems in the activated bacterial solution for 15 min, take them out and blot them dry on filter paper (sterilized), and transfer them to co-culture medium (500 mL MS + 25 μL 2 mg / mL KT + 100 μL 1 mg / mL 2,4-D + 735 μL 10 mg / mL ACE) and culture for 2 days.

[0054] (5) Screening culture: The co-cultivated cotyledons and stems were transferred to K55 screening medium and cultured for 20 days, then replaced with Kana resistance gradient until K70 screening medium.

[0055] (6) Rooting culture: Seedlings that grow normally on K70 screening medium are transferred to rooting medium (500 mL MS + 2500 μL 100 mg / mL sb (carbenicillin) + 1000 μL 1 mg / mL IAA + 700 μL 50 mg / mL Kana + 0.5% agar), and after rooting, they are acclimated and transplanted into soil.

[0056] 3. Experimental Results

[0057] After positive identification, a homozygous "tomato 35S::AcHAIR strain" was obtained. By observing the stems and leaves of transgenic tomato plants, it was found that compared with wild-type tomatoes WT, the stems and leaves of tomato seedlings transformed with 35S::AcHAIR had more epidermal hairs, such as Figure 3 .

[0058] Experimental Example 2

[0059] AcHAIR stable transformation of Arabidopsis:

[0060] 1. Experimental Materials

[0061] Wild-type Arabidopsis thaliana (Columbia) in flowering stage.

[0062] 2. Experimental Methods

[0063] (1) Activating "35S::AcHAIR (recombinant bacteria constructed in Example 4) with OD = 0.8" by soaking and infecting wild-type Arabidopsis thaliana in the flowering stage for 30 seconds using the floral immersion method; after culturing in the dark for 12 hours, the seeds were continued to be cultured under normal conditions until they matured.

[0064] (2) The seeds received were Kana (kanamycin) resistant 1 / 2MS culture medium (200mL 1 / 2MS+220μL 50mg / mL Kana) until homozygous. The homozygous overexpressing Arabidopsis seeds obtained were sown on MS medium without Kana resistance, transplanted into soil, and the phenotype was observed.

[0065] (3) Total RNA from leaves of the experimental group (overexpression) and the control group (wild type) was respectively obtained and inverted into a single strand of cDNA, which was used as a template to semi-quantitatively detect the expression of the overexpressed genes.

[0066] 3. Experimental Results

[0067] After positive identification, a stably transformed homozygous "Arabidopsis 35S::AcHAIR strain" was obtained. By observing the transgenic seedlings, it was found that compared with the wild-type Arabidopsis WT, the trichomes on the leaves of 35S::AcHAIR-transformed Arabidopsis were significantly increased, and the increase in trichomes in the central area was the most significant, such as Figure 4 .

[0068] Experimental Example 3

[0069] Microscopic observation and statistics of AcHAIR overexpression Arabidopsis:

[0070] 1. Experimental Materials

[0071] Wild-type Arabidopsis thaliana (Columbia) in flowering stage.

[0072] 2. Experimental Methods

[0073] The number of epidermal hairs with different branch numbers in the first pair of true leaves of 15-d-old WT and AcTCP4 transgenic lines was counted using a British fluorescence microscope, and the statistical data was analyzed using SPSS software to determine whether AcHAIR also has the function of regulating epidermal hair branching.

[0074] 3. Experimental Results

[0075] After observation and statistics, the epidermal hairs with two branches in the first pair of true leaves of WT plants accounted for 21.4% of the total number of epidermal hairs, the epidermal hairs with three branches accounted for 55.7%, and the rest were epidermal hairs with four branches. In the three biological replicates of AcHAIR overexpressing transgenic plants, the proportions of epidermal hairs with two branches were 14.6%, 14.2% and 15.8%, respectively, the proportions of epidermal hairs with three branches were 41.4%, 40.7% and 43.2%, respectively, and the proportions of epidermal hairs with four branches were 43.4%, 42.1% and 43.2%, respectively. In addition, we also detected a small number of epidermal hairs with five branches in the AcHAIR overexpressing transgenic lines, which were not found in WT plants. Figure 5 .

[0076] In summary, the AcHAIR gene of the present invention also plays an important role in the development of epidermal hairs. After overexpression of AcHAIR and stable transformation of Arabidopsis thaliana, the epidermal hairs on the leaves of Arabidopsis thaliana increased significantly and even five-branched trichomes appeared. The present invention provides a reference for the future breeding of high-quality kiwifruit varieties.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene for regulating kiwifruit epidermal hair development, AcHAIR, characterized in that: The nucleotide sequence of the gene AcHAIR is shown in SEQ.ID.NO.1, and the encoded amino acid sequence is shown in SEQ.ID.NO.

2.

2. The gene AcHAIR for regulating kiwifruit epidermal hair development according to claim 1, characterized in that: The gene AcHAIR is used to positively regulate the development of epidermal hairs in kiwifruit plants.

3. A recombinant expression vector, characterized in that: Contains the gene AcHAIR for regulating kiwifruit epidermal hair development as described in any one of claims 1 to 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is specifically pCAMBIA2300-AcHAIR-HA, and the construction method is as follows: inserting the nucleotide sequence of the gene AcHAIR into the eukaryotic expression vector pCAMBIA2300-HA.

5. A recombinant Agrobacterium, characterized in that: Contains the gene AcHAIR for regulating kiwifruit epidermal hair development as described in any one of claims 1 to 2.

6. The recombinant Agrobacterium according to claim 5, characterized in that The construction method is as follows: the vector pCAMBIA2300-AcHAIR-HA constructed by the gene AcHAIR regulating the development of kiwifruit epidermal hairs is introduced into Agrobacterium GV3101 bacteria.

7. Use of the gene AcHAIR for regulating kiwifruit epidermal hair development as claimed in any one of claims 1 to 2 in cultivating high-quality kiwifruit.

8. The use according to claim 7, characterized in that: By overexpressing the gene AcHAIR in kiwifruit plants, the growth of epidermal hairs on plant leaves is promoted, thereby enhancing the plant's defense ability against environmental stress.

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