CN115651073B - EjWUSa gene for advancing loquat flowering time, encoding protein and application thereof - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjWUSa gene related to loquat flowering phase regulation, and a coded protein and application thereof. The full length of cDNA sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2. The EjWUSa gene is expressed in flower buds of a diploid late-flowering variety and a triploid early-flowering variety of the loquat, and the expression quantity is higher in the first two periods and the last period of flower bud differentiation. The expression vector pFGC5941-EjWUSa is introduced into wild Arabidopsis thaliana by an Agrobacterium-mediated inflorescence infection method, and transgenic Arabidopsis thaliana 35S is subjected to EjWUSa overexpression and blooming in advance. The transgenic arabidopsis plant material obtained by using the loquat EjWUSa gene can be used for the early flowering and fruiting time, is used for the breeding of new early flowering and fruiting varieties, and has good application prospect.

Description

EjWUSa gene for advancing loquat flowering time, encoding protein and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to loquat EjWUSa protein, and a coding gene and application thereof.

Background

Loquat belongs to Eriobotrya (Eriobotrya japonica) plant of Rosaceae, and is native to subtropical evergreen tree in China, and its mature period is in early summer of light season. The length of the shelf life influences the economic benefit of the loquat, and the promotion of the premature loquat of the loquat by advancing the flowering phase is an important measure for prolonging the shelf life, so that the research of the expression characteristics of the flowering phase regulating key genes in the development process of regulating the loquat flower has important significance, and the loquat variety can be used for directionally and genetically improving the flowering phase of the loquat variety to accumulate gene resources.

The adult flower organ develops from a apical meristem (SAM), the vegetative meristem produces an inflorescence meristem, the inflorescence meristem produces a Flower Meristem (FM), the flower meristem produces a flower organ primordia, and finally the flower organ is produced. WUSCHEL (WUS) is a characteristic gene in SAM tissue center, involved in coordinating division and differentiation of stem cells in CZ region, regulating the occurrence of collateral organs such as flower organs, leaf organs, etc. Studies in Arabidopsis have found that when the AtWUS gene is mutated, it causes the number of stem cells to be maintained, resulting in premature termination of shoot apical meristems and inflorescence meristems, and ectopic expression of AtWUS can form ectopic meristems and processes. Nevertheless, the specific effect of the WUS gene on FM development and molecular mechanisms are not well understood. Second, many studies of flower-related genes have focused on herbaceous plants, especially Arabidopsis and ornamental plants, with less research in woody plants and less related reports in loquat. Therefore, the research on the influence and molecular mechanism of the WUS gene of the loquat on FM development is very necessary, which is helpful for breeding early-flowering loquat varieties and provides reference for the research on flowering mechanisms of other plants.

Disclosure of Invention

In order to solve the problems, ejWUSa genes with higher expression in flowers are obtained through screening, cloning, expression characteristics and functional characteristics are analyzed, and the loquat EjWUSa protein, the encoding gene and the application thereof are provided.

First, the present invention provides loquat EjWUSa protein, which is:

1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)

2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention also provides a gene for encoding the loquat EjWUSa protein.

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

The invention also provides an over-expression vector containing the gene, a host cell and engineering bacteria.

The invention also provides application of the gene in promoting flowering of plants.

In one embodiment of the invention, the gene is transferred into the genome of a plant and overexpressed in the transgenic plant, and the flowering and fruiting time of the plant is advanced.

According to the invention, 1 EjWUSa gene closely related to loquat flower development regulation is separated from loquat flower buds, and subcellular localization shows that the EjWUSa gene is expressed in cell nuclei. Real-time fluorescence quantitative PCR shows that EjWUSa gene is expressed in early and late flower varieties of diploid and triploid loquat, and the expression quantity is mainly concentrated in the first two periods and the last period of flower development, which shows that EjWUSa is favorable for FM differentiation and plays an important role in physiological differentiation and morphological differentiation of flower buds. The plant over-expression vector of the EjWUSa gene is constructed by utilizing a genetic engineering means, and is transferred into wild arabidopsis for over-expression, so that the flowering time of the arabidopsis can be shortened, and the fruiting time is further promoted. The invention provides a good application prospect for the transformation of the flowering phase of plants.

Drawings

FIG. 1 shows an electrophoresis photograph of a clone of the coding region sequence of the EjWUSa gene. Wherein M is DL2000 DNA marker,1 is PCR product of EjWUSa gene ORF; the arrow indicates the gene band of interest amplified by PCR.

FIG. 2 shows subcellular localization of the Eriobotrya EjWUSa gene transiently expressed in tobacco leaves, showing that the expression product of the gene is localized to the nucleus. GFP: green fluorescent protein; BF: bright field imaging; mered: pooled images of GFP and BF.

FIG. 3 shows that the expression of the Eriobotrya japonica EjWUSa gene in early and late flower varieties of diploid and triploid Eriobotrya japonica shows significant differences. Wherein different lower case letters in the same breed indicate significant differences (P < 0.05). S1, physiological differentiation period of flower buds; s2, flower bud morphology differentiation period; s3, an inflorescence main shaft differentiation period; s4, inflorescence fulcrum differentiation period; s5, rapidly extending the inflorescence lateral growing support shaft; s6, a floret differentiation period; s7, bud white period; and S8, full bloom stage.

FIG. 4 is PCR identification of transgenic Arabidopsis thaliana 35S: ejWUSa positive plants. Wherein M is DNA molecular mass standard (DL 2000), WT is wild type Arabidopsis negative control, and P is pFGC5941-EjWUSa plasmid.

FIG. 5 is a photograph of the bolting times of overexpressed Arabidopsis thaliana and wild type Arabidopsis thaliana, the overexpressed Arabidopsis thaliana being 10d earlier than the wild type Arabidopsis thaliana.

FIG. 6 is a statistical plot of the number of rosette leaves of overexpressing Arabidopsis.

FIG. 7 is a photograph of flowering times of overexpressed Arabidopsis and wild-type Arabidopsis. The over-expression Arabidopsis thaliana is about 9d earlier than the flowering phase of the wild Arabidopsis thaliana.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 cloning of the cDNA sequence of the Eriobotrya japonica EjWUSa Gene

Collecting flower buds with fresh length of about 0.5cm in the differentiation period, rapidly sampling, placing into a freezing tube, quick-freezing in liquid nitrogen for 2 hours, placing into an ultralow temperature refrigerator at-80 ℃ for standby, and extracting total RNA in the flower buds of the loquat by using an RNA extraction kit.

Based on transcriptome sequencing data of loquat flower buds in the early stage of the team in different development periods, specific primers EjWUSaF 5'-ATGGACCCTCAACAGAAC-3' and EjWUSaR 5'-TCAATATGATCTG AAGTA-3' are designed at two ends of the full length of the coding region sequence of the loquat EjWUSa gene, and the reaction condition is 94 ℃ for 5min; carrying out 35 cycles at 94 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 1 min; and at 72℃for 10min. After completion of the PCR reaction, the target band was excised (FIG. 1) and the PCR product was recovered using an agarose gel DNA recovery kit. After being connected to a pMD19-T vector, the pMD19-EjWUSa plasmid is constructed, transferred into competent cells of escherichia coli, and monoclonal is selected for sequencing, and the coding region sequence of the EjWUSa gene is verified.

And (3) performing sequence splicing analysis on the PCR sequencing result of the coding region sequence verification experiment by using DNAMAN software to obtain the coding region sequence (SEQ ID No. 1) of the EjWUSa gene cDNA. The coding region sequence of cDNA of EjWUSa gene is translated by DNAstar software, and the sequence is shown as SEQ ID No. 2.

EXAMPLE 2 subcellular localization analysis of the EjWUSa Gene of Eriobotrya japonica

Primer design using Snapgene software:

pCAMBIA1300-EjWUSaF：5'-GCCATGGAGGCCAGTGAATTCATGGACCCTCAACAGAAC-3'；

pCAMBIA1300-EjWUSaR：5'-CAGGTCGACTCTAGAGGATCCATATGATCTGAAGTAATC-3'; the plasmid pCAMBIA1300 was subjected to double digestion with restriction enzymes EcoR1 and BamH1, and recovered by agarose gel electrophoresis. And 5. Mu.L, 1. Mu.L and 4. Mu.L of the enzyme, the amplification product and the pCAMBIA1300 vector subjected to enzyme digestion are prepared into a 10. Mu.L system by using ClonExpress II One Step Cloning Kit, the 10. Mu.L system is connected at 50 ℃ for 30min, the recombinant vector is transferred into competent cells of escherichia coli, and then bacterial liquid PCR and double enzyme digestion verification are performed, and sequencing is performed to ensure that the target gene sequence is successfully connected to the vector. The vector plasmid is extracted and transferred into competent cells of agrobacterium GV3101 by a freeze thawing method.

Monoclonal colonies of Agrobacterium were picked from solid LB medium plates, inoculated into 10mL of liquid medium (containing Rif+kan), and cultured at 28℃and 250rpm to OD ₆₀₀ =0.5. The cells were collected by centrifugation for 10min in 5mL of the culture broth, then resuspended in 2mL of the permeate, and then centrifuged for 10min in 2mL of the permeate (10 mM MgCl2, 10mM MES-KOH, pH=5.6, 150. Mu.M acetosyringone). Finally dilute to OD ₆₀₀ After 0.03 to 0.1, tobacco leaves were transformed, and after 1-2d of dark culture of the transformed tobacco, observation of GFP fluorescence was performed. The results indicated that the EjWUSa protein was localized to the nucleus (fig. 2).

Example 3 real-time fluorescent quantitative PCR analysis of Eriobotrya japonica EjWUSa Gene

Extracting total RNA of flower buds of a loquat diploid late-flowering variety 'normally white No. 1' and a triploid early-flowering variety 'Huayuwu' in 8 periods respectively, wherein the 8 periods are respectively as follows: the method comprises a flower bud physiological differentiation stage (S1), a flower bud morphological differentiation stage (S2), an inflorescence main shaft differentiation stage (S3), an inflorescence support shaft differentiation stage (S4), an inflorescence side support shaft rapid elongation stage (S5), a floret differentiation stage (S6), a flower bud white-out stage (S7) and a full-bloom stage (S8), wherein S1 belongs to an FM physiological differentiation stage, and S2-S8 belong to an FM morphological differentiation stage. After detection of its integrity and concentration, it was reverse transcribed into cDNA. The loquat cDNA sequence is used as a template, and the real-time fluorescence quantitative PCR primers qRTEjWUSaF:5'-TGGATCATGGGTTGGTGTTGA-3' and qRTEjWUSaR:5'-AGCCATGGATGTCTTCACCG-3' are designed by using Oligo7.0 software. The loquat Ejacin gene is taken as an internal reference gene, and the primer sequence is qRTEjacin F:5'-AATGGAACTGGAATGGTCAAGGC-3' and qRTEjactinR:5'-TGCCAGATCTTCTCCATGTCATC-3' the specificity is detected by PCR, and on the premise of ensuring the specific amplification of the PCR, a real-time fluorescent quantitative PCR experiment is carried out, wherein 3 biological repeats are arranged for each reaction. The PCR reaction procedure is 94 ℃ pre-denaturation for 5min; after 94℃for 20s,56℃for 20s,72℃for 20s,41 cycles, a dissolution profile was collected: adjusting the temperature to 60 ℃ for 90s, and pre-dissolving; then heating at a speed of 1.0 ℃/s, and preserving heat for 5s at 1 ℃ when the temperature is raised to 95 ℃.

The EjWUSa gene is expressed in flower buds of a diploid late-flowering variety and a triploid early-flowering variety of the loquat, and the expression level is higher in the first two periods and the last period of flower bud differentiation (figure 3). This indicates that EjWUSa is favorable for FM differentiation and plays an important role in physiological differentiation and morphological differentiation of flower buds.

Example 4 transformation of EjWUSa Gene of Arabidopsis thaliana

Primers were designed using software Oligo7.0, pGC5941-EjWUSaF:5' -ACAATTACCATGGGGCGCGCCATGGACCCTCAACAGAAC-3'；pGC5941-EjWUSaR：5'-CTCTAGACTCACCTAGGATCCTCAATATGATCTGAAGTA-3'; the pMD19-EjWUSa plasmid with correct sequence was used as a template for amplification, pGC5941 plasmid was subjected to double digestion reaction with restriction enzymes Asc1 and BamH1, and recovered by agarose gel electrophoresis. And ClonExpress II One Step Cloning Kit, preparing the enzyme, the amplification product and the pCAMBIA1300 vector subjected to enzyme digestion into a 10u system according to 5u, 1u and 4u, connecting for 30 minutes at 50 ℃, transferring the recombinant vector into competent cells of escherichia coli, and then carrying out bacterial liquid PCR and double enzyme digestion verification and sequencing to ensure that the target gene sequence is successfully connected to the vector. The correct plasmid was verified to be designated as FGC5941-EjWUSa plasmid.

Taking 1 mug of pFGC5941-EjWUSa plasmid, adding 100 mug of agrobacteria competent cells, and uniformly mixing; ice bath for 5min, transferring into liquid nitrogen, rapidly freezing for 5min, rapidly placing in 37deg.C, and water bath for 5min; 800. Mu.L of LB liquid medium is added, and the mixture is oscillated at 28 ℃ and 250rpm for 5 hours; the bacterial liquid is transferred to LB (50 mL LB+50. Mu.g/mL Kan+50. Mu.g/mL Rif) solid selection medium, evenly coated, and inversely cultured for 48 hours at 28 ℃.

Agrobacterium containing pFGC5941-EjWUSa positive clones was streaked onto 25mL of solid plate medium (containing 25. Mu.g/mL Kan+25. Mu.g/mL Rif), cultured upside down at 28℃for 48h; picking single colony to 5mL LB liquid medium (50 mu g/mL Kan and 50 mu g/mL Rif), shaking culturing at 200rpm in shaking table at 28deg.C for 15-20h, taking 100 mu L to 10mL LB liquid medium (50 mu g/mL Kan and 50 mu g/mL Rif), culturing overnight, taking 2mL bacterial liquid to 100mL LB liquid medium (50 mu g/mL Kan and 50 mu g/mL Rif), culturing to OD ₆₀₀ The bacterial solution was transferred to 250 mL centrifuge tubes, centrifuged at 5 000rpm for 5min at 4 ℃, the supernatant was decanted, and 50mL of infection buffer (0.5% silwet L-77,5% sucrose, 1/2MS medium) was added per tube.

Placing Arabidopsis seeds on wet filter paper, placing at 4 ℃ for 48 hours, then sowing to nutrient soil (perlite: vermiculite: nutrient soil=1:4:5), and culturing under the conditions of 22 ℃ and 70% humidity and 14 hours illumination/10 hours darkness; watering wild arabidopsis plants thoroughly before transgenosis; cutting off the existing horns on the Arabidopsis plants to be used when the Arabidopsis plants are infected, and allowing flower buds to invade pFGC5941-EjWUSa Agrobacterium dip for 60s; covering a black plastic bag, keeping high temperature and high humidity environment in the film, and after dark culturing for 24 hours, uncovering the black plastic bag and placing for normal culturing; the method is infected 3 times, and the interval time is 7d.

The EjWUSa transgenic Arabidopsis mature seeds were harvested and the seeds were cleaned. Placing in a refrigerator at 4 ℃ for vernalization treatment 14d; uniformly sowing the vernalized seeds on culture soil, spraying water, covering with a preservative film for preserving moisture, removing the preservative film after germination, and managing according to conventional water and fertilizer. Spraying 20mg/L glufosinate once every three days when the seedlings grow to about ten days, and spraying three times in total. Culturing surviving Arabidopsis plants for about 20d, taking a certain amount of leaves from each plant, extracting EjWUSa transgenic Arabidopsis DNA, taking 1 small Arabidopsis leaf, placing in a 2.0mL eppendorf tube, placing in liquid nitrogen for quick freezing, and grinding; adding 600 mu L of extraction buffer, vortex shaking, and placing on ice; after all samples are treated, placing the samples in a water bath at 65 ℃ for 25min; taking out the sample from the water bath, standing to room temperature, cooling to room temperature, adding 340 mu L of potassium acetate solution, and carrying out vortex vibration for 20min in an ice bath; 13000rpm, high speed centrifugation for 5min, transferring the supernatant to a new eppendorf tube; adding equal volume of isopropanol, centrifuging at 4deg.C and 13000rpm for 10min, removing clear liquid, and rinsing with ice absolute ethanol (absolute ethanol is put into-20deg.C refrigerator 2 hr in advance); rinsing the precipitate with 70% ethanol and 100% ethanol in sequence; after drying the precipitate, it was dissolved in 50. Mu.L of sterile water.

The EjWUSa gene was confirmed on positive plants of transgenic Arabidopsis thaliana by using DNA of non-transgenic wild Arabidopsis thaliana as a control. PCR identification was performed on Arabidopsis plants against glufosinate with 2 XMix taq, 1. Mu.L of DNA according to the Eriobotrya japonica EjWUSa gene cloning primers (EjWUSaF: 5'-ATGGACCCTCAACAGAAC-3' and EjWUSaR: 5'-TCAATATGATCTGAAGTA-3'), and the gel recovery purified solution was sent to the Rheumatoid company for sequencing; 9 positive EjWUSa overexpressing T0 generation Arabidopsis plants were obtained altogether (FIG. 4).

EXAMPLE 5 phenotypic identification of transgenic Arabidopsis thaliana of the Eriobotrya EjWUSa Gene

We observe, count and photograph the bolting time, rosette number and flowering time of Arabidopsis. Under the same planting conditions, overexpressing arabidopsis was bolting at 21 days after germination, while wild arabidopsis was bolting at 31 days (fig. 5); the former research shows that the number of rosette leaves of arabidopsis thaliana is inversely related to the flowering time and the flowering time, and the number of rosette leaves of the wild arabidopsis thaliana is generally 12 or 13. Therefore, we counted the number of rosette leaves when the arabidopsis flower bolt was 1cm high: the number of rosette leaves in the process of over-expression arabidopsis bolting is only 7 and 8, and the number of few plants is 9, which is obviously less than 12 or 13 (figure 6). Finally, we counted the flowering time of arabidopsis: overexpressing arabidopsis flowers 28d after germination, whereas wild type arabidopsis flowers only 37 days (fig. 7). Notably, over-expressed arabidopsis flowers only when the flowering bolts are high compared to wild type arabidopsis.

From the results, ejWUSa is a characteristic gene of the flower meristem, is responsible for the initial differentiation and formation of the flower meristem, and the transgenic arabidopsis thaliana material can be used for the improvement of the flowering time of plants, so that the plants bloom early and the fruit ripeness time are prolonged, and the breeding of early maturing varieties is facilitated.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (9)

1. Loquat EjWUSa protein, which is a protein consisting of the amino acids shown in SEQ ID No. 2.

2. A gene encoding the loquats EjWUSa protein of claim 1.

3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.

4. A vector comprising the gene of claim 2 or 3.

5. An engineered bacterium comprising the gene of claim 2 or 3.

6. Use of a gene according to claim 2 or 3 for promoting flowering in plants.

7. The use according to claim 6, wherein the gene is transferred into the genome of a plant and overexpressed in the transgenic plant, and the flowering and fruiting time of the plant is advanced.

8. A method for constructing transgenic plant, which comprises transferring the vector containing the gene of claim 2 or 3 into plant genome by agrobacterium-mediated method, and screening to obtain transgenic plant.

9. The method of claim 8, wherein the transgenic plant has earlier flower bud differentiation and flowering time than the wild type plant, and further earlier fruiting time.