CN108467868B

CN108467868B - Application of soybean sucrose transporter important gene GmSWEET6

Info

Publication number: CN108467868B
Application number: CN201810441170.1A
Authority: CN
Inventors: 王秀荣; 陈阿; 赵少鹏; 陈康
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-05-10
Filing date: 2018-05-10
Publication date: 2021-02-19
Anticipated expiration: 2038-05-10
Also published as: CN108467868A

Abstract

The invention discloses an application of a soybean sucrose transporter important gene GmSWEET6, and relates to the field of plant genetic engineering and biotechnology. The invention identifies a sucrose transporter gene GmSWEET6 induced and expressed by mycorrhiza in soybean by a real-time fluorescent quantitative PCR method, and the expression of the gene is regulated and controlled by the infection of mycorrhizal fungi. Under the condition of mycorrhizal fungi inoculation, the transgenic soybean plant with the excessive or interference expression GmSWEET6 has the effects of regulating and controlling plant growth and phosphorus absorption, and has obvious influence on the beneficial symbiosis of soybean and arbuscular mycorrhizal fungi, so that the method has important significance for clarifying the biological function of a SWEET gene in the symbiosis of leguminous crops and mycorrhizal fungi, and further regulating and controlling the beneficial symbiosis between plants and mycorrhizal fungi.

Description

Application of soybean sucrose transporter important gene GmSWEET6

Technical Field

The invention relates to the field of plant genetic engineering and biotechnology, in particular to application of an important gene GmSWEET6 of a soybean sucrose transporter.

Background

Higher plants are autotrophic organisms consisting of autotrophic and heterotrophic organs. Heterotrophic organs require the photosynthetic production of autotrophic organs to sustain growth and development. 80% of photosynthetic assimilate synthesized in the 'source' organs such as mature leaves of plants is transported to the 'sink' organs such as roots, flowers and fruits through a plant vascular system. Sucrose is one of green plant photosynthetic assimilates, and is the main form of transport and distribution of photosynthetic products in the bodies of most higher plants (Yangshang et al, 2005; white plum et al, 2006). The mode of directional transportation and distribution of sucrose in plants not only regulates the whole growth and development process of plants, but also determines the yield and quality of crops. Sweet (sugar wil eventualy be Exported transporters) as a new sugar transporter, has the function of sucrose and monosaccharide efflux, which may be the key point for the unloading of sucrose from phloem to the root and other reservoir organs. Numerous studies have shown that SWEET genes are involved in a variety of physiological processes, including phloem loading, influencing reproductive development, participating in disease and stress responses of host-pathogen interactions, ion transport, and the like. MtSWEET11 that has been reported to be associated with rhizobium symbiosis; the expression of 12 StSWEETs genes in potatoes is up-regulated after inoculation of arbuscular mycorrhizal fungi, which indicates that SWEET transport proteins are probably involved in the process of sugar efflux from sucrose to symbiotic interfaces.

Arbuscular mycorrhizal Fungi (amb) form a symbiotic relationship with plants 4 hundred 5 million years ago (Wang et al, 2006). Now, more than about 80% of terrestrial plants can form a symbiotic system with the AM fungus (Smith et al, 2008). Spores of the AM fungus germinate in soil, hyphae invade into the root system of the plant to cortical cells, and then form an arbuscular structure. The developed extra-root hypha of the AM fungi can reach the area which can not be contacted with the root system of the plant to absorb nutrient substances (especially phosphorus) and is transferred to the plant through the mycorrhizal path of a symbiotic system, so that the nutrient efficiency of the host is improved. Moreover, the formation of a symbiotic system can also effectively relieve the poison of heavy metals such as aluminum, manganese and the like, and improve the capability of the plant to resist abiotic stress. In return, plant material provides a carbon source for AM fungi, including sugars, for the normal development and life cycle of the basic structures of the fungi, such as their rosettes, hyphae, spores and vesicles (Varma et al, 1995; Read et al, 1997). Thus, the sucrose transporter must also play a crucial role in the symbiotic system.

