Regulation of hair development gene acHAIR on kiwi fruit peel and recombinant vector, agrobacterium and application thereof

Abstract

The application provides a kind of regulation kiwi skin hair development gene AcHAIR, its nucleotide sequence is as shown in SEQ.ID.NO.1, the amino acid sequence as shown in SEQ.ID.NO.2 is encoded.The application also provides a kind of recombinant expression vector containing above-mentioned AcHAIR gene and agrobacterium bacteria.In addition, the application also provides a kind of above-mentioned AcHAIR gene in the application of cultivating high-quality kiwi fruit.The excess expression of the application AcHAIR gene can significantly promote the growth of epidermal hair on leaf and stem of plant, and has important significance for exploring the regulation mechanism of kiwi skin hair development and cultivating high-quality kiwi fruit varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant molecular genetic engineering, and particularly relates to a kiwi fruit epidermal hair development gene AcHAIR, a recombinant vector, an agrobacterium and application thereof. BACKGROUND

[0002] Kiwi fruit (Actinidia chinensis Planch), also known as Tengli, Yangtao, Muzi, Maotongguo, Qiguo, etc., is a kind of fruit with fresh quality, rich nutrition and delicious flavor, which is generally oval in shape and greenish brown in appearance, and the surface is covered with epidermal hair.

[0003] In addition to kiwi fruit, epidermal hair can be seen on the leaves, stems and flower organs of many terrestrial plants. These epidermal hairs are derived from the proliferation of epidermal cells, which undergo cell division, differentiation and growth to produce tissues extending from the surface of the epidermis. The resulting hairy protruding structure is the first key barrier against biological stress and environmental changes, which participates in most processes of plant adaptation to external environmental changes (biotic and abiotic stress), and is irreplaceable in physical protection and chemical defense. Plant epidermal hair can reduce ultraviolet radiation, low temperature, excessive transpiration, and the consumption and damage of insects to plants, reduce the incidence of plant infection by pathogenic bacteria, and also help the growth and development of plants.

[0004] In view of the fact that epidermal hair can provide a strong physical and chemical defense system for plants, it is of great theoretical and practical significance to study the genes regulating epidermal hair development and the development mechanism of epidermal hair. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a kiwi fruit epidermal hair development gene AcHAIR, a recombinant vector, an agrobacterium and application thereof. Overexpression of the gene AcHAIR can significantly promote the growth of epidermal hair on the leaves of the plant, which has important theoretical and practical significance for exploring the regulation mechanism of kiwi fruit epidermal hair development.

[0006] The present application solves the above technical problems by adopting the following technical solutions:

[0007] A kiwi fruit epidermal hair development gene AcHAIR, 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 modes of the present application, the gene AcHAIR is used to positively regulate the epidermal hair development of kiwi fruit plants.

[0009] A recombinant expression vector containing the above-mentioned kiwi fruit epidermal hair development gene AcHAIR.

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

[0011] A recombinant Agrobacterium contains the above-mentioned regulatory kiwifruit skin hair development gene AcHAIR.

[0012] As one of the preferred modes of the present application, the construction method of the recombinant Agrobacterium is as follows: the vector pCAMBIA2300-AcHAIR-HA constructed by the regulatory kiwifruit skin hair development gene AcHAIR is introduced into the Agrobacterium GV3101 bacterial body.

[0013] The above-mentioned regulatory kiwifruit skin hair development gene AcHAIR is applied to cultivate high-quality kiwifruit.

[0014] As one of the preferred modes of the present application, the growth of the plant leaf skin hair is promoted by overexpressing the gene AcHAIR in the kiwifruit plant, and the defense ability of the plant to environmental stress is enhanced.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] (1) The present application screens and obtains a regulatory gene AcHAIR affecting the development of skin hair by analyzing the expression difference genes between different skin hair kiwifruits; AcHAIR belongs to the C2H2-GATA transcription factor (The zinc finger gene family), wherein HAIR is a positive regulator of skin hair biosynthesis; in order to further explore whether AcHAIR affects the function of skin hair development, the present application further constructs a corresponding overexpression recombinant expression vector, obtains a recombinant plasmid, and reveals the function of AcHAIR promoting the development of kiwifruit skin hair by biochemical and molecular biological means and transgenic biological technology (the number of tomato leaf skin hairs is significantly increased after overexpression pCAMBIA2300-AcHAIR-HA stable transformation MicroTom tomato; at the same time, the number of skin hairs on the Arabidopsis thaliana leaf is also significantly increased after overexpression pCAMBIA2300-AcHAIR-HA stable transformation Arabidopsis thaliana, and even a five-branch hair-like body appears);

[0017] (2) The present application reveals a new molecular regulation mechanism for promoting the accumulation of kiwifruit skin hair, and discloses a new functional gene for promoting the development of kiwifruit skin hair, which provides a basis for enhancing the defense ability of the plant to environmental stress and cultivating high-quality kiwifruit varieties;

