NL2031841B1 - Application of Pectin Methylesterase Inhibitor Gene GhPMEI39 and its Encoded Protein in Plant Inflorescence Regulation - Google Patents
Application of Pectin Methylesterase Inhibitor Gene GhPMEI39 and its Encoded Protein in Plant Inflorescence Regulation Download PDFInfo
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Abstract
The present invention relates to the field of plant performance gene regulation, in particular to the application of pectin methylesterase inhibitor gene GhPME/39 and its encoded protein in plant inflorescence regulation. The gene sequence of pectin methylesterase inhibitor gene GhPME/39 is shown in SEQIDNO:1. The invention screens the target gene by public database, designs primers to clone the target gene, constructs the cotton and Arabidopsis transgenic materials, analyzes the plant development phenotype after overexpression and oonstitutive expression of pectin methylesterase inhibitor protein, and verifies the role of pectin methylesterase inhibitor gene GhPME/39 in plant flowering and inflorescence morphology.
Description
Application of Pectin Methylesterase Inhibitor Gene GhPMEI39 and its
Encoded Protein in Plant Inflorescence Regulation
The present invention relates to the field of plant performance gene regulation, in particular to the application of pectin methylesterase inhibitor gene
GhPMEI39 and its encoded protein in plant inflorescence regulation.
BACKGROUND TECHNOLOGY
Vascular bundle is an important transport channel for plants, which is mainly composed of phloem and xylem cells. It is responsible for transporting water, inorganic salts, organic substances, various proteins and amino acids during plant development, thereby regulating vegetative and reproductive growth of plants. Previous studies have shown that CLE25 APL and other genes play important roles in regulating plant phloem initiation and development.
PMElIs are cell wall pectin methylesterase inhibitors, which play important roles in regulating cell wall toughness, permeability and biochemical properties, thereby affecting the various plant development such as seed germination, pathogen invasion, and the interaction with external factors.
HG is the most abundant pectin polymer. HG methyl esterification has a significant impact on the rheological properties of cell wall and plant development. Demethylated HG can be easily hydrolyzed by pectin-degrading enzymes {e.g., galactosidase), or by promoting the formation of intermolecular calcium bonds and the so-called egg box model structure to form rigid gels, which means that demethylated HG has a dual role. At the same time, HG methyl esterification can also hinder the establishment of calcium bridge and increase the flexibility of cell wall. The degree of methyl esterification (DM) of
HG is considered to be mediated by the reaction of pectin methylesterases (PME) and PME/invertase inhibitors (PMEI) through methyl transfer and release,
which forms a large plant sequence family, named PMEl-related proteins (PMEI-RP). It directly interacts through the indispensable hairpin motif.
Flowering is an important trait of plants, which is related to the yield of crops and the economic value of ornamental plants, but its specific regulatory mechanism is unclear.
In view of this, the invention is proposed.
Existing studies have shown that pectin methylesterases mainly affect plant resistance, such as bacterial or fungal diseases. The invention verifies the function of a cotton pectin methylesterase inhibitor gene GhPMEI39 and its encoded protein in plants by using gene cloning technology and plant transgenic technology.
Based on the foregoing, the present invention provides the following technical schemes:
The pectin methylesterase inhibitor gene GhPME/39 and its gene sequence are shown in SEQ ID NO: 1.
The invention provides the pectin methylesterase inhibitor gene GhPMEI39, which is cloned from cotton by biological means.
The invention screens the target gene by public database, designs primers to clone the target gene, and constructs cotton and Arabidopsis transgenic materials, analyzes plant development phenotype after overexpression and constitutive expression of pectin methylesterase inhibitor protein, verifies the role of pectin methylesterase inhibitor gene GhPMEI39 in plant flowering and inflorescence morphology.
Specifically, the invention also provides the application of pectin methylesterase inhibitor gene GhPMEI39 or its homolog or encoded protein in regulating one or more of the following properties of plants: (a) Vascular tissue differentiation;
(b) Vascular tissue growth; (Cc) Inflorescence differentiation; (d) Inflorescence growth; (e) Flower bud differentiation;
The gene sequence of pectin methylesterase inhibitor gene GhPMEI39 is shown in SEQID NO:1.
Further, the vascular bundle tissue differentiation of plants includes the number of vascular bundle tissue;
Vascular bundle tissue development includes the number of cells in phloem and xylem of vascular bundle;
The inflorescence differentiation includes the number of inflorescences;
The inflorescence development includes the number of cells in the vascular bundle of the inflorescence;
The bud differentiation includes the number of buds;
Further, the vascular tissue includes vascular tissue in stem and leaf organs.
The pectin methylesterase inhibitor gene GhPMEI39 provided by the invention can affect the number of vascular bundle tissues and change the number of cells in the phloem and xylem of vascular bundle in terms of vascular bundle tissues; In terms of inflorescence, it can affect the number of flowers; in terms of buds, it can change the number of buds.
Wherein, the stems include primary stems and branching stems.
Specifically, the described plant is cotton, and the overexpression of the pectin methylesterase inhibitor gene GhPMEI39 promotes the development of the cotton fruit stems and increases the number of buds.
If the plant is a similar economic crop such as cotton, the overexpression of pectin methylesterase inhibitor gene GhPMEI39 can promote the development of flowers and increase the number of buds, thus achieving the effect of increasing cotton yield.
It should be noted that the cotton in the invention can be various planted cottons, such as Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum, and the variations of these cotton varieties. That is to say, cotton varieties with the same or similar genes as
GhPMEI39 are also within the protection scope of the invention. Similar genes such as the sequence similarity with the gene GhPMEI39 of the present invention can reach more than 80%, or the similarity can reach more than 85% or more than 90% or more than 95% or more than 96% or more than 97% or more than 98% or more than 99%, etc.
The plant is Arabidopsis thaliana, and the overexpression of the pectin methylesterase inhibitor gene GhPMEI39 increases the number of inflorescences, increases the diameter of main stem, and increases the number of buds;
Further, the plant is Arabidopsis thaliana, and the overexpression of pectin methylesterase inhibitor gene GhPMEI39 increases the number of phloem cells in the first rosette leaf, increases the number of vascular tissues in the primary stem, and increases the amount of phloem in a single vascular tissue.
If the plant is a cruciferous plant such as Arabidopsis, overexpression of the pectin methylesterase inhibitor gene GhPMEI39 can improve the stem and leaf thickness of Arabidopsis thaliana, increase the number of inflorescences and flower buds, and increase the yield of such plants.
The application of the pectin methylesterase inhibitor gene GhPMEI39 provided by the present invention is not limited to the above-mentioned plants, and can also be used in the regulation of inflorescence traits of other plants such as ornamental plants and the regulation of hardness of woody plants.
Further, the plants include cruciferous plants, ornamental plants, cash crops and woody plants;
Further, the cruciferous plants include Arabidopsis, radish, rape, and cabbage;
The ornamental plants include chrysanthemum, rose, rose and violet;
The economic crops include cotton, oil, sugar, tobacco, hemp and alfalfa;
The woody plants include poplar, pine and cypress.
In the present invention, when the pectin methylesterase inhibitor gene 5 GhPMEI39 is applied to different plants, the gene sequence used can be the pectin methylesterase inhibitor gene GhPMEI39 or its homolog, and the homologues include not only the pectin methylesterase inhibitor genes in different plants, but also the pectin methylesterase inhibitor genes in their own plants. Generally, the sequence similarity of common homologous genes is above 80 %, if in different plants, the similarity can reach 85 % or 90 % or 95 % or 96 % or 97 % or 98 % or 99 %, etc.
The invention also provides a method for detecting whether the sample contains overexpressed pectin methylesterase inhibitor gene GhPMEI39 or the product produced by overexpressed pectin methylesterase inhibitor gene
GhPMEI39;
If these substances are detected, the tested sample is judged to have higher cotton yield performance;
Wherein, the gene sequence of the pectin methylesterase inhibitor gene
GhPMEI39 is shown in SEQ ID NO:1.
Since the overexpressed pectin methylesterase inhibitor gene GhPMEI39 has the above-mentioned effect of increasing cotton yield, in different cotton varieties or in breeding, if you want to select the overexpressed pectin methylesterase inhibitor gene GhPMEI39 plant, it is necessary to detect whether the sample to be tested contains the overexpressed pectin methylesterase inhibitor gene GhPMEI39. The detection is not limited to the detection of pectin methylesterase inhibitor gene GhPMEI39, but also can detect its overexpressed markers, overexpressed products. The detection of products produced by pectin methylesterase inhibitor gene GhPMEI39 can be carried out by various means, such as ELISA kit.
Further, the test samples are detected by primer pairs or probes or chips designed by overexpressing the elements of the pectin methylesterase repressor gene GhPMEI39.
Further, the nucleic acid sequence of the primer pair is shown in SEQ ID
NO.2 and SEQ ID NO.3.
Further, the sample to be tested includes a material suitable for sexual reproduction, asexual reproduction or tissue culture of regenerable cells;
Materials suitable for sexual reproduction, such as selected from pollen, ovary, ovule, embryo sac, etc.;
Materials suitable for asexual reproduction materials such as cuttings, roots, stems, protoplasts;
Materials suitable for asexual reproduction can be selected from cuttings, roots, stems, protoplasts, etc.;
Materials suitable for tissue culture of regenerable cells can be selected from leaves, pollens, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
Further, the sample to be detected includes any of the following materials: seeds, leaves, roots, stems, radicles, and embryos.
Further, the invention also provides a breeding method for increasing the cotton yield by recombining fragments containing an overexpressed pectin methesterase inhibitor gene into the cotton genome;
Screen to obtain plants containing overexpressed pectin methylesterase inhibitor gene element;
The gene sequence of the pectin methylesterase inhibitor gene GhPMEI39 is shown in SEQ ID NO:1.
In the invention, the target fragment is recombined into the cotton genome, and the process involves some transformation methods, such as common
Agrobacterium-mediated method, gene gun method, PEG method, laser microbeam puncture method, microinjection method and pollen-mediated method. These methods can be finished according to the technical steps in this area.
Screen to obtain a plant containing the overexpressed pectin methylesterase inhibitor gene GhPMEI39 element, the plant here can represent either the propagation material of the plant, or the non-propagative material of the plant, that is, part of the plant itself after gene transformation. These materials include but not limited to tubers, branches, roots, protoplasts, leaves, embryos, cotyledons, hypocotyls, meristem cells, root tips, anthers, flowers, seeds, pollens, ovary, ovules, and blastocysts.
Plants with overexpression of pectin methylesterase inhibitor gene
GhPMEI39 can be screened by various means, such as molecular biological methods, including but not limited to the detection of the tested samples by primer pairs or probes or chips of pectin methylesterase inhibitor gene
GhPMEI39.
Screening to obtain plants containing overexpressed pectin methylesterase inhibitor gene GhPMEI39 can be achieved by various means, such as molecular biological means, including but not limited to the detection of the tested samples by primer pairs or probes or chips of pectin methylesterase inhibitor gene GhPMEI39.
For example, to detect the product produced by overexpression of the pectin methylesterase inhibitor gene GhPMEI38, some visible markers can be set on the overexpression vector, such as those detected by ELISA kit or marked by fluorescence under specific conditions.
Utilize the pectin methylesterase inhibitor gene GhPMEI39 of the invention to assist the breeding of cotton fiber with high quality performance can screen out the target cotton at an early stage and save time.
Compared with existing technologies, the beneficial effects of the invention include at least the following aspects: (1) The present invention provides the pectin methylesterase inhibitor gene
GhPMEI39. The target gene is screened by using a public database, and primers are designed to clone the target gene. (2) By constructing its cotton and Arabidopsis transgenic materials, and analyzing the plant developmental phenotype after excessive and constitutive expression of pectin methylesterase inhibitory protein, it is first found that the pectin methylesterase inhibitory factor GhPMEI39 is involved in plant flowering and inflorescence morphology and other related functions. (3) The pectin methylesterase inhibitor gene GhPMEI39 provided by the invention can be applied to a variety of plants, such as economic crops such as cotton, and other similar crops. It can also be used for the regulation of inflorescence traits of other plants, such as ornamental plants, and the regulation of hardness of woody plants. (4) The pectin methylesterase inhibitor gene GhPMEI39 provided by the invention is expected to find genes with similar functions in other plants, providing good technical support for further research.
In order to more clearly illustrate the technical scheme in the embodiment or existing technology of the invention, the attached drawings of the embodiment or existing technology description will be described below.
Fig. 1 is the electrophoresis image of recombinant GhPMEI39 protein expressed by SDS-PAGE analysis of Escherichia coli BL21 (DE3) in embodiment 1 of the present invention;
Fig. 2 is a bar graph of the in vitro inhibitory activity of GhPMEI39 protein on Arabidopsis thaliana stem PME crude extract in embodiment 1 of the invention;
Fig. 3 is the subcellular localization micrographsof GhPMEI39 protein in embodiment 1 of the invention;
Fig. 4 is a graph of gene identification and expression status of GhPMEI39 overexpressed Arabidopsis plants in embodiment 2 of the invention,
Figure 5 is a phenotype diagram of GhPMEI39 overexpressed Arabidopsis plants in embodiment 2 of the invention;
Fig. 6 is the first rosette leaf and main stem phloem structure correlation diagram of GhPMEI39 overexpressed Arabidopsis in embodiment 2 of the present invention;
Fig. 7 is the immunofluorescence derived from GhPMEI39 OE and wild- type (WT) with the LM19 and LM20 antibodies in the vascular tissue of
Arabidopsis thaliana primary stem cross-section in embodiment 2 of the present invention;
Fig. 8 is a correlation diagram for the identification of overexpressed transgenic cotton in embodiment 3 of the present invention;
Fig. 9 is an observation diagram of the reproductive growth phenotype of the overexpressed GhPMEI39 transgenic cotton in embodiment 3 of the present invention.
SPECIFIC IMPLEMENTATION METHODS
The implementation scheme of the invention is described in detail in combination with the embodiments below, but technicians in this field will understand that the following embodiments are only used to illustrate the invention and should not be regarded as limiting the scope of the invention. If no specific conditions are specified in the embodiments, they shall be subject to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products that can be purchased from the market if the manufacturer is not specified.
Embodiment 1 1. Cloning of cotton pectin methylesterase inhibitory coding gene (GhPMEI39)
The GhPMEI39 cDNA is based on the total cDNA of cotton SDPA ovule of
China Cotton Research Institute 24 (ZM24), and 5'-
CACCATGGGAATTCTCTTGCCTCCT-3' (sense) and 5'-
TACAAACCTATCAACTATTCT-3' (antisense) are used as primers to obtain a 553bp gene sequence. (As shown in SEQ ID NO:1). The pENTR-GhPMEI39 entry clone is constructed by gateway and verified by sequencing. 2. Expression, purification and enzyme activity verification of GhPMEI39 protein in Escherichia coli (1) Prokaryotic induction and purification of GhPMEI39 protein
Using Infusion technology and pColdTF expression vector, the specific
PCR product of GhPMEI39 is amplified so that the 5’ and 3’ ends are the same sequences as the two ends of the linearized vector (BamHI/ECORI double- enzyme digestion linearization). pColdTF-GhPMEI39 recombinant plasmid is obtained by referring to Ultra One Step Cloning Kit (Vazyme, Cat. #115-02) seamless cloning method and it contains 6xHis-Tag tag. The pColdTF-
GhPMEI39 recombinant plasmid is transformed into Escherichia coli BL21{DE3) competent cells. The transformed cells are activated and cultured in Luria-
Bertani (LB) medium containing 50 pg/ml ampicillin. When the cell density at 600nm (OD600) reaches 0.4-0.6, the final concentration of 0.5mM IPTG is added to LB medium. After overnight incubation at 16°C, the cells are centrifuged at 5000g for 10mins at 4°C, and the pelleted cells are resuspended in binding buffer (50mM Tris-HCI, 150mM NaCl, pH7.5). The suspension is sonicated, and the lysate is centrifuged at 12,000 g for 10 mins at 4°C.
Referring to the purification method of Ni SepharoseTM6 Fast Flow (GE
Healthcare, Cat. #11-0008-87), the supernatant is added to the his-binding column with nickel, and the column solution is collected and washed with 30 mM, 50 mM and 100 mM imidazole, respectively. Remove the buffer to elute the target protein, and collect the eluate. The purification effect detected by
SDS-PAGE is shown in Figure 1.
In Fig. 1, M is the protein marker; non-induction represents the cell lysate of pColdTF-GhPMEI39 transformed bacteria before IPTG induction; induction represents the cell lysate of pColdTF-GhPMEI39 transformed bacteria under
IPTG induction; The supernatant represents the cell lysate supernatant of pColdTF-GhPMEI39 transformed bacteria under IPTG induction; The precipitation represents the cell lysate precipitation of pColdTF-GhPMEI39 transformed bacteria induced by IPTG; Purification shows purified GhPMEI39 protein; Empty vector shows purified pColdTF empty protein; WB shows
Westernblot analysis of purified GhPMEI39 protein using GhPMEI39 autoantibody; It represents IPTG-induced GhPMEI39 protein. (2) Enzyme activity detection
In order to obtain the crude extract of PME, 1 g of A. thaliana stems (1 month) is grinded with liquid nitrogen and dissolved in 1 mL of extraction buffer (PBSpH7.4, 1 M NaCl) to form homogenate. After placing on ice for 1 h (three times of up-down mixing), the homogenate is centrifuged at 11000 g for 10 min, repeat once, and the supernatant is collected and stored in ice. Protein concentration is measured by NanoDrop2000 spectrophotometer (Thermo
Science, USA). PMEI purified protein and PME crude extract are placed in 25 °C water bath for 30 mins before enzyme activity assay. The mixture of reaction solution is added to each reaction tube, including 1 mL of 0.5 % (w/v) pectin, 400 pL of 0.01 % bromothymol blue, 1.50 mL of distilled water (mixture pH=7.5), and then 100 pl of different proportions of PME: GhPMEI39purified protein mixture (1:0, 1:0.5, 1:1 and 1:2) are added. The mixture is heated at 30 °C for 16 h to detect the OD value of the reactants at the wavelength of 620 nm. The calculation method is to calculate the AOD value of different proportions of PME: PMEI reactants within 1 s. In this experiment, pColdTF empty-cvector-induced protein is used as theinternal control. The results are shown in Figure 2. As the ratio of PME:GhPMEI39 changes from 1:0 to 1:2, the
PME enzyme activity in the corresponding reaction solution also decreases from 100% to 24%, while the PME activity in the negative control pColdTF empty vector induced protein did not change, indicating that GhPMEI39 protein has the ability to inhibit PME activity.
In Fig. 2, the crude protein of pColdTF-GhPMEI39 or empty vector (pColdTF-, EV) is extracted from Escherichia coli BL21 cells expressing pColdTF-GhPMEI39 or empty vector (pColdTF, EV) by IPTG induction. The error bar represents S.E.M. (n 2 3). 1:0, 1:0.5, 1:1, and 1:2 represent the ratio of crude PME extraction from
Arabidopsis stems to purified protein extracted from Escherichia coli BL21 pColdTF-GhPMEI39 or empty vector pColdTF. The white column represents the reaction solution of PME: pColdTF-GhPMEI39, and the black column represents the reaction solution of PME: pColdTF-. 3. Subcellular localization analysis of GhPMEI39 protein
The construction of the vector pCAMBIA2300: GhPMEI39: YFP, the
GhPMEI39 CDS PCR product is cloned into the upstream of the Kpn1/Asc1
YFP (yellow fluorescent protein) gene, and the GhPMEI39: YFP fusion protein is generated and transferred into GV3101 Agrobacterium. 35S:GhPMEI39: YFP overexpression transgenic Arabidopsis is obtained by Floral-dip method. The
Arabidopsis thaliana-positive seedlings grown for 4 days and the roots of WT are used as the observation objects, and are observed by LSM 510 laser confocal scanning microscope. Green (YFP) is GhPMEI39 protein localization, and red (FM4-64, cell membrane dye) is fluorescently labeled for the cell membrane (Figure 3). The results of the study are shown in the figure below.
The GhPMEI39 protein is located between the cell wall and the cell membrane, which is an exocrine protein.
In Figure 3, Before represents pre-positioning of seedlings treated with 0.4M NaCl, and After represents post-positioning of seedling roots treated with 0.4M NaCl.
Embodiment 2 1. Construction of overexpressed GhPMEI39 vector
GhPMEI39 cDNA fragment is cloned into the target vector PGWB2 (CaMV 358 promoter) by using pENTR-GhPMEI39 and Gateway LR reaction. 355:GhPMEI39 overexpression transgenic Arabidopsis thaliana is obtained by
Floral-dip method after pGWB2-GhPMEI39 Ti plasmid is transformed into
Agrobacterium tumefaciens strain GV3101. 2. DNA extraction and detection of GhPMEI39 expression in transgenic
Arabidopsis thaliana
A total of 12 transgenic plants of GhPMEI39 are obtained, and DNA of 10 transgenic and WT plants are extracted. DNA is amplified by primers
CaMV3585 -AACACGGGGGACTCTAGA-3 (sense) and GhPMEI395' -
TACAAACCTATCAACTATTCT-3 (antisense), and 12 transgenic plants of
GhPMEI39 shows bands (Figure 4A). The expression levels of GhPMEI39 in 9
Arabidopsis thaliana stems are detected. Compared with the negative control
WT, the expression levels of 6 transgenic plants are up-regulated (Figure 4B, 4C).
In Figure 4, A represents the DNA identification figure of twelve
GhPMEI3%transgenic Arabidopsis thaliana, B and C represent the semi- quantitative RNA expression level of GhPMEI39 transgenic Arabidopsis plants and the qRT-PCR identification results respectively, and Bar value represents the standard deviation of three repetitive PCRs. 3. Phenotype observation of GhPMEI39 transgenic Arabidopsis
Further, three lines of GhPMEI39-5, 8, and 9 are selected to obtain single- copy homozygotes for phenotypic observation. The results are shown in Figure 5. In Figure 5, A represents the phenotype of GhPMEI3S0E-5, 8, 9 transgenic
Arabidopsis thaliana and WT plants, B- E represent the primary inflorescence, flower bud, the first rosette leaf and the main stem respectively. F is the silique phenotype of transgenic and WT Arabidopsis.
According to the observation of the three transgenic lines and WT plants, it is found that the phloem cellss of the main stem and rosette leaves in the overexpression GhPMEI39 transgenic plants increases (Fig. 5A, 5D, 5E). More importantly, due to the overexpression of GhPMEI39 gene, the inflorescence of the primary main stem of Arabidopsis thaliana changes from raceme to umbel, and the number of buds also increases significantly (Fig. 5A-5C), which leads to the simultaneous production of two or even three silique phenotypes at a pod growth point on the main stem at the same time (Fig. 5F). 4. Overexpression of GhPMEI39 causes an increase in the number of vascular tissue cells in the first rosette leaf and primary stem of Arabidopsis thaliana.
In order to explore the reasons for the increase of transverse diameter of leaves and main stem of GhPMEI39 transgenic lines, the vascular tissue structure of main stem and rosette leaves of over-expression GhPMEI39 transgenic plants OE-5,8,9 and WT plants are observed by paraffin section.
The results are shown in Figure 8. In Figure 6, A represents the cross-section of thefirst rosette leaf of the transgenic lines and WT plant and t, B is the enlarged view of the vascular tissue in Figure A, and C is the vascular tissue of main stem in the WT and OE lines. D is the single vascular tissue in C diagram, E is the number of phloem cells in the first rosette leaf, the total number of vascular tissues in primary inflorescence and the total number of phloem is also calculated statistically in GhPMEI39 transgenic Arabidopsis and wild-type plants. wherein n=6.
Due to the overexpression of GhPMEI39, the number of phloem cells in the first rosette leaf increases from 69 of WT to 108, 117, 121 of OE lines (Fig. 6A, 6B, 6E). At the same time, the number of vascular tissues in the primary stem also increases from 6 — 8 of WT to 12 — 15 of OE transgenic materials, and the number of phloem cells in a single vascular tissue also increases significantly, from 330 of WT to 1079 — 1181in OE lines (Fig. 6C, 6D, 6E). 5. Increased Methylation Degree (DM) in primary stem of GhPMEI39 overexpressing Arabidopsis thalianaByimmuno-observation of cross-sections of primary stem vascular tissue of three OE lines and WT Arabidopsis plants with two pectin antibodies LM19 and LM20 representing different degrees of methylesterification, it is found that in the OE line, LM19 fluorescencerepresenting a low methyl ester pectin was weak, and the high methyl ester pectin label LM20 was stronger, while WT showed the opposite trend (Figure 7). In Figure 7, LM19 represents low methyl ester pectin, while
LM20 represents high methyl ester pectin, Bar=300 um in all images.
The results show that the content of methylated pectin is higher in the OE line, that is, the methylated degree (DM) of primary stem of GhPMEI39 overexpression Arabidopsis increases.
Embodiment 3 1. Construction of GhPMEI39 transgenic cotton
GhPMEI39 cDNA fragment is cloned into the target vector pGWB2 (CaMV35S promoter) by using pENTR-GhPMEI39 and GatewayLR reaction.
The pGWB2 - GhPMEI39 Ti plasmid is transformed into Agrobacterium tumefaciens strain LBA4404, and the hypocotyls of 7 - day ZM24 sterile cotton seedlings (5 - day dark treatment and 2 - day light) are used as explants for immersion cultivation to obtain transgenic overexpression cotton. 2. DNA extraction and detection of GhPMEI39 expression
The plant genomic DNA extraction kit (Bioteke, Cat#DP3112) is used to extract the genomic DNA of the young leaves of all the quasi-transgenic and
ZM24 cottons obtained from tissue culture. DNA is used as a template, and
CAMV35S-F :5 '-AACACGGGGGactcTAGa-3' (Sense) and GHPMEI39-R :5 -
TacaaaccTATCAactATTCT-3' (Antisense) are used for PCR amplification. The expression level of the plants with 562 bp fragment detected by gel is further detected.
In order to determine the expression level of GhPMEI39 in transgenic and wild-type cotton plants, the 5 DPA ovules of transgenic GhPME/39 and wild-type
ZM24 cotton are collected from 8:00 to 9:00 a.m, soaked in liquid nitrogen and stored in a refrigerator at -80 °C, or it is immediately ground and RNA is isolated from all samples according to the instructions of polysaccharide polyphenol plant total RNA extraction kit (Tiangen, Cat. # DP441). Invitrogen (Cat. # 18080- 093) is used to reverse transcription about 1ug total RNA to obtain cDNA.
GhUBQ7 (DQ116441) is used as the reference gene by qRT-PCR. The primers are 5 -CCCGAAACCTGCATAAAATG-3' (sense) and 5 -
GCGATCAAATTGGTTTTCGT-3 (antisense).
The 5 DPA ovules of transgenic and wild-type cotton stored at -80 °C are removed and ground into powder in liquid nitrogen, then add protein extract (150mM NaCl; 1M Tris-HCI, pH = 7.5; 0.1% NP40; 0.1% TritonX-100). After fully mixing, place it on ice for 30 mins. Then it is centrifuged at 12000g for 30mins at 4°C, and the supernatant is collected. GhPMEI39 polyclonal antibody is produced by Shanghai Youke Company. Westernblotting experiment is carried out according to previous reports.
A total of 146 GhPMEI39-overexpression transgenic cotton plants are obtained, and the DNA of all transgenic and WT plants is extracted. The DNA is amplified and detected by using CaMV35S5 -AACACGGGGGACTCTAGA-3' (sense) and GhPMEI39 - R:5' -TACAAACCTATCAACTATTCT-3 (antisense) as primers. As shown in Fig. 8, a total of 10 over-expression transgenic GhPMEI39 cotton lines show bands.
By detecting the expression of GhPME/39 in the 5 DPA ovules (RNA in Fig. 8), compared with the negative control WT, the expression of all transgenic plants is up-regulated. Randomly select five lines of GhPMEI39-8, 9, 13, 15, and 17 with low, medium and high expression levels of SDPA ovule proteins, and perform western blot detection with GhPMEI39 autoprotein antibody. It is found that compared with WT, four strains GhPMEI39-9, 13 ‚15,17 protein levels are also up-regulated (Protein in Fig. 8), indicating that these four lines are indeed transgenic GhPME/39 overexpressing plants. 3. Phenotypic analysis of the transgenic plants
By observing the vegetative growth and reproductive growth stages of three transgenic lines and ZM24 plants, it is found that the vegetative growth of
OE-13,15,17 transgenic plants with overexpression of GhPMEI39 is not different from that of ZM24. However, in the reproductive growth stage, overexpression of GhPMEI39 promotes the appearance of flower bud clusters in fruiting branches (Figure 9A, 9B), and flower bud clusters could further develop into flowers and even mature bolls (Figure 9C). Meanwhile, the observation on the structures of three OE lines and ZM24 fruiting branches shows that overexpression of GhPMEI39 can promote the development of phloem in fruiting branches (Fig. 9D).
In Figure 9, A is the whole plant phenotype of ZM24 and GhPMEI39 transgenic cotton lines OE-13,15,17 in the bud stage from left to right. B is the magnifying figure of bud differentiation of different lines in Figure A. Fig. C is the vascular tissue structure observation of ZM24 and OE branches and stems, wherein bar = 600 um. Fig. D is the bolls and mature fiber formed by transgenic line andZM24.
Based on the above studies, it is found that the overexpression of
GhPMEI39 promotes the development of plant vascular tissues, especially the development of phloem, thereby changing inflorescence differentiation and inflorescence characteristics.
Wherein, DPA is the abbreviation of days post anthesis, which means the number of days after flowering.
Although the invention has been explained and described by specific embodiments, it should be realized that many other changes and modifications can be made without deviating from the spirit and scope of the invention.
Therefore, it means that all these changes and modifications falling within the scope of the invention are included in the attached claims.
Sequence List <110> INSTITUTE OF COTTON RESEARCH OF CAAS <120> Application of Pectin Methylesterase Inhibitor Gene GhPMEI39 and its
Encoded Protein in Plant Inflorescence Regulation <130> 2021 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 549 <212> DNA <213> INSTITUTE OF COTTON RESEARCH OF CAAS24() <400> 1 atgggaattc tcttgcctcc ttgctatctt actctttttc tcctectttt catctcattc 60 aacaatgtcg gccatcgaaa cctcgttttc gccaacgatg ccctgattga agcccaatgt 120 cacaatgctg aagtccccga aacctgcata aaatgcgtaa aatctgattc tcgaagtcaa 180 tcagccaata aagttggcat tgctgccatc atcataactt gtcttagcaa caaagccacg 240 accttgataa acaacatgac gactcttgct tcgggcgctc gcgacaagaa cttgaaagtg 300 gctctccgag gttgcgagaa agggttttac tacacgaaaa ccaatttgat cgctgcgacg 360 aaccgattga aggggaaaga atatgatcaa acgaatcttc tggtgaaaca agcgctcgaa 420 gaagaatttg tttgtaagat gaaagtggag gttttacgat ttaactttcc gagtagtgtc 480 acgtttgaca tgggagttta cgaggaactt tcaactgcag taatgagaat agttgatagg 540 tttgtatga 549 <210> 2 <211> 18 <212> DNA <213> Artificial sequence() <400> 2 aacacggggg actctaga 18 <210> 3 <211> 21 <212> DNA <213> Artificial sequence() <400> 3 tacaaaccta tcaactattc t 21
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