Soybean is an important grain and oil crop in China. Soybeans cultivated in all countries in the world are directly or indirectly transmitted from China (how to go, 2009). A total of 53 members of the gmsheets gene family are currently identified in soybean (Patil et al, 2015). To date, no mycorrhiza-induced SWEET transporter responsible for the export of sucrose to the symbiotic interface has been found in mycorrhizal plants, and no relevant studies on mycorrhizal-induced SWEET transporter have been found in soybean.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the soybean sucrose transporter important gene GmSWEET 6.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of a soybean sucrose transporter important gene GmSWEET6 in mycorrhizal symbiosis.

The nucleotide sequence of the soybean sucrose transporter important gene GmSWEET6 is shown in Glyma.04G198600, and the amino acid sequence of the protein coded by the GmSWEET6 gene is shown in Glyma.04G198600.

The invention identifies a sucrose transporter gene GmSWEET6 induced and expressed by mycorrhiza in soybean by a real-time fluorescent quantitative PCR method, and the expression of the gene is regulated and controlled by the infection of mycorrhizal fungi. Under the condition of mycorrhizal fungi inoculation, the transgenic soybean plant with the excessive or interference expression GmSWEET6 has the functions of regulating and controlling the plant growth and phosphorus absorption, and has important significance for regulating and controlling the beneficial symbiosis between plants and mycorrhizal fungi.

The invention also provides application of the soybean sucrose transporter important gene GmSWEET6 in plant breeding, and further application in cultivation of transgenic plants.

The plant is a dicotyledonous leguminous plant.

The dicotyledonous leguminous plant is soybean.

The gene GmSWEET6 and the protein provided by the invention can regulate the growth and phosphorus absorption of a transgenic plant containing the gene GmSWEET 6.

The primer pair for amplifying the whole length of the GmSWEET6 gene or any fragment thereof belongs to the protection scope of the invention.

The invention also provides a recombinant expression vector containing the GmSWEET6 gene, and the recombinant expression vector containing the GmSWEET6 gene can be constructed by using the existing plant expression vector. Such plant expression vectors include binary agrobacterium vectors and the like, such as ptf101.1(Wang et al, 2009) or other derived plant expression vectors.

The invention also provides a genetic engineering bacterium containing the recombinant expression vector.

The invention also relates to a cell which contains the GmSWEET6 gene or the recombinant expression vector. The cell may be a plant cell, such as a leguminous plant cell, or a microbial cell, such as a bacterial or fungal cell. The cell may be isolated, ex vivo, cultured, or part of a plant.

The invention also relates to plants or plant parts, plant material, plant seeds, which comprise the cells of the invention. The plant may be a leguminous plant, such as beans and soybeans, but also other plants, such as monocotyledonous plants like rice, wheat, barley, maize, sorghum, sugarcane, oats, rye, etc., or other dicotyledonous plants like tobacco, sunflower, sugar beet, capsicum, potato, tomato, etc. Also relates to transgenic seeds derived from said plants.

The present invention also relates to a method of producing a plant, the method comprising: regenerating a transgenic plant from a plant cell of the invention, or crossing a plant of the invention with another plant.

The invention also relates to plants produced by the methods of the invention.

The invention also provides application of the soybean sucrose transporter important gene GmSWEET6 in regulation and control of formation of beneficial symbiotic relationship between plants and mycorrhizal fungi; further, the application in the preparation of a symbiotic preparation for regulating and controlling plants and mycorrhizal fungi.

The plant is a dicotyledonous leguminous plant.

The dicotyledonous leguminous plant is soybean.

The invention also relates to application of the GmSWEET6 gene or the recombinant vector in regulation and control of formation of beneficial symbiotic relationship between plants and mycorrhizal fungi, which comprises preparation of transgenic plants and preparation of a preparation for changing formation of beneficial symbiotic relationship between plants and mycorrhizal fungi.

The invention also relates to a method for regulating the beneficial symbiotic relationship between plants and mycorrhizal fungi, which comprises preparing plants containing the GmSWEET6 gene or the recombinant vector. For example, the method may comprise regenerating a transgenic plant from a plant cell of the invention or crossing a plant of the invention with another plant.

The invention provides a preferred embodiment, the gene GmSWEET6 is introduced into a root system induced by soybean hypocotyl injection to obtain a transgenic plant line; the growth of the transgenic lines, including biomass and phosphorus content, changed significantly.

