EjCLE1 gene related to development regulation and control of loquat plants as well as encoding protein and application of EjCLE1 gene

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjCLE1 gene related to loquat plant development, a protein coded by the EjCLE1 gene and application of the EjCLE1 gene. The full length of the cDNA sequence of the gene is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the gene is as shown in SEQ ID NO.2. The EjCLE1 gene disclosed by the invention contains a typical CLE motif, and the genetic relationship between the EjCLE1 gene and the AtCLE1 gene is relatively close to that of the Arabidopsis thaliana. An overexpression vector pFGC5941-EjCLE1 is introduced into wild type arabidopsis thaliana through an agrobacterium tumefaciens-mediated inflorescence infection method, a 35S:: EjCLE1 transgenic plant develops asymmetrically, cotyledons and euphylla are deformed, and lotus leaf-shaped leaves and a plurality of disordered growing points grow. The EjCLE1 gene of the loquat can participate in stem cell maintenance and stem end meristem formation, so that clues are provided for culturing loquat varieties with better plant types and higher ornamental value, and the EjCLE1 gene of the loquat has a very good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and specifically relates to a loquat EjCLE1 protein and a coding gene and application thereof. Background Art

[0002] Loquat is a subtropical evergreen fruit tree belonging to the genus Eriobotrya in the Rosaceae family. Its fruit is rich in nutrients and has the effects of quenching thirst, moisturizing, clearing the lungs, and relieving coughs. Loquat leaves can also be used to treat stomach problems. However, loquat plants are tall and difficult to pick, and there is a lack of dwarf and horticultural ornamental varieties. CLAVATA3 / ESR-RELATED (CLE) peptides, as hormone regulators of plant development, play an important role in regulating plant growth, development and environmental response, especially in maintaining the balance between proliferation and differentiation of stem cells in the apical meristem. The CLE peptide-leucine-rich repeat receptor kinase (LRR-RLK)-WUSCHEL (WUS) gene signaling pathway plays an important role in regulating the division and differentiation of stem cells. The WUS-CLV3 and WUS-CLE40 negative feedback loops are the basis for maintaining the homeostasis of stem cells in the shoot apical meristem. The latest research shows that the functionally redundant CLE1-7 polypeptide also acts through the CLV1 / BAM1 receptor to inhibit WUS gene expression and participate in regulating the regeneration ability of the apical meristem. However, the functional identification and mechanism of the CLE gene in perennial woody plants have not been reported, and it is still unclear whether it is involved in regulatory functions. Therefore, it is very necessary to study the effects and molecular mechanisms of the loquat CLE1 gene on the development of the apical meristem, which may provide some clues for breeding loquat varieties with better plant types and higher ornamental value. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a loquat EjCLE1 protein and its encoding gene and application.

[0004] First, the present invention provides loquat EjCLE1 protein, which is:

[0005] 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

[0006] 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2.

[0007] The present invention also provides a gene encoding the loquat EjCLE1 protein.

[0008] Preferably, the sequence of the gene is shown as SEQ ID No.1.

[0009] The invention also provides an overexpression vector, a host cell and an engineering bacterium containing the gene.

[0010] The invention also provides the use of the gene in regulating plant development.

[0011] In a specific embodiment of the present invention, the gene is transferred into the plant genome and overexpressed in the transgenic plant to participate in regulating the plant development of the plant. Compared with the wild type, the transgenic plant has asymmetrical development, malformed cotyledons and true leaves, lotus-like leaves and / or multiple disordered growth points.

[0012] The present invention isolates an EjCLE1 gene closely related to the development and regulation of loquat leaves from loquat flower buds. The loquat EjCLE1 protein is most closely related to the Arabidopsis AtCLE1 protein, and both contain a typical CLE motif at their C-termini. A plant overexpression vector of the EjCLE1 gene was constructed by genetic engineering, and it was transferred into wild-type Arabidopsis for overexpression. The transgenic plants developed asymmetrically, with deformed cotyledons and true leaves, lotus-shaped leaves, and multiple disordered growth points. The loquat EjCLE1 gene may be involved in the maintenance of stem cells and the formation of stem apical meristems, which provides some clues for cultivating loquat varieties with better plant types and higher ornamental value, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows the electrophoresis photos of the coding region sequence clone of the EjCLE1 gene, where Maker is DL2000 DNA marker.

[0014] Figure 2 The phylogenetic tree of loquat EjCLE1 gene and 32 Arabidopsis AtCLE genes is shown. The results show that loquat EjCLE1 gene and Arabidopsis AtCLE1 gene are most closely related.

[0015] Figure 3 The figure shows the sequence alignment of loquat EjCLE1 protein and Arabidopsis AtCLE1 protein. Loquat EjCLE1 protein and Arabidopsis AtCLE1 protein have high similarity, and both contain a typical CLE motif at their C-termini.

[0016] Figure 4 This is the PCR identification of transgenic Arabidopsis 35S::EjCLE1 positive plants. Maker is DL2000 DNA marker, and WT is the wild-type Arabidopsis negative control.

[0017] Figure 5Photographs of phenotypic identification of transgenic Arabidopsis thaliana expressing the loquat EjCLE1 gene. Compared with wild-type Arabidopsis thaliana, 35S::EjCLE1 transgenic plants showed asymmetric leaf development, malformed cotyledons and true leaves, lotus-shaped leaves, and multiple disordered growth points. DETAILED DESCRIPTION

[0018] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular cloning laboratory manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0019] Example 1 Cloning of loquat EjCLE1 gene cDNA sequence

[0020] Fresh loquat flower buds of about 0.5 cm in length at the differentiation stage were collected, quickly sampled, placed in a cryovial, quickly frozen in liquid nitrogen for 2 h, and then placed in a -80°C ultra-low temperature refrigerator for use. Total RNA in the loquat flower buds was extracted using an RNA extraction kit.

[0021] Based on the genome sequencing data of loquat flower buds at different developmental stages in the previous stage of our team, specific primers were designed at both ends of the full-length sequence of the loquat EjCLE1 gene coding region: EjCLE1F 5'-ATGGGTAGCCTCAAATTCTGG-3' and EjCLE1R5'-CTAATGATGTCGCGGATCA-3'. The reaction conditions were 94℃5min; 94℃30s, 56℃30s, 72℃30s, 35 cycles; 72℃10min; 94℃5min; 16℃∞. After the PCR reaction was completed, the target band ( Figure 1 ), and the PCR product was recovered using an agarose gel DNA recovery kit. After ligation to the pMD19-T vector, the pMD19-EjCLE1 plasmid was constructed and transformed into competent E. coli cells. Single clones were picked and sequenced to verify the coding region sequence of the EjCLE1 gene.

[0022] Snapgene software was used for sequence splicing analysis and protein translation, and finally the coding region sequence (SEQ ID No.1) and protein sequence (SEQ ID No.2) of the EjCLE1 gene cDNA were obtained. MEGAX software was used to construct a phylogenetic tree, and the results showed that the loquat EjCLE1 gene was most closely related to the Arabidopsis AtCLE1 gene. DNAMAN software and Interpro website (https: / / www.ebi.ac.uk / interpro / entry / InterPro / IPR039616 / ) were used for sequence alignment and conserved domain analysis, respectively. The results showed that the loquat EjCLE1 protein had a high degree of sequence similarity with the Arabidopsis AtCLE1 protein, and both contained a typical CLE motif at their C-termini.

[0023] Example 2 Transformation of loquat EjCLE1 gene into Arabidopsis thaliana

[0024] Using the correctly sequenced pMD19-EjCLE1 fusion plasmid as a template, specific primers were designed using the software Oligo7.0: pGC5941-EjCLE1F5'-ACAATTACCATGG ggcgcgcc ATGGGTAGCCTCAAATTCTGG-3';pGC5941-EjCLE1R:5'-CTCTAGACTCACCTA ggatcc CTAATGATGTC GCGGATCA-3'; The pGC5941 plasmid was double-digested with restriction endonucleases AscI and BamHI, and recovered after agarose gel electrophoresis. The enzyme, amplified product, and pCAMBIA1300 vector after digestion were connected at 50°C for 30 minutes according to the system of 5μ, 1μ, and 4μ to 10μ, and the recombinant vector was transferred into E. coli competent cells. After bacterial liquid PCR and double enzyme digestion verification, sequencing was performed to ensure that the target gene sequence was successfully connected to the vector. The verified correct plasmid was named FGC5941-EjCLE1 plasmid.

[0025] Take 1 μg of pFGC5941-EjCLE1 plasmid, add 100 μL Agrobacterium competent cells, and mix well; place in an ice bath for 5 minutes, transfer to liquid nitrogen for rapid freezing for 5 minutes, and quickly place in a 37°C water bath for 5 minutes; add 800 μL LB liquid culture medium, and shake at 28°C and 250 rpm for 5 hours; transfer the bacterial liquid to LB (50 mL LB + 50 μg / mL Kana + 50 μg / mL Rif) solid selection medium, spread evenly, and invert and culture at 28°C for 48 hours.

[0026] Streak the Agrobacterium containing pFGC5941-EjCLE1 positive clones on 25 mL solid plate medium (containing 25 μg / mL Kana + 25 μg / mL Rif) and invert and culture at 28°C for 48 h; pick a single colony to 5 mL LB liquid medium (50 μg / mL Kana and 50 μg / mL Rif), shake and culture at 200 rpm in a 28°C shaker for 15-20 h, take 100 μL to 10 mL LB liquid medium (50 μg / mL Kana and 50 μg / mL Rif) and culture overnight, then take 2 mL of bacterial solution to 100 mL LB liquid medium (50 μg / mL Kana and 50 μg / mL Rif) and culture until OD 600 =0.8~1.5, transfer the bacterial solution to two 50mL centrifuge tubes, centrifuge at 4℃, 5000rpm for 5min, discard the supernatant, and add 50mL of invasion and infection buffer (0.5% Silwet L-77, 5% sucrose, 1 / 2MS culture medium) to each tube.

[0027] Arabidopsis seeds were placed on moist filter paper and kept at 4°C for 48 hours, then sown in nutrient soil (perlite: vermiculite: nutrient soil = 1:4:5), and cultured under temperature of 22°C, humidity of 70%, and 14h light / 10h dark conditions; the wild-type Arabidopsis plants were watered thoroughly before transgenic; when infecting, the existing siliques on the Arabidopsis plants to be used were cut off, and the flower buds were infiltrated with pFGC5941-EjCLE1 Agrobacterium infection solution for 60s; covered with a black plastic bag, and the high temperature and high humidity environment inside the film was maintained. After dark culture for 24 hours, the black plastic bag was uncovered and placed for normal culture; the above method was used for infection 3 times with an interval of 7d.

[0028] Collect mature seeds of EjCLE1 transgenic Arabidopsis and clean them. Place them in a 4℃ refrigerator for vernalization for 14 days; evenly sow the vernalized seeds on the culture soil, spray them with water and cover them with plastic wrap to keep them moist. Remove the plastic wrap after germination and follow the conventional water and fertilizer management. When the seedlings grow to about ten days old, spray them with 20 mg / L of glufosinate ammonium, spray them once every three days, and spray them three times in total. The surviving Arabidopsis plants were cultured for about 20 days, and a certain amount of leaves were taken from each plant to extract the EjCLE1 transgenic Arabidopsis DNA. A small piece of Arabidopsis leaf was placed in a 2.0 mL eppendorf tube, frozen in liquid nitrogen, and ground; 600 μL of extraction buffer was added, vortexed, and placed on ice; after all samples were processed, they were placed in a 65°C water bath for 25 minutes; the samples were taken out of the water bath and placed at room temperature. After cooling to room temperature, 340 μL of potassium acetate solution was added, vortexed, and ice bathed for 20 minutes; 13000 rpm, high-speed centrifugation for 5 minutes, the supernatant was transferred to a new eppendorf tube; an equal volume of isopropanol was added, 4°C, 13000 rpm, centrifuged for 10 minutes, the clear liquid was poured out, and rinsed with ice anhydrous ethanol (the anhydrous ethanol was placed in a -20°C refrigerator 2 hours in advance); the precipitate was rinsed with 70% and 100% ethanol in turn; after the precipitate was blown dry, it was dissolved in 50 μL sterile water.

[0029] The DNA of non-transgenic wild-type Arabidopsis was used as a control to confirm the EjCLE1 gene in the positive transgenic Arabidopsis plants. According to the cloning primers of the loquat EjCLE1 gene (EjCLE1F'-ATGGGTAGCCTCAAATTCTGG-3' and EjCLE1R'-CTAATGATGTCGCGGATCA-3'), 2×Mix taq, 1μL DNA, PCR identification of Arabidopsis plants resistant to glufosinate was performed, and the gel recovery and purification solution was sent to Qingke Company for sequencing; a total of 10 positive EjCLE1 overexpressing T0 generation Arabidopsis plants ( Figure 4 ).

[0030] Example 3 Phenotypic Identification of Transgenic Arabidopsis thaliana Expressing the Loquat CLE1 Gene

[0031] Using the above method, we obtained multiple T3 generation homozygous 35S::EjCLE1 transgenic lines ( Figure 5 ). 35S::EjCLE1 transgenic plants have asymmetric leaf development ( Figure 5 BI), cotyledon ( Figure 5 E) and true leaf deformity ( Figure 5 F) grow lotus-shaped leaves ( Figure 5 GI) and with multiple disordered growth points ( Figure 5 J).

[0032] Combined with existing research, the loquat EjCLE1 gene is involved in the maintenance of stem cells and the formation of stem apical meristem, which provides some clues for breeding loquat varieties with better plant shape and higher ornamental value, and has good application prospects.

[0033] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Loquat EjCLE1 protein, which is: 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No.

2.

2. A gene encoding the loquat EjCLE1 protein according to claim 1.

3. The gene according to claim 2, It is characterized in that The sequence is shown as SEQ ID No.

1.

4. A vector containing the gene according to claim 2 or 3.

5. A host cell containing the vector according to claim 4.

6. An engineered bacterium containing the gene according to claim 2 or 3.

7. Use of the gene according to claim 2 or 3 in regulating plant development.

8. The use according to claim 7, It is characterized in that The gene is transferred into the plant genome and overexpressed in the transgenic plant to participate in regulating the plant development.

9. A method for constructing a transgenic plant, comprising: transferring a vector containing the gene of claim 2 or 3 into a plant genome by Agrobacterium-mediated method, and screening to obtain a transgenic plant.

10. The construction method according to claim 9, It is characterized in that Compared to the wild type, the transgenic plants have asymmetrical development, malformed cotyledons and true leaves, lotus-shaped leaves and / or multiple disordered growth points.

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