CN117447574A - Application of cucumber CsSWEET5a gene and encoding protein thereof in regulation and control of plant pollen fertility - Google Patents
Application of cucumber CsSWEET5a gene and encoding protein thereof in regulation and control of plant pollen fertility Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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Abstract
The invention discloses application of a cucumber CsSWEET5a gene and a coding protein thereof in regulating plant pollen fertility, belonging to the technical field of biological breeding. The invention aims to improve the pollen fertility of plants, and provides application of CsSWEET5a protein in regulating and controlling the pollen fertility of plants in order to solve the technical problem. The present invention also provides a method of preparing a plant with increased pollen fertility, the method comprising introducing into a recipient plant a coding sequence for CsSWEET5a protein to obtain a plant with increased pollen fertility. The result of the invention shows that the over-expression of the CsSWEET5a protein can obviously improve the pollen activity, the pod length and the seed yield of the Arabidopsis, which shows that the CsSWEET5a protein can regulate the pollen fertility of plants. These results indicate that the cucumber CsSWEET5a protein can be used in cucumber breeding, and has important significance for cucumber seed high quality and high yield or induction of male sterile line for hybrid seed production.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a cucumber CsSWEET5a gene and a coding protein thereof in regulating and controlling plant pollen fertility.
Background
Most plants complete a series of sexual processes through the flower organs to fertilize, fruiting, produce seeds, etc., to reproduce the offspring. Clearly, pollen (male germ cells of plants) development plays an extremely important role in plant ontogeny. Pollen generally takes days to complete from formation in anthers to development into mature pollen grains, and a large amount of sugar is required for the process, so that abnormal pollen development and even abortion can be caused if sugar supply occurs or the sugar transportation process is blocked, and the yield and quality of crop fruits or seeds are seriously reduced.
Pollen is a whole heterotrophic organ, sugar synthesized in leaves is transported to anthers through long distance, and is unloaded from anther tissues into pollen cells for pollen to grow and develop. Throughout pollen development, from its formation within the anther to its development into mature pollen grains, they have been devoid of intercellular fibrils with surrounding anther tissue, completely in a symplastically isolated state. Thus, sugar in anther tissue can only be offloaded into pollen cells via the apoplast pathway, a process involving the involvement of two types of sugar transporters: sugar in anther tissue is firstly excreted to apoplast between anther tissue and pollen cells by first type sugar transport proteins, which is the first key step in carrying out sugar unloading in apoplast pathway; next, sugar in the apoplast is taken up into pollen cells by a second type of sugar transporter. Three types of sugar transporters, SUT/SUC (sucrose transporter/cross carrier), MST (monosaccharide transporter) and SWEET (Sugars Will Eventually be Exported Transporter), were found in plants so far, only SWEET had the function of sugar excretion, and all three of SUT/SUC, MST and SWEET had the function of sugar absorption.
Cucumber is a worldwide important vegetable crop and is also one of main cultivated vegetable crops in China. Currently, 17 CsSWEET protein gene family members are identified in cucumber, but until now, there is no report that the CsSWEET protein gene family members of cucumber are involved in sugar supply in the pollen development process. Therefore, the function of the cucumber CsSWEET protein gene in regulating pollen fertility is revealed to have important significance for high quality and high yield of cucumber seeds and induction of male sterile lines for hybrid seed production.
Disclosure of Invention
The invention aims to solve the technical problems that: how to regulate plant fertility, for example how to regulate pollen fertility of cucumber.
To solve the technical problem, in a first aspect, the present invention provides the use of a CsSWEET5a protein or a substance regulating the expression of a gene encoding the CsSWEET5a protein or a substance regulating the activity or content of the CsSWEET5a protein in any one of the following,
a1 Use in regulating plant fertility;
a2 The application of the plant fertility regulator in the preparation of products for regulating and controlling plant fertility;
a3 Application in regulating plant pollen fertility;
a4 The application of the plant pollen fertility regulator in preparing a product for regulating and controlling plant pollen fertility;
a5 Use in plant breeding or plant assisted breeding;
a6 Use in the preparation of a product for plant breeding or plant-assisted breeding;
the CsSWEET5a protein may be any one of the following a 1) to a 3):
a1 A protein having an amino acid sequence of SEQ ID No. 2;
a2 A protein related to plant fertility obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No. 2;
a3 A) a protein which has 80% or more identity with the protein represented by a 1) or a 2) and which is related to plant fertility.
In the present invention, the plant breeding or the index of auxiliary breeding includes pollen fertility.
The purpose of the plant breeding or assisted breeding may be to obtain plants with increased fertility.
In particular, the object of the plant breeding or assisted breeding may be to obtain plants with increased pollen fertility.
In the present invention, the improvement in pollen fertility may be specifically expressed as an improvement in pollen viability.
In the present invention, the improvement in pollen fertility may be specifically expressed as an increase in the length of the seed pod.
In the present invention, the improvement of pollen fertility may be specifically expressed as an increase in seed grain number.
In the present invention, the CsSWEET5a protein may be derived from cucumber.
In the present invention, SEQ ID No.2 consists of 238 amino acid residues.
The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.
The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.
Further, in the application, the regulating plant fertility may be improving plant fertility, the regulating CsSWEET5a protein encoding gene expression may be promoting the CsSWEET5a protein encoding gene expression, and the regulating CsSWEET5a protein activity or content may be promoting or improving the CsSWEET5a protein activity or content.
Further, in the application, the substance that promotes or increases the expression of the CsSWEET5a protein-encoding gene is a biological material, and the biological material may be any one of the following:
b1 A nucleic acid molecule encoding the CsSWEET5a protein;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
b5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;
b6 A) a transgenic plant tissue comprising the nucleic acid molecule of B1), or a transgenic plant tissue comprising the expression cassette of B2), or a transgenic plant tissue comprising the recombinant vector of B3);
b7 A transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2), or a transgenic plant organ containing the recombinant vector of B3).
Further, in the application, the nucleic acid molecule encoding the CsSWEET5a protein may be a DNA molecule as set forth in any one of the following:
b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;
b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;
b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
In the present invention, identity refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the present invention, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Further, in the application, the plant may be any of the following:
p1), cucurbitaceae plants;
p2), cucumis plants;
p3), cucumber (culumis sativus l.);
p4), crucifers;
p5), arabidopsis plants
P6), arabidopsis thaliana (Arabidopsis thaliana (l.) heynh.).
The Arabidopsis thaliana may be an Arabidopsis thaliana attunet 8 mutant.
The arabidopsis thaliana attunet 8 mutant may be an atspeet 8 knockout mutant.
In the above-mentioned related biological materials, the expression cassette of B2) refers to DNA capable of expressing the CsSWEET5a protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the CsSWEET5a gene, but also a terminator for terminating transcription of the CsSWEET5a gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock starterA mover (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed specific promoters such as the millet seed specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein specific promoters (e.g., phaseolin, napin, oleosin and soybean beta-glucose promoters (Beachy et al (1985) EMBO J.4:3047-3053) which may be used alone or in combination with other plant promoters all references cited herein are incorporated by reference in their entirety suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, tml terminator, the pea rbcS E9 terminator and the nopaline and octopine synthase terminators (see, e.g., odell et al (I) 985 ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).
In the above related biological material, B3) the recombinant vector may contain a DNA molecule for encoding CsSWEET5a protein shown in SEQ ID No. 1.
The recombinant vector containing the CsSWEET5a protein coding gene expression cassette can be constructed by using a plant expression vector.
Of the above-mentioned related biological materials, the recombinant microorganism of B4) may be specifically yeasts, bacteria, algae and fungi.
In the above related biological materials, the plant tissue of B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
In the above related biological material, the transgenic plant organ of B7) may be the root, stem, leaf, flower, fruit and seed of the transgenic plant.
Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation material.
To solve the above technical problem, in a second aspect, the present invention provides a method for regulating fertility of a plant, the method comprising regulating expression of a gene encoding the CsSWEET5a protein in a recipient plant or regulating activity or content of the CsSWEET5a protein in a recipient plant.
Further, in the method, the regulating plant fertility may be improving plant fertility, and the regulating expression of a gene encoding CsSWEET5a protein or regulating activity or content of CsSWEET5a protein may be promoting expression of a gene encoding the CsSWEET5a protein in a recipient plant or promoting or improving activity or content of the CsSWEET5a protein in a recipient plant.
Further, in the method, the method comprises introducing into the recipient plant a coding sequence of the CsSWEET5a protein, which may be a DNA molecule as set forth in any one of the following:
b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;
b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;
b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
In order to solve the above technical problems, in a third aspect, the present invention provides a method for preparing a plant with improved fertility, the method comprising introducing a CsSWEET5a protein coding sequence into a recipient plant to obtain a plant of interest with improved fertility, wherein the CsSWEET5a protein coding sequence may be a DNA molecule as shown in any one of the following:
b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;
b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;
b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
In order to solve the above technical problems, in a fourth aspect, the present invention provides the CsSWEET5a protein in the above application or the biological material in the above application.
The beneficial technical effects obtained by the invention are as follows:
1. the invention proves that the CsSWEET5a protein has the function of regulating and controlling the plant pollen fertility by introducing the coding gene of the CsSWEET5a protein into the Arabidopsis thaliana. Specifically, csSWEET5a protein positively regulates plant pollen fertility.
2. The invention reveals the function of CsSWEET5a protein for regulating plant pollen fertility for the first time, and provides technical support for cucumber breeding or auxiliary breeding.
Drawings
FIG. 1 shows the analysis of the expression level of CsSWEET5a gene at different tissue parts of cucumber, different development stages of male flower buds and different parts of male flower buds. A is the expression level of CsSWEET5a gene in different tissue parts of cucumber. B is a morphological diagram of male flower buds of cucumbers in different development stages. Ruler = 5mm. C is the expression level of CsSWEET5a gene in male flower buds of cucumber at different development stages. D is the expression level of CsSWEET5a gene in different parts of male flower buds of cucumber in period 11, period 12 and period 13.
FIG. 2 is a cucumber CsSWEET5a subcellular localization analysis. The first row in the figure is tobacco epidermal cells transformed with empty vector pX-eyfp_gw; the second row is tobacco epidermal cells transformed with CsSWEET5a-YFP recombinant vector and CD3-1007 (plasma membrane marker).
FIG. 3 shows semi-quantitative RT-PCR identification of cucumber CsSWEET5a to Arabidopsis atssweet 8 mutant plants. WT: columbia wild type Arabidopsis thaliana; attsweet 8: arabidopsis thaliana attsweet 8 mutant plants; 1-17: arabidopsis thaliana attunet 8 mutant strain 1-17 transformed with CsSWEET5a.
FIG. 4 shows the pollen viability assay of cucumber CsSWEET5a to Arabidopsis attsweet 8 mutant plants. attsweet 8: arabidopsis thaliana attsweet 8 mutant plants; csSWEET5 a/attsweet 8-OE3 and CsSWEET5 a/attsweet 8-OE6: t of Arabidopsis atssweet 8 mutant plants lines 3 and 6 transformed with CsSWEET5a 3 Generating plants; WT: columbia wild type Arabidopsis thaliana. Scale = 100 μm.
FIG. 5 shows the pod phenotype of cucumber CsSWEET5a to Arabidopsis attsweet 8 mutant plants. A is a morphological diagram of seed pods on the main stems of each strain. Scale = 1cm. B is the total length of the first 40 seed pods on the main stems of each plant systemDegree. C is the total number of seeds of the first 40 pods on the main stems of each strain. The different letters (a-C) above the bar graphs in B and C represent the difference significance (P<0.01). attsweet 8: arabidopsis thaliana attsweet 8 mutant plants; csSWEET5 a/attsweet 8-OE3 and CsSWEET5 a/attsweet 8-OE6: t of Arabidopsis atssweet 8 mutant plants lines 3 and 6 transformed with CsSWEET5a 3 Generating plants; WT: columbia wild type Arabidopsis thaliana.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative tests in the examples below were repeated three times, and the results averaged, unless otherwise specified.
Example 1, acquisition of cucumber CsSWEET5a Gene and its encoded protein
1. Material
The tested cucumber material is a C49 inbred line, and is provided by a cucumber breeding subject group of vegetable institute of the national academy of sciences of agriculture and forestry in Beijing, and the material is hermaphrodite and heteroflower. The cucumber seeds are sown in a mixed matrix filled with turf and vermiculite (turf: vermiculite=2:1, volume ratio), and when seedlings grow to three leaves and one heart, the seedlings are planted in a sunlight greenhouse, and conventional cultivation management is performed. After the plants bloom and bear fruits, taking male flower buds, quick-freezing with liquid nitrogen, and storing in a refrigerator at-80 ℃ for standby.
2. Extraction of Total RNA and cDNA Synthesis
(1) Extraction of Total RNA
Total RNA of male flower buds is extracted by using a rapid universal plant RNA extraction kit (purchased from Beijing Hua Vietnam Biotechnology Co., ltd., product No. 0416-50 gk), DNA remained in the RNA sample is removed by DNase I (purchased from Tiangen Biotechnology (Beijing) Co., ltd., product No. RT 411), and the concentration of RNA is measured by using Nanodrop, and the specific operation steps are referred to the kit instruction.
(2) Synthesis of first strand cDNA
The first strand of cDNA was synthesized using a reverse transcription kit (available from Bao Ri doctor materials technology (Beijing) Co., ltd., cat# 6110A) using 1. Mu.g of cucumber male bud RNA as a template (obtained in step 1), and the specific procedure was as described in the kit. The cDNA first strand is synthesized and then stored at-20 ℃ for later use.
3. Cloning of CsSWEET5a Gene
PCR amplification was performed using cucumber male bud cDNA (obtained in step 2) as a template, and the forward primer QC-CsSWEET5a-F and the reverse primer QC-CsSWEET5 a-R. The PCR reaction system is as follows: primeSTAR Max DNA Polymerase (available from Bao Ri doctor technology (Beijing), cat# R045A) 10. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M concentration), 1. Mu.L of template (cucumber male bud cDNA (obtained in step 2)), ddH 2 O was replenished to 20. Mu.L. The PCR conditions were as follows: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, renaturation at 56℃for 5s, extension at 72℃for 30s for 30 cycles, and extension at 72℃for 10min to give 1139bp PCR product. Sequencing the PCR product, wherein the sequencing result shows that the PCR product is a DNA molecule with a nucleotide sequence of SEQ ID No.1, the DNA molecule is named as CsSWEET5a gene, and the open reading frame of the CsSWEET5a gene is the 176 th-892 th site of the SEQ ID No. 1; the protein encoded by the CsSWEET5a gene is designated CsSWEET5a protein, and the amino acid sequence of the CsSWEET5a protein is SEQ ID No.2.
Wherein the nucleotide sequences of the forward primer and the reverse primer are as follows:
QC-CsSWEET5a-F：5’-ATCTCAAAAATATTTATTAAAAAAAACCCAC-3’；
QC-CsSWEET5a-R：5’-AAATGAAGTAAGACTATATTTCAAAAAGAAT-3’。
example 2 expression level of cucumber CsSWEET5a Gene in different tissues, different stages of Male flower bud development and different parts of Male flower bud
In order to understand the expression condition of the CsSWEET5a gene in each tissue part of the cucumber, qRT-PCR analysis is carried out on each tissue part of the cucumber, and the method is specifically as follows:
1. material
The following materials were taken from cucumber C49 plants:
different tissue sites: female flowers (day of flowering, FF), mature leaves (L), male flowers (day of flowering (instant 13), MF), ovary (fruit 2 days before flowers, O), fruit 3 days after flowers (3 DAA), fruit 6 days after flowers (6 DAA), root (R), stem (S) and tendril (T) on the same cucumber plant.
Different developmental stages of male flower buds: male flowers at developmental stages 9, 10, 11, 12 and 13 (i.e. day of flowering).
Different parts of male flower buds: the male flowers at developmental stages 11, 12 and 13 (day of flowering) were divided into four parts, petals, sepals, anthers and pollen.
Each of the above samples was taken in 3 biological replicates.
2. qRT-PCR detection of CsSWEET5a Gene expression level
Extracting total RNA of the materials, synthesizing first-strand cDNA, detecting the expression condition of CsSWEET5a gene by adopting a qRT-PCR method (taking an action gene as an internal reference gene), detecting the expression of the CsSWEET5a gene of the cucumber by adopting a primer pair consisting of a primer qCsSWEET5a-F and a primer qCsSWEET5a-R, and detecting the expression of the CsActin gene of the cucumber by adopting a primer pair consisting of the primer qCsActin-F and the primer qCsActin-R.
qCsSWEET5a-F：5’-AGTCATCAAAACAAGGAGTGTGAA-3’
qCsSWEET5a-R：5’-GACGTTAGGATCGAACTTGAGAAT-3’
qCsActin-F：5’-CCACGAAACTACTTACAACTCCATC-3’
qCsActin-R：5’-GGGCTGTGATTTCCTTGCTC-3’
The results are shown in FIG. 1. The results showed that CsSWEET5a gene was specifically expressed in male flowers (day of flowering, i.e., period 13), and the expression levels in other parts (female flowers, leaves, ovaries, fruits, roots, stems and tendrils) were significantly lower than those in male flowers or undetectable (fig. 1 a). To further understand the expression change of CsSWEET5a gene in the development process of male flowers of cucumber, male flowers from the 9 th period to the 13 th period (day of flowering) (fig. 1B) were taken for expression analysis, and the result shows that the expression level of CsSWEET5a gene increases rapidly with the development of male flowers, reaches the highest value in the 11 th period, remains at a higher level in the 12 th period, and decreases rapidly from the 13 th period (fig. 1C), but the expression level of CsSWEET5a gene in the male flowers from the 13 th period is still much higher than that of female flowers, leaves, ovaries, fruits, roots, stems, tendrils and other tissue sites (fig. 1A). Next we sub-divided the male flowers at 11, 12 and 13 stages and analyzed the expression of CsSWEET5a gene in petals, sepals, anthers and pollen, which indicated that CsSWEET5a was expressed very high in anthers, especially pollen, while the expression in petals and sepals was relatively reduced (D in fig. 1). The above results indicate that CsSWEET5a gene specificity is expressed in cucumber male flower buds, especially highly expressed in anthers and pollen of male flower buds.
EXAMPLE 3 subcellular localization analysis of the cucumber CsSWEET5a Gene in tobacco leaf epidermal cells
Amplifying an open reading frame sequence (without a stop codon, SEQ ID No. 3) of a CsSWEET5a gene by using a primer pair consisting of a primer YFP-CsSWEET5a-F and a primer YFP-CsSWEET5a-R by using cucumber male flower bud cDNA as a template, and introducing attB sites at two ends of the SEQ ID No. 3; SEQ ID No.3 containing the attB site was cloned into the Gateway donor vector pDONR221 (available from Invitrogen, cat. No. 12536017) by a Gateway BP reaction; after sequencing confirmation, SEQ ID No.3 was transferred into a target vector pX-EYFP_GW (benefit of the national academy of sciences and forestry, beijing, academy of agricultural and forestry, ren Yi, reference Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon, parts. The Plant Cell,2021, 33:1554-1573) by using Gateway LR reaction to obtain a recombinant vector CsSWEET5a-YFP. Plasma membrane position was indicated using empty vector pX-eyfp_gw as negative control and CD3-1007 (professor to the dawn bud of the university of china agriculture, ref Hexose transporter CsSWEET a in cucumber mediates phloem unloading in companion cells for fruit development.plant Physiology,2021, 186:640-654) as marker. pX-EYFP_GW, CD3-1007 and CsSWEET5a-YFP were transferred to Agrobacterium GV3101 (available from Beijing Bomaide Gene technologies Co., ltd., product number BC 304-01), respectively, and injected into tobacco leaves of 3 to 4 weeks old. After 3 days of transformation, the expression of the CsSWEET5a-YFP fusion protein was observed using a laser confocal microscope (Leica TCS SP 8) and the results are shown in FIG. 2. In the figure, YFP is green fluorescence, plasma membrane marker (CD 3-1007) is red fluorescence, chloroplast autofluorescence is blue, and Merge is a superposition result, and the result shows that CsSWEET5a is positioned on the plasma membrane.
The nucleotide sequences of the primers YFP-CsSWEET5a-F and YFP-CsSWEET5a-R are as follows:
YFP-CsSWEET5a-F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGTGAATACAGAGACAGCAAGAA-3’；
YFP-CsSWEET5a-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTACACATTATCTGTCATTTGCACCT-3’。
example 4 functional study of cucumber CsSWEET5a Gene
1. Construction of the overexpression vector
Amplifying an open reading frame of a CsSWEET5a gene by using a cucumber male flower bud cDNA as a template and a primer pair consisting of primers O-CsSWEET5a-F and primers O-CsSWEET5a-R, introducing attB sites at two ends, wherein an amplified product is a DNA molecule with a nucleotide sequence of SEQ ID No. 4; SEQ ID No.4 shows attB1 site sequence at positions 1-25, csSWEET5a gene open reading frame sequence at positions 28-744, attB2 site sequence at positions 746-770. The open reading frame of CsSWEET5a gene containing attB site (SEQ ID No.4, positions 28-744) was cloned into Gateway donor vector pDONR221 (from Invitrogen, cat. No. 12536017) by Gateway BP reaction; after sequencing and confirmation, the open reading frame (SEQ ID No.4, positions 28-744) of the CsSWEET5a gene is transferred into a destination vector pMDC32 (purchased from ABRC and having the product number of CD 3-738) of a destination vector to obtain a recombinant vector 35S:: csSWEET5a. The structure of the recombinant vector 35S is that CsSWEET5a is as follows: the DNA fragment between attR1 and attR2 of vector pMDC32 (small fragment between attR1 and attR 2) was replaced with the DNA molecule shown in SEQ ID No.4, and the other nucleotide sequences of vector pMDC32 were kept unchanged to obtain a recombinant expression vector. Recombinant vector 35S. CsSWEET5a contains the open reading frame of the CsSWEET5a gene (SEQ ID No.4, positions 28-744), transcription of the open reading frame of the CsSWEET5a gene is driven by the 35S promoter. The recombinant vector was transferred into Agrobacterium GV3101 (Beijing Bomaide Gene technology Co., ltd., accession number BC 304-01).
O-CsSWEET5a-F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGTGAATACAGAGACAGCAAGAA-3’；
O-CsSWEET5a-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTACTACACATTATCTGTCATTTGCACCT-3’。
2. Obtaining transgenic Arabidopsis thaliana
The overexpression vector 35S was transformed by the dip-in method with CsSWEET5a into the Arabidopsis attsweet 8 mutant (available from ABRC under the accession number SALK_ 142803C) and T harvested from the infected Arabidopsis thaliana 0 Seed generation. Will T 0 The arabidopsis seeds of the CsSWEET5a gene are uniformly broadcast on a 1/2MS solid culture medium containing 25 mug/mL hygromycin for screening after being disinfected, and the arabidopsis plants which can normally grow on the hygromycin resistant culture medium are transplanted into a nutrition pot for culturing to form seedlings, thus obtaining 17T 0 Arabidopsis thaliana transformed with CsSWEET5a gene.
RNA of Arabidopsis thaliana Columbia Wild Type (WT), attunet 8 mutant and 17 transformants (CsSWEET 5 a/attunet 8-OE) were extracted, respectively, and first-strand cDNA was synthesized, and the expression levels of AtSWEET8 and CsSWEET5a were detected by RT-PCR. The Arabidopsis internal reference gene selects AtActin, and the primers are adopted as follows:
AtSWEET8-F:5’-ATGGTTGATGCAAAACAAGTTCG-3’
AtSWEET8-R:5’-CTAAACCCTCTCCGTAGCAGAAATC-3’
CsSWEET5a-F:5’-ATGGTGAATACAGAGACAGCAAGAA-3’
CsSWEET5a-R:5’-CTACACATTATCTGTCATTTGCACCT-3’
AtActin-F:5’-TCCAAGCTGTTCTCTCCTTG-3’
AtActin-R:5’-GAGGGCTGGAACAAGACTTC-3’
the results are shown in FIG. 3. CsSWEET5a is a gene in cucumber, which is not expressed in the wild type of Arabidopsis thaliana and in the control plant of the atssweet 8 mutant which is not transformed with CsSWEET5a gene, and is heterologously expressed in 17 Arabidopsis thaliana atssweet 8 mutant plants which are transformed with CsSWEET5a gene, wherein the expression level of CsSWEET5a gene is the most in the strain 3 and the strain 6High. Seeds of the 2 transgenic lines are harvested by a single plant, and sowing and screening are continued until T is obtained 3 The generation homozygous lines were used for subsequent experiments, both T 3 The generation lines were named CsSWEET5 a/attheat 8-OE3 and CsSWEET5 a/attheat 8-OE6, respectively.
3. Pollen fertility detection of transgenic arabidopsis plants
Seed of WT Arabidopsis thaliana, attunet 8 mutant Arabidopsis thaliana, csSWEET5 a/attunet 8-OE3 and CsSWEET5 a/attunet 8-OE6 were sterilized and then sown in 1/2MS solid medium, and after 3 days of treatment at 4℃they were placed in an illumination incubator under the following conditions: 16h light/8 h dark, light intensity 125 mu mol m -2 s -2 The temperature was 22 ℃. After 7-10 days, 50 plants are planted in each plant line, the plants continue to grow in an illumination incubator, and pollen fertility detection is carried out after bolting of the plants.
Pollen viability detection: flowers 50 on the day of flowering (from main stem section 10-20) were taken, anthers were removed under the stereoscope and placed on a glass slide, 1-2 drops of 1% triphenyltetrazolium chloride (TTC) solution (0.2 g TTC,12g sucrose dissolved in 20mL distilled water. TTC was purchased from Sigma Co., cat. No. T8877; sucrose was purchased from Sigma Co., cat. No. V900116) were added dropwise, and after 2 hours at room temperature, observed using a Zeiss optical microscope (Zeiss Axio Imager Z2). Viable pollen may be colored red or pale red and non-viable pollen may not be colored. The results are shown in FIG. 4, and indicate that overexpression of the CsSWEET5a gene in the attunet 8 mutant Arabidopsis provides a significant increase in pollen viability in the attunet 8 mutant plants.
Pod phenotype observation: 1 plant was randomly selected from each line, the main stem was removed, and a digital camera was used to photograph. In addition, 10 plants were randomly selected for each line, the lengths of the first 40 pods on the main stems of the plants were measured with a vernier caliper, the seed numbers contained in the pods were recorded, and the total length and total number of seeds of the first 40 pods on the main stems of the plants were counted. The results are shown in FIG. 5, and demonstrate that overexpression of the CsSWEET5a gene in the attunet 8 mutant Arabidopsis significantly increases pod length and seed yield in the attunet 8 mutant plants. FIGS. 4 and 5 illustrate that CsSWEET5a can regulate plant pollen fertility.
Table 1 sequences of SEQ ID Nos. 1-4 in the examples
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
- The use of a CsSWEET5a protein or a substance regulating the expression of a gene encoding said CsSWEET5a protein or a substance regulating the activity or content of said CsSWEET5a protein,a1 Use in regulating plant fertility;a2 The application of the plant fertility regulator in the preparation of products for regulating and controlling plant fertility;a3 Application in regulating plant pollen fertility;a4 The application of the plant pollen fertility regulator in preparing a product for regulating and controlling plant pollen fertility;a5 Use in plant breeding or plant assisted breeding;a6 Use in the preparation of a product for plant breeding or plant-assisted breeding; the CsSWEET5a protein is any one of the following a 1) to a 3):a1 A protein having an amino acid sequence of SEQ ID No. 2;a2 A protein related to plant fertility obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No. 2;a3 A) a protein which has 80% or more identity with the protein represented by a 1) or a 2) and which is related to plant fertility.
- 2. The use according to claim 1, characterized in that: the regulation of plant fertility is to improve plant fertility, the regulation of CsSWEET5a protein coding gene expression is to promote the CsSWEET5a protein coding gene expression, and the regulation of CsSWEET5a protein activity or content is to promote or improve the CsSWEET5a protein activity or content.
- 3. Use according to claim 1 or 2, characterized in that: the substance that promotes or increases the expression of the CsSWEET5a protein-encoding gene of claim 1 is a biological material, which is any one of the following:b1 A nucleic acid molecule encoding the CsSWEET5a protein;b2 An expression cassette comprising the nucleic acid molecule of B1);b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);b5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;b6 A) a transgenic plant tissue comprising the nucleic acid molecule of B1), or a transgenic plant tissue comprising the expression cassette of B2), or a transgenic plant tissue comprising the recombinant vector of B3);b7 A transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2), or a transgenic plant organ containing the recombinant vector of B3).
- 4. A use according to claim 3, characterized in that: the nucleic acid molecule encoding the CsSWEET5a protein is a DNA molecule as set forth in any one of the following:b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
- 5. The use according to claim 1, characterized in that: the plant is any one of the following:p1), cucurbitaceae plants;p2), cucumis plants;p3), cucumber (culumis sativus l.);p4), crucifers;p5), arabidopsis plantsP6), arabidopsis thaliana (Arabidopsis thaliana (l.) heynh.).
- 6. A method for regulating plant fertility, comprising: the methods comprise modulating expression of a gene encoding the CsSWEET5a protein in a recipient plant or modulating activity or content of the CsSWEET5a protein in a recipient plant.
- 7. The method according to claim 6, wherein: the regulation of plant fertility is to improve plant fertility, and the regulation of the expression of a gene encoding CsSWEET5a protein or the regulation of the activity or content of CsSWEET5a protein is to promote the expression of a gene encoding CsSWEET5a protein in a recipient plant or to promote or improve the activity or content of CsSWEET5a protein in the recipient plant.
- 8. The method according to claim 7, wherein: the method comprises introducing into the recipient plant a gene encoding the CsSWEET5a protein, wherein the gene encoding the CsSWEET5a protein is a DNA molecule as shown in any one of the following:b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
- 9. A method of preparing a plant with increased fertility, characterized by: the method comprises the steps of introducing a CsSWEET5a protein coding gene into a receptor plant to obtain a target plant with improved fertility, wherein the CsSWEET5a protein coding gene is a DNA molecule shown in any one of the following:b1 A DNA molecule with a coding chain shown as SEQ ID No. 1;b2 A DNA molecule with the coding sequence of the coding strand shown in the 176 th to 892 th positions of SEQ ID No. 1;b3 A DNA molecule which has more than 80% identity with the DNA molecule described in b 1) or b 2) and which encodes the same functional protein.
- 10. The CsSWEET5a protein in the use of claim 1 or the biomaterial in the use of claim 2.
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2023
- 2023-11-30 CN CN202311620428.1A patent/CN117447574A/en active Pending
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