The gene GmSWEET6 can be introduced into a receptor soybean hypocotyl root system through the recombinant expression vector.

The plant expression vector carrying the gene GmSWEET6 of the invention can be transformed into soybean hypocotyl root system by, for example, Agrobacterium-mediated transformation.

Compared with the prior art, the invention has the following advantages and effects:

the gene GmSWEET6 belongs to the SWEET gene family which is cloned and reported in Arabidopsis thaliana, alfalfa and the like, but the biological functions of the gene in the beneficial symbiosis of leguminous crops and arbuscular mycorrhizal fungi are not clear. The gene GmSWEET6 has obvious influence on the beneficial symbiosis of soybean and arbuscular mycorrhizal fungi, and has important significance for clarifying the biological function of the SWEET gene in the symbiosis of leguminous crops and mycorrhizal fungi so as to regulate and control the beneficial symbiosis between plants and mycorrhizal fungi.

Drawings

FIG. 1 is the response of GmSWEETs family genes to AM fungal infestation at roots; the plants were cultivated in sand and soil, and two different soybean genotypes BD2 and BX10 were selected and cultivated in a low phosphorus (LP, 50 μ M KH)₂PO₄) Or high phosphorus (HP, 500. mu.M KH)₂PO₄) Under the treatment condition, the plant is harvested without inoculation (NM) or after inoculation with arbuscular mycorrhizal fungi Ri 45 d. Ri: rhizophagus irregularis. a-h: expression level of GmSWEETs gene in roots; data in the figure are mean ± SE of four biological replicates. Asterisks indicate that the difference between inoculated and non-inoculated plants was significant for the same genotype and the same phosphorus treatment.

FIG. 2 is a co-localization analysis of GUS expression driven by the GmSWEET6 promoter and mycorrhizal structures; wherein, FIG. 2a shows the expression of GUS driven by GmSWEET6 promoter, and FIG. 2b shows the fluorescence of mycorrhizal structure after WGA488 staining.

FIG. 3 is a subcellular localization analysis of GmSWEET 6; the first row in the figure is tobacco epidermal cells transformed with empty vector, followed by tobacco cells transformed with GFP-GmSWEET6 vector, and the figure is fluorescence co-localization by imaging GFP and membrane localization Marker mCheerry under confocal microscope observation.

FIG. 4 is the expression level detection of transgenic plants expressing GmSWEET6 in excess and interference. An overexpression vector GmSWEET6-OX-pTF101.1s and an interference vector GmSWEET6-pFGC5941 are transferred into a phosphorus high-efficiency variety YC03-3 by adopting an hypocotyl injection method, and low phosphorus (50 mu M KH)₂PO₄) Without inoculation (NM) or with treatment with arbuscular bacteria (Ri). a, b are the data of the gene expression level of the overground part; and c and d are root data. Wherein CK1 represents a control transformed into pTF101.1s empty; 6-OX represents transgenic plants overexpressing GmSWEET 6; CK2 represents the control unloaded into pFGC 5941; 6-RNAi indicates the interference expression of GmSWEET6 transgenic plants. The data in the figure are the mean and standard error of five biological replicates. Indicates that the difference between different transgenic strains is significant under the treatment of the same inoculum (P)<0.05)。

FIG. 5 is a graph showing the effect of excessive and interfering expression of GmSWEET6 on phosphorus content (a, b) and plant growth (c, d) of soybean transgenic plants. An overexpression vector GmSWEET6-OX-pTF101.1s and an interference vector GmSWEET6-pFGC5941 are transferred into a phosphorus high-efficiency variety YC03-3 by adopting an hypocotyl injection method, and low phosphorus (50 mu M KH)₂PO₄) Without inoculation (NM) or with treatment with arbuscular bacteria (Ri). a and b are the phosphorus content of the root; c and d are root biomass. Wherein CK1 represents a control transformed into pTF101.1s empty; 6-OX represents transgenic plants overexpressing GmSWEET 6; CK2 represents the control unloaded into pFGC 5941; 6-RNAi indicates the interference expression of GmSWEET6 transgenic plants. The data in the figure are the mean and standard error of five biological replicates. Indicates that the difference between different transgenic strains is significant under the treatment of the same inoculum (P)<0.05)。

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

Analysis of expression pattern of GmSWEETs gene family in soybean root system: the soybean SWEET gene family has 53 members (Patil et al, 2015), the third 28 members related to sucrose transport and microbial symbiosis are screened as candidate genes according to the evolutionary homologous relationship, and then the expression conditions of the Gmsweet family member genes in different soybean varieties, different inoculation treatments and different phosphorus levels are determined by the quantitative PCR technology.

With 2 soybean varieties, phosphorus efficient brazil 10(BX10) and phosphorus inefficient local 2(BD2), 2 AMF inoculations were set for no inoculation (NM) and inoculation with the AM fungus Rhizophagus irregularalis (Ri), respectively. Two phosphorus treatments were set, 50 μ M KH₂PO₄As Low Phosphorus (LP) and 500 μ M KH₂PO₄As High Phosphorus (HP), low phosphorus treated K⁺By K₂SO₄And (5) filling. The seeds are cultivated in sand by adopting 1/2Hoagland nutrient solution. Red plastic round pots with the specification of 2kg are selected, 1.6kg of quartz sand and 0.2kg of acid soil are filled into each round pot, the sand soil is autoclaved at 121 ℃ for 40min, and the cycle is carried out twice. When the soybean seeds are potted, 0.2kg (10%) of corresponding AMF microbial inoculum or-AMF simulation inoculum is added, the soybean seeds are wetted by 150mL of corresponding nutrient solution after being completely mixed, the soybean seeds are sterilized by a chlorine dry heat sterilization method, 3 seeds are dibbled in each pot, and then 100mL of corresponding nutrient solution is supplemented in each pot. Seedlings were thinned 1 week after emergence and 1 plant was kept per pot. The corresponding (LP/HP) 1/2Hoagland nutrient solution was applied once a week. 4 biological replicates were set for each treatment. The expression pattern of GmSWEETs was further determined by quantitative PCR, after routine manipulation, harvesting after 7 weeks, extraction of aerial and root RNA, reverse transcription into cDNA. The soybean housekeeping gene EF-1a is used as an internal reference. The primers for quantitative PCR detection of gene expression are respectively:

the primers of the soybean EF-1a gene are as follows:

EF-1a F：5′-TGAACCACCCTGGTCAGATT-3′(SEQ ID NO:1)

EF-1a R：5′-TCCAGCATCACCATTCTTCA-3′(SEQ ID NO:2)

the primers of the GmSWEET6 gene are as follows:

GmSWEET6F：5′-CGTTCGGGAGCGTAACATAG-3′(SEQ ID NO:3)

GmSWEET6R：5′-TCGGACCAAAAGCGTAGAGT-3′(SEQ ID NO:4)

other GmSWEETs gene quantitative primer sequences are described in Patil et al (2015).

qPCR reaction system: 10 mu LPowerUp^TM

Green Master Mix，0.6μL Primer R，0.6μL Primer F，2μL cDNA，ddH₂O Up to 20μL。

Wherein the cDNA is diluted by 20 times and used for experiments, cDNA stock solution is randomly taken from each different experimental treatment to prepare standard koji, at least one standard koji is ensured to be mixed in each treatment, and the standard koji is diluted by 5 times in a gradient way.

PCR conditions were 95 ℃ denaturation for 1 min, followed by 95 ℃ denaturation for 15 sec, 60 ℃ annealing and extension for 60 sec, and 40 cycles.

And (3) data analysis: a suitable threshold value is chosen in the ABI Step OnePlus biosystems such that the R of the standard curve²The value of E is between 85% and 110% as much as possible, the CT values of the target gene and the reference gene are automatically calculated by ABI Step One Plus biosystems software according to a standard curve to obtain an expression quantity, and the ratio of the concentration of the target gene to the expression quantity of the reference gene is the relative expression quantity of the target gene. In order to ensure the accuracy of the experimental results, at least 4 biological replicates were set for each treatment, ensuring consistent response trends. The results are shown in FIG. 1 (genes not listed in the figure are not expressed by low phosphorus and/or mycorrhiza induction).

The results show that: GmSWEET6 is strongly induced by mycorrhiza expression, especially under low phosphorus conditions.

The results suggest that GmSWEET6 may play an important role in the beneficial symbiotic relationship of plants with mycorrhizal fungi.

Example 2

1. Cloning of the GmSWEET6 gene: using soybean YC03-3 leaf cDNA as template, and using upstream specific primer 5' -ATCGCCCGGGATGTCGTCCCACAGTCATCTAA-3 '(SEQ ID NO:5) and downstream specific primer 5' -ATCGCCCCGGGTCAAACTTCGCAACTGATCACCC-3' (SEQ ID NO:6), amplifying the ORF full-length sequence 864bp of GmSWEET6 gene, and sequencing and aligning to obtain the coding sequence of GmSWEET6 shown in Glyma.04G198600 and the corresponding protein sequence shown in Glyma.04G198600.

2. Cloning and constructing a vector of a GmSWEET6 gene promoter and analyzing tissue expression positioning: promoter analysis construction of expression vectors: extracting leaf genome DNA of soybean YC03-3 genotype according to conventional method, using soybean leaf genome DNA as template, and using upstream specific primer 5' -CTATGACATGATTACGAATTCCCACCTTGTTATACCTCATT-3 '(SEQ ID NO:7) and downstream specific primer 5' -GACTGACCTACCCGGGGATCCGGAATTTCTCTCTCTCTCTCT-3' (SEQ ID NO:8) amplifies 2176bp fragment of GmSWEET6 promoter, after recovery of PCR fragment, the target vector is subjected to double enzyme digestion by EcoR I and BamH I, and the GmSWEET6 promoter gene is connected to the target vector pTF102 by a one-step cloning method. Coli DH10B, and Agrobacterium tumefaciens EHA101 for Agrobacterium tumefaciens-mediated transformation of whole soybean cotyledonary node strains after sequencing (Wang et al, 2009).

For the tissue expression positioning analysis of GmSWEET6, a sand culture test is adopted, a promoter whole strain is selected to transform a strain proGmSWEET6, the rhizomycorrhizal fungi of the tested root plexus is Ri, and the phosphorus content is 50 mu M KH₂PO₄A nutrient solution. Low phosphorus treated K⁺By K₂SO₄And (5) filling. 10 biological replicates were set up. And (4) performing conventional management, collecting a fresh root sample in a flowering period, cleaning, and performing GUS activity staining. Then, the blue-stained roots were cut into small root segments, embedded in low-melting agar and then longitudinally cut, the infected structure of the AM fungus was stained with WGA488, and then photographed by observation with a fluorescence microscope at the bright field and at the wavelength of 488nm, respectively. The results are shown in FIG. 2. The results show that: GmSWEET6 was induced to express in the arbuscular structure of mycorrhiza.

Example 3

Construction of subcellular localization experiment vector: soybean YC03-3 leaf cDNA is used as a template, an ORF full-length sequence 864bp of the GmSWEET6 gene is amplified by an upstream specific primer 5'-GGGGacaagtttgtacaaaaaagcaggcttcATGTCGTCCCACAGTCATCTAA-3' (SEQ ID NO:9) and a downstream specific primer 5'-GGGGaccactttgtacaagaaagctgggtcTCAAACTTCGCAACTGATCACCC-3' (SEQ ID NO:10), a fragment is recovered by PCR, and the GmSWEET6 gene is connected to a target vector pMDC43 by a Gatway technology. Transforming Escherichia coli DH10B, and transforming Agrobacterium GV3101 after sequencing for subcellular localization experiment.

For the subcellular localization analysis of GmSWEET6, a transient transformation method of tobacco epidermal cells is adopted. Firstly, the GV3101 bacterial liquid fused with GmSWEET6 gene and the GV3101 bacterial liquid transferred into membrane Marker plasmid 1008 are shaken overnight, then centrifuged, and the mixture is treated by using the mixture containing 10mM MES and 10mM MgCl₂And 150 μ M AS in heavy suspension to resuspend the bacteria. Culturing in the dark at 22-24 ℃ for 3-4 h, mixing the two in equal volumes, transforming the mixture into 3-4 weeks old tobacco leaves, and observing fluorescence at 488nm (GFP) and 587nm (mCherry) of laser confocal point after 3 days of transformation. The results of the experiment are shown in FIG. 3. The results show that: GmSWEET6 was localized on the plasma and nuclear membranes.

Example 4

1. Construction of overexpression GmSWEET6 vector: using leaf cDNA of soybean YC03-3 genotype as template, and using upstream specific primer 5' -GCGAGCTCGGTACCCGGGATGTCGTCCCACAGT CATCTAA-3 '(SEQ ID NO:11) and downstream specific primer 5' -CTCTAGAGGATCCCCGGGTCAAACTTCGCAACTGATCACCC-3' (SEQ ID NO:12), amplifying the ORF full-length sequence 864bp of the GmSWEET6 gene, recovering the fragment by PCR, digesting the target vector through Sma I, connecting the GmSWEET6 gene to the target vector pTF101.1s, and obtaining the overexpression vector GmSWEET 6-OX-pTF101.1s. Transforming Escherichia coli DH10B, and transforming Agrobacterium rhizogenes K599 after sequencing without errors, and using the Agrobacterium rhizogenes-mediated soybean hypocotyl injection transformation (Li et al, 2014).

2. Construction of the interference GmSWEET6 vector: using leaf cDNA of soybean YC03-3 genotype as template, and using upstream specific primer 5' -TTACCATGGGGCGCGCCGATATCTATGTTACGCTCCC GA-3 '(SEQ ID NO:13) and downstream specific primer 5' -CATCGATTGGGCGCGCCAACTT CGCAACTGATCACCCTTTC-3' (SEQ ID NO:14), amplifying a 390bp specific target fragment, carrying out enzyme digestion on a target vector by using Asc I, and connecting a forward fragment of a GmSWEET6 gene with a one-step cloning methodAnd (4) receiving a destination vector pFGC 5941. Then the upstream specific primer 5' -GCAGGTATTT is used againGGATCCAACTTCGCAACTGATCACCCTTTC-3 '(SEQ ID NO:15) and downstream specific primer 5' -GACTCACCTAGGATCCGATATCTATGTTACGCTCCCGA-3' (SEQ ID NO:16), amplifying a reverse fragment, digesting a target vector by using BamH I, connecting the reverse fragment to a target vector pFGC5941 containing a forward fragment by using a one-step cloning method to obtain an interference vector GmS WEET6-pFGC5941, transforming escherichia coli DH10B, transforming agrobacterium rhizogenes K599 after sequencing is free, and using the transformed agrobacterium rhizogenes for agrobacterium rhizogenes-mediated soybean hypocotyl injection transformation (Li et al, 2014).

3. Obtaining of soybean transgenic plants: and transforming the constructed excessive and interference GmSWEET6 expression vector plasmid into agrobacterium rhizogenes, adopting agrobacterium rhizogenes-mediated soybean hypocotyl injection transformation to obtain a transgenic chimeric plant (Li et al, 2014), and using a transgenic chimeric strain for subsequent phenotype identification.

4. Detection of soybean transgenic plants

Excess or interference with the detection of GmSWEET6 hypocotyl injection of transformation material: a part of adventitious roots growing at a hypocotyl wound of a single plant are cut, RNA is extracted and is inverted into cDNA as a template, plasmid DNA is used as a positive control, root system cDNA of non-transgenic soybean is used as a negative control, and an upstream specific primer 5'-CAACCACTACATCGAGACAAGCA-3' (SEQ ID NO:17) and a downstream specific primer 5'-TCATCAGATCTCGGTGACGGG-3' (SEQ ID NO:18) are used for amplifying herbicide resistance gene (Bar gene) fragments for PCR detection. In addition, quantitative PCR is further used for detecting the excessive or interference effect, and in the quantitative PCR test, the soybean housekeeping gene EF-1a is used as a reference gene (primer sequence: SEQ ID NO:1, 2) as described above, and the relative expression quantity is the ratio of the expression quantity of the target gene GmSWEET6 (primer sequence: SEQ ID NO:3, 4) to the expression quantity of the housekeeping gene. Different effective transgenic strains are obtained through PCR detection and quantitative PCR confirmation. The results are shown in FIG. 4.

5. Excessive or interfering with the effect of GmSWEET6 on the phosphorus content of soybean transgenic plants: harvesting 45-day-old different transgenic lines inoculated with arbuscular mycorