Application of polygonatum kingianum KNOX-BP gene in increasing number of plant compound leaves

Abstract

The invention discloses application of a polygonatum kingianum KNOX-BP gene in increasing the number of plant compound leaves, and belongs to the technical field of gene engineering. The invention provides a polygonatum kingianum gene KNOX-BP related to plant compound leaf formation, a recombinant plasmid containing the KNOX-BP gene is constructed, the recombinant plasmid is transformed to obtain agrobacterium carrying the KNOX-BP gene, and the agrobacterium is utilized to infect tomato leaves to obtain a tomato plant expressing the KNOX-BP gene. According to phenotype observation and statistics of overexpression of the polygonatum kingianum KNOX-BP gene in a model plant tomato, compared with a wild tomato plant, the KNOX-BP gene enables branches and compound leaves of the tomato plant to be increased, so that the meristematic ability of polygonatum kingianum is expected to be regulated and controlled, branches and compound leaves of polygonatum kingianum are increased, and the number of parts of available tender shoots and flowers is increased.

Description

Technical Field

[0001] The invention relates to application of polygonatum sibiricum KNOX-BP gene in increasing the number of compound leaves of plants, belonging to the technical field of genetic engineering. Background Art

[0002] Plant leaves are important nutritional organs of higher plants, the main body of photosynthesis, and also important organs for plants to perceive changes in the external environment, receive and transmit environmental signals. In flowering plants, leaf morphology varies, but according to the morphology of mature leaves, leaves can basically be divided into two types: simple leaves and compound leaves. For plants whose branches have important medicinal value, or plants whose branches and leaves are important sources of economic crops, increasing the number of compound leaves is beneficial to enhancing their economic value.

[0003] KNOX belongs to an important class of homeobox genes. The homeobox proteins encoded by them can bind to the cis-regulatory regions of target genes, widely participate in the development process of organisms, and are closely related to the differentiation of plants and the number of compound leaves. KNOXⅠ family members include four types in the Arabidopsis genome, SHOT MERISTEMLESS (STM), BREVIPEDICELLUS (BP), KNAT2 and KNAT6. At present, the research on promoting plant compound leaves includes that in Arabidopsis overexpressing BP, the leaves are extremely serrated and even form notches, and overexpression of KNOX genes in legumes increases the number of plant compound leaves. However, there are few studies on KNOX genes in medicinal plants, and increasing the medicinal branches of medicinal plants is currently a shortcoming.

[0004] Polygonatum sibiricum is a very excellent perennial medicinal plant, and its medicinal value has been confirmed. It has the effects of tonifying the kidney, nourishing yin, and moistening dryness. It is suitable for symptoms such as weak spleen and stomach, lung deficiency and dry cough, insufficient blood and semen, internal heat and thirst, fatigue, dry mouth and poor appetite. Therefore, the exploration of the Polygonatum sibiricum KNOX transcription factor family and the key genes that affect the formation of plant compound leaves have important application value. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the invention provides an application of Polygonatum sibiricum KNOX-BP in increasing the number of compound leaves of plants, with the aim of solving the technical problem that the effects and influences of the Polygonatum sibiricum KNOX transcription factor family on plant compound leaves are currently unknown, and whether Polygonatum sibiricum with medicinal value can be used to increase the number of plant compound leaves is unclear.

[0006] The first technical solution provided by the present invention is a Polygonatum sibiricum KNOX-BP gene, the nucleotide sequence of the KNOX-BP gene is shown in SEQ ID NO:1.

[0007] In certain embodiments, the amino acid sequence of the protein encoded by the KNOX-BP gene is shown in SEQ ID NO:2.

[0008] The second technical solution provided by the present invention is an expression vector carrying the gene described in the first technical solution.

[0009] The third technical solution provided by the present invention is a recombinant cell containing the gene described in the first technical solution, or transformed with the expression vector described in the second technical solution.

[0010] In certain embodiments, the recombinant cell is a plant cell or a microbial cell.

[0011] The fourth technical solution provided by the present invention is a method for increasing the number of compound leaves of a plant, wherein the method comprises connecting the Polygonatum sibiricum KNOX-BP gene described in the first technical solution to a vector and transforming the vector into the plant through Agrobacterium-mediated infection.

[0012] In certain embodiments, the plant is tomato.

[0013] The fifth technical solution provided by the present invention is a method for preparing a transgenic plant, the method comprising the following steps:

[0014] S1, connecting the Polygonatum cyrtonema KNOX-BP gene described in the first technical solution to a vector to obtain an expression vector;

[0015] S2, the expression vector in S1 is transferred into Agrobacterium to obtain recombinant cells;

[0016] S3, infecting plants with the recombinant cells obtained in S2 to obtain transgenic strains.

[0017] In certain embodiments, the plant is tomato.

[0018] In certain embodiments, in step S1, the vector is a 1380-Cas9-HA plasmid.

[0019] In certain embodiments, in step S2, the Agrobacterium is GV3101.

[0020] In certain embodiments, in step S3, the infection method is to infect plants by leaf dipping mediated by Agrobacterium.

[0021] The use of the gene described in the first technical solution, or the expression vector described in the second technical solution, or the recombinant cell described in the third technical solution in increasing the number of compound leaves of plants.

[0022] In certain embodiments, the plant is tomato.

[0023] The technical effects of the present invention are as follows:

[0024] The present invention provides a polygonatum gene KNOX-BP related to the formation of plant compound leaves, constructs a recombinant plasmid containing the KNOX-BP gene, transforms it to obtain Agrobacterium carrying the KNOX-BP gene, and uses the Agrobacterium to infect tomato leaves to obtain a tomato plant expressing the KNOX-BP gene. According to the phenotypic observation and statistics of the overexpression of the polygonatum KNOX-BP gene in the model plant tomato, compared with the wild-type tomato plant, the KNOX-BP gene increases the branches and compound leaves of the tomato plant, and is expected to regulate the division ability of polygonatum, increase the branches and compound leaves of polygonatum, and increase the number of the part of tender shoots and flowers that can be used. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the evolutionary tree diagram for the Knox gene family analysis of Polygonatum cyrtonema in the present invention.

[0026] Figure 2 This is the map of the Polygonatum sibiricum KNOX-BP plant expression vector of the present invention.

[0027] Figure 3 This is the electrophoresis diagram of the plant expression vector Agrobacterium tumefaciens liquid of the Polygonatum cyrtonema KNOX-BP of the present invention after PCR.

[0028] Figure 4 This is a resistance screening diagram of the Polygonatum sibiricum KNOX-BP transgenic tomato plants of the present invention.

[0029] Figure 5 This is a comparison diagram of the positive seedling stage of the Polygonatum cyrtonema KNOX-BP transgenic tomato of the present invention and the wild type.

[0030] Figure 6 This is a comparison chart of the leaf complexity of the Polygonatum sibiricum KNOX-BP transgenic tomato of the present invention and that of the wild type.

[0031] Figure 7 This is a comparison diagram of the branches of the whole plant leaves of the Polygonatum sibiricum KNOX-BP transgenic tomato of the present invention and the wild type. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] The raw materials used in the embodiment:

[0034] 1. The tomato variety Micro Tom comes from the National Key Laboratory of Zhejiang A&F University.

[0035] 2. 1380-Cas9-HA plasmid is a commercial plasmid and is deposited in the National Key Laboratory of Zhejiang A&F University.

[0036] Agrobacterium competent cells (GV3101 Chemically Competent Cell) were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0037] 3. LB medium formula: 1% tryptone, 0.5% yeast extract, 1% sodium chloride.

[0038] 4. YEP medium formula: 1% tryptone, 1% yeast extract, 0.5% sodium chloride.

[0039] 5. The seeding medium is MS: 2.21g, agar 7.2g, water 1000ml, pH: 5.8-6.0.

[0040] 6. The screening basal medium is: MS: 4.43 g, sucrose: 30 g, agar 7.2 g, water 1000 ml, pH: 5.8-6.0;

[0041] A1 screening medium: basal medium + ZT 2.0mg / L + IAA 0.1mg / L;

[0042] A2 screening medium: basic medium + ZT 2.0mg / L + IAA 0.1mg / L + Kanamycin 100mg / L + Timentin 300mg / L;

[0043] A3 screening medium: basic medium + ZT 1.0mg / L + IAA 0.1mg / L + Kanamycin 50mg / L + Timentin 300mg / L;

[0044] A4 screening medium: basic medium + IAA 0.3 mg / L + Kanamycin 30 mg / L + Timentin 150 mg / L.

[0045] Example 1 Cloning of Polygonatum cyrtonema KNOX-BP gene

[0046] The genome data of rice (PRJNA1014593), Arabidopsis thaliana (GCA_000001735.2), and tomato (GCA_000188115.4) were obtained from the NCBI database. Based on the transcriptome data of Polygonatum cyrtonema, the hidden Markov model of the KNOX conserved domain was retrieved through the pfam database (http: / / :pfam.xfam.org / ) (database number PF03789 / PF05920). The sequences containing the KNOX domain in the proteins encoded by the whole genome of Polygonatum cyrtonema were searched by HMMER software as candidate members of the KNOX gene family of Polygonatum cyrtonema. In order to obtain more accurate results, the blast sequence alignment was performed using the sequence information of the KNOX protein that has been well studied in Arabidopsis thaliana. The candidate members of the Polygonatum KNOX gene family obtained by the two methods were merged, and after removing duplicate values, the NCBI CDD (http: / / :www.ncbi.nlm.nih.gov / cdd) was used to further verify the searched protein sequence domain, and finally the members of the Polygonatum KNOX gene family and the KNOX gene family members in other plants were screened out. The BP member of the Polygonatum KNOX gene family was selected, and its nucleotide sequence is shown in SEQ ID NO:1, and the protein sequence is as follows:

[0047] PC06G02273 (SEQ ID NO: 2)

[0048] MEEFSHLGGGGGGGGGGGDGGSSSNSNSRPTASFMYFPSPPTPTTPSTQPQAFSFYSPNP

[0049] NPNPNSLQPKPEAAGSLIPYARRLVSADDDAIKAKIVSHPQYSALLGAYMDCQKVGTPPEV

[0050] ASRLSATAREVEARQQQLATAGCRRDASAGADPELDQFMEAYCNMLVKYREELTRPLQEA

[0051] MDFLRRVESQLSSLTHGATASIFNNTDEKCEGVSSSEEDQDGSGGEAEHPEIDPRAEDKELK

[0052] HHLLKKYSGYLSSLRHELSKKKKKGKLPKEARQKLLNWWELHYKWPYPSENEKVALAES

[0053] TGLDQKQINNWFINQRKRHWKPSEDMQFVVMDAFHPQNAAALYMGGQFIGDGSYRLGP.

[0054] like Figure 1 The results of phylogenetic analysis showed that it was most closely related to the BP gene of Arabidopsis thaliana, so PC2273 was selected as the BP gene of Polygonatum sibiricum.

[0055] The above-mentioned Polygonatum sibiricum KNOX-BP gene was inserted between the XbaI restriction site and the SalI restriction site of the plant expression vector 1380-Cas9-HA to obtain the fusion vector plasmid (recombinant plasmid) 1380-Cas9-HA-KNOX-BP (PC06G02273_1380-Cas9-HA). The plasmid map is shown in Figure 2 shown.

[0056] Example 2 Preparation of Agrobacterium carrying KNOX-BP gene

[0057] The recombinant plasmid 1380-Cas9-HA-KNOX-BP obtained in Example 1 was transformed by freeze-thaw method as follows:

[0058] (1) Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while until it partially thaws. When it is in a mixture of ice and water, insert it into ice.

[0059] (2) Add 1 μg of plasmid DNA (recombinant plasmid 1380-Cas9-HA-KNOX-B) per 100 μL competent medium, flick the bottom of the tube to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0060] (3) Add 700 μL of LB medium without antibiotics and culture at 28°C in the dark with shaking (220 rpm) for 2 to 3 h.

[0061] (4) Collect the bacteria by centrifugation at 6000 rpm for 1 min. Take about 100 μL of the supernatant and gently blow to resuspend the bacteria. Apply the suspension to an LB medium plate containing 1 / 1000 kanamycin. Place the plate upside down in a 28°C incubator and culture for 2-3 days.

[0062] PCR amplification was performed using gene-specific primers to identify the transformed Agrobacterium. Figure 3 As shown, the length of the inserted gene is 1095 bp, which further indicates that the transgenic was successful, that is, Agrobacterium carrying the KNOX-BP gene was obtained.

[0063] Example 3 Preparation of transgenic tomatoes

[0064] 1. Seed sterilization

[0065] In the clean bench, pour the weighed tomato seeds (200 seeds) into a sterilized 100ml conical flask, add 75% alcohol (just enough to cover the seeds), shake for 30 seconds, wash twice with sterile water, add 27ml of sterile water and 3ml of bleach, shake on a shaker (200rpm) for 1 hour (the seed coat will be slightly discolored), wash 5 times with sterile water, leave a little water in the conical flask to keep the seeds moist, and finally mark the seed variety and date of sterilization on the conical flask and store in a refrigerator at 4 degrees.

[0066] 2. Sowing

[0067] The seeds were sown on the second day after sterilization. In the clean bench, the sterilized seeds were sown into 1 / 2MS culture medium, about 50 seeds per bottle, and the sown seed variety and sowing date were marked on the sown bottle.

[0068] 3. Cut the leaves

[0069] Five days after sowing, when the two cotyledons of the tomato are fully expanded and no true leaves have grown, you can cut the leaves.

[0070] In the clean bench, use sterilized tweezers to pull the seedlings out of the 1 / 2MS medium, put them in a glass dish filled with sterilized water, use scissors to cut the leaf tip and the position near the petiole twice, put the cut leaves in a glass dish filled with MSO (MSO contains AS, which can induce the leaf state and improve the infection efficiency of Agrobacterium). After all the leaves are cut, pour out the MSO in the glass dish, spread a piece of sterilized filter paper on the A1 medium, use tweezers to put the leaves on A1 with the leaf surface facing up, mark the variety and time of the cut leaves on the cover, and finally wrap them with tin foil and place them in dark conditions.

[0071] IV. Infection

[0072] The infection was carried out on the second day after the leaves were cut. The OD value of the bacterial solution was first detected (spectrophotometer λ=600), and the infection requirement was met when it was between 0.7 and 0.8. The bacterial solution of Agrobacterium carrying the KNOX-BP gene in Example 2 was poured into a sterilized 50 ml centrifuge tube, centrifuged at room temperature and 4000 rpm for 10 min, the supernatant was discarded, the bacterial solution was resuspended with MSO and then poured into a glass dish, the cut leaves were also placed in the glass dish, and infected for 15 min (the bacterial solution should be shaken several times during the period to make the bacterial solution evenly mixed and to make the tomato leaves fully contact with the bacterial solution), and then the bacterial solution was poured out, and the infected leaves were placed in A1 with the back facing up. After one A1 was filled, the A1 cover was marked with: A1 / infected receptor / infected plasmid / infected date, and finally wrapped with tin foil.

[0073] 5. Reverse board

[0074] The next day after infection, turn the plates over. Place the leaves in A1 into A2, with the leaves facing upward. After filling up one A2 (there should be appropriate gaps between the leaves), write on the A2 cover: A2 / infected receptor / infected plasmid / infection date / turning date.

[0075] VI. Dedifferentiation and redifferentiation process of leaves (infected with Agrobacterium)

[0076] The leaves infected with Agrobacterium need to be cultured in A2 medium for one and a half to two months. During this process, the leaves must first undergo dedifferentiation to generate callus tissue, and then differentiate to form tomato seedlings with growth points. Then the seedlings are trimmed from the callus tissue (the seedlings are generally 2 cm tall, and there should not be too much callus tissue at the base of the seedlings, otherwise it will affect their growth in A3 medium), placed in A3 medium, and cultured in A3 medium for about a month. When the seedlings grow to 5 cm, the seedlings can be transferred to A4 medium for rooting culture. Generally, the seedlings only need about 10 days to grow small roots. When the roots of the seedlings grow strong enough, the seedlings can be transplanted into nutrient soil. When the leaves are dedifferentiated and redifferentiated in A2 medium, it takes about 20 days to turn the plate. The purpose of A2 medium is to make the leaves infected with Agrobacterium dedifferentiate first, and then differentiate to grow transgenic seedlings; the purpose of A3 medium is to make the transgenic seedlings grow a little bigger and reach a state where they can be cultured for rooting; the purpose of A4 medium is to make the transgenic seedlings take root. Figure 4 This is the A2 screening medium. The green ones are callus tissue, which differentiates into complete plants (i.e. transgenic tomatoes), while the white ones are leaves that failed the genetic modification and were screened out.

[0077] Example 4 Cultivation of transgenic tomatoes and phenotypic analysis thereof

[0078] The transgenic tomatoes obtained in Example 3 were inoculated into soil and cultured in simulated culture bottles for 2 weeks. The seedling cover was removed and the tomatoes were cultured for about two months to grow into adult tomatoes. The complexity of their leaves was compared with that of the wild type.

[0079] like Figure 5 As shown, compared with wild-type tomatoes, leaf differentiation is more complex at the seedling stage, with more branches and more leaves.

[0080] like Figure 6 As shown, adult wild-type tomatoes generally have only 5 leaves on one branch, while transgenic tomatoes have 15 leaves and more complex branching.

[0081] like Figure 7As shown, from the overall perspective of the plant, the wild-type tomato branch development is simpler, with 6 main branches, while the transgenic tomato has as many as 10 main branches, and the branches are more complex and have more compound leaves.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Polygonatum sibiricum KNOX-BP gene, characterized in that: The nucleotide sequence of the KNOX-BP gene is shown in SEQ ID NO:

1.

2. The gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by the KNOX-BP gene is shown in SEQ ID NO:

2.

3. An expression vector carrying the gene according to claim 1 or 2.

4. A recombinant cell containing the gene according to claim 1 or 2, or transformed with the expression vector according to claim 3.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell is a plant cell or a microbial cell.

6. A method for increasing the number of compound leaves of a plant, characterized in that: The method comprises connecting the Polygonatum cyrtonema KNOX-BP gene of claim 1 or 2 to a vector and transforming the vector into a plant through Agrobacterium-mediated infection.

7. A method for preparing a transgenic plant, characterized in that: The method comprises the following steps: S1, connecting the Polygonatum cyrtonema KNOX-BP gene described in claim 1 or 2 to a vector to obtain an expression vector; S2, the expression vector in S1 is transferred into Agrobacterium to obtain recombinant cells; S3, infecting plants with the recombinant cells obtained in S2 to obtain transgenic strains.

8. The method according to claim 7, characterized in that The plant is tomato.

9. The method according to claim 7, characterized in that: In step S1, the vector is a 1380-Cas9-HA plasmid; In step S2, the Agrobacterium is GV3101; In step S3, the infection method is to infect plants by leaf dipping mediated by Agrobacterium.

10. Use of the gene according to claim 1 or 2, or the expression vector according to claim 3, or the recombinant cell according to claim 4 in increasing the number of compound leaves of a plant.

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