[0018] (3) The present application proposes a new index for quantitatively calibrating the epidermal hair development, i.e. the branch index (WT, transgenic lines and percentage of hairy branch on the first pair of true leaves of Arabidopsis thaliana; the average value of three pairs of leaves is taken);

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

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

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

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

[0023] Figure 4is the result chart of stable transformation of 35S::AcHAIR in Arabidopsis thaliana to promote epidermal hair development in experimental example 2 (in the figure, a figure is the appearance of wild type and three 35S::AcHAIR overexpression plants and enlarged leaf blades grown for 15d, and the left figure is the appearance of WT wild type plant, and the right figure is the appearance of three 35S::AcHAIR transformed plants, the scale is 1mm; b figure is the number of epidermal hair on the first pair of true leaves of WT and transgenic Arabidopsis thaliana, the average value of 15 pairs of leaves; c figure is the semi-quantitative detection of gene expression in transgenic plants; wherein, Actin is the internal reference gene, WT indicates wild type plant, 35S::AcHAIR-1, 35S::AcHAIR-3, 35S::AcHAIR-4 indicates three independent 35S::AcHAIR overexpression transgenic Arabidopsis thaliana lines; and wherein, "**" indicates P<0.01);

[0024] Figure 5 is the micrograph and statistical chart of wild type and 35S::AcHAIR transformed Arabidopsis thaliana in experimental example 3 (in the figure, a figure is the leaf cross-section epidermal hair microscope observation chart of wild type and 35S::AcHAIR transformed plants grown for 15d, and the left figure is the appearance of WT wild type plant, and the right figure is the appearance of three 35S::AcHAIR transformed plants, the scale is 1mm; b figure is the percentage of hair branching on the first pair of true leaves of WT and transgenic Arabidopsis thaliana, the average value of 3 pairs of leaves; wherein, WT indicates wild type plant, 35S::AcHAIR-1, 35S::AcHAIR-3, 35S::AcHAIR-4 indicates 3 independent 35S::AcHAIR overexpression transgenic Arabidopsis thaliana lines; and wherein, "br" indicates the number of branch branches). DETAILED DESCRIPTION

[0025] The following detailed description of the embodiments of the present application is given on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0026] Meanwhile, the reagents and culture media used in the present application are not particularly described, and are all conventional reagents and culture media in the art, which will not be described herein. The experimental conditions and experimental methods used in the present application are not particularly described, and are all conventional conditions and methods in the art, for example, the conditions and methods described in Sambrook, Russell's Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions and methods recommended by the manufacturer, which will not be described herein.

[0027] Example 1

[0028] Screening and determination of epidermal hair development gene AcHAIR:

[0029] The application determines a gene regulating epidermal hair development by analyzing different epidermal hair kiwifruit expression difference genes. The gene belongs to C2H2-GATA gene family through analysis, positively regulates epidermal hair biosynthesis, and thus participates in regulating epidermal hair development. The gene is named AcHAIR, the nucleotide sequence is shown as SEQ. ID. NO. 1, and the encoded amino acid sequence is shown as SEQ. ID. NO. 2.

[0030] Example 2

[0031] Cloning of AcHAIR gene:

[0032] According to the cDNA sequence (Gene ID: Actinidia32415) of 'Hongyang' kiwifruit in the Kiwifruit Genome Database, PCR specific primers (AcHAIR F, AcHAIR R, sequences shown as SEQ. ID. NO. 3 and SEQ. ID. NO. 4, respectively) were designed by using primer design software Primer Premier 5.0; total RNA was extracted from Hongyang kiwifruit (using an RNA extraction kit), and the gene sequence of AcHAIR was isolated from Hongyang kiwifruit by 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, and the results are shown in Figure 1 , and the target band size is 678bp.

[0034] Example 3

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

[0036] (1) Extract the plasmid DNA containing the "target gene AcHAIR", and extract the expression vector pCAMBIA2300-HA plasmid at the same time. The endonuclease Sac1 and Spe1 are selected to double-enzyme cut pCAMBIA2300-HA, and the linearized pCAMBIA2300-HA vector is obtained after enzyme cutting and purification.

[0037] (2) The linearized pCAMBIA2300-HA vector and AcHAIR are connected and transformed by using homologous recombinase, and the recombinant plasmid pCAMBIA2300-AcHAIR-HA is finally obtained.

[0038] (3) After PCR detection and double enzyme digestion verification, sequencing is performed, and the enzyme digestion verification results are shown in Figure 2.

[0039] Example 4

[0040] Recombinant vector transformed Agrobacterium tumefaciens GV3101:

[0041] (1) 100 μL of Agrobacterium GV3101 competent cells, thawed on ice.

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

[0043] (3) Quick-freeze in liquid nitrogen for 5 min, 37°C water bath for 5 min, then add 1 mL of YEP liquid medium without antibiotics, and cultivate at 28°C for 4 h.

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

[0045] Experimental Example 1

[0046] AcHAIR stable transformation of tomato (MicroTom):

[0047] I. Experimental materials

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

[0049] II. Experimental method

[0050] (1) Seed disinfection: wash tomato seeds with 75% ethanol for 30 sec, immerse in 10% NaClO solution for 8 min, then wash with sterile water for 7-8 times, and place the washed seeds evenly on 1 / 2MS solid medium.

[0051] (2) Vernalization treatment and light culture: place the seeds in 4°C dark low-temperature vernalization treatment for 12 h, then perform light culture, cultivate at 22°C with 16 h light and 8 h darkness for 7 days.

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

[0053] (4) Co-cultivation: Activate "OD=0.6 of 35S::AcHAIR (recombinant bacteria constructed in Example 4)", and put the pre-cultured cotyledon and stem into the activated bacteria solution for 15 min, take out and absorb on filter paper (sterilized), and transfer to co-cultivation medium (500 mL MS + 25 μL 2 mg / mL KT + 100 μL 1 mg / mL 2,4-D + 735 μL 10 mg / mL ACE) for 2 d.

[0054] (5) Selection culture: transfer the co-cultured cotyledon and stem to K55 selection medium for 20 d, and then replace with Kana resistance gradient until K70 selection medium.

[0055] (6) Rooting culture: transfer the seedlings normally grown on K70 selection medium 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 acclimate and transplant into soil after rooting.

[0056] III. Experimental results

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

[0058] Experimental Example 2

[0059] Stable transformation of AcHAIR into Arabidopsis:

[0060] I. Experimental materials

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

[0062] II. Experimental methods

[0063] (1) Activate "OD=0.8 of 35S::AcHAIR (recombinant bacteria constructed in Example 4)", and use the flower dipping method to soak the wild-type Arabidopsis thaliana in the flowering stage for 30 sec; after 12 h of dark culture, continue to culture under normal conditions until the seeds mature.

[0064] (2) The received seeds were cultured on Kana (kanamycin) resistant 1 / 2MS medium (200 mL 1Screening on MS+220 μL 50 mg / mL Kana until homozygous. The homozygous overexpression Arabidopsis seeds were sowed on MS medium without Kana resistance, transplanted into soil and observed for phenotype.

[0065] (3) Total RNA was extracted from leaves of the experimental group (overexpression) and the control group (wild type) respectively, and cDNA was synthesized. The expression of the overexpression gene was detected by semi-quantitative PCR.

[0066] III. Experimental results

[0067] After positive identification, the stable transformed homozygous "Arabidopsis 35S::AcHAIR strain" was obtained. By observing the transgenic seedlings, it was found that compared with wild type Arabidopsis WT, the trichomes on the leaves of 35S::AcHAIR transformed Arabidopsis increased significantly, among which the increase of trichomes in the central region was most significant, as shown in Figure 4 .

[0068] Experimental Example 3

[0069] Microscopic observation of AcHAIR overexpression Arabidopsis

[0070] I. Experimental materials

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

[0072] II. Experimental methods

[0073] The number of epidermal hairs of different branches in the first pair of true leaves of 15d seedlings of WT and AcTCP4 transgenic strains was counted by a British fluorescence microscope, and the statistical data were analyzed by SPSS software to determine whether AcHAIR also has the function of regulating epidermal hair branching.

[0074] III. Experimental results

[0075] After observing and counting, it was found that the two-branch epidermal hairs of WT plants accounted for 21.4% of the total number of epidermal hairs, the three-branch epidermal hairs accounted for 55.7%, and the rest were four-branch epidermal hairs. In the three biological replicates of AcHAIR overexpression transgenic plants, the proportion of two-branch epidermal hairs was 14.6%, 14.2% and 15.8% respectively, the proportion of three-branch epidermal hairs was 41.4%, 40.7% and 43.2% respectively, and the proportion of four-branch epidermal hairs was 43.4%, 42.1% and 43.2% respectively. In addition, a small amount of five-branch epidermal hairs was detected in the AcHAIR overexpression transgenic strains, but not in the WT plants. As shown in Figure 5 .

[0076] In summary, the AcHAIR gene plays an important role in the development of epidermal hair, and overexpression of AcHAIR stably transformed Arabidopsis makes the epidermal hair on the leaves of Arabidopsis significantly increase and even appear five-branch hair body phenomenon. The present application provides a reference for breeding high-quality kiwi varieties in the future.

[0077] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gene regulating the development of kiwifruit epidermal hairs LAND Its application in cultivating high-quality kiwifruit is characterized by... The gene LAND The nucleotide sequence is shown in SEQ.ID.NO.1, and the encoded amino acid sequence is shown in SEQ.ID.NO.2; By overexpressing the gene in kiwifruit plants LAND It promotes the growth of epidermal hairs on plant leaves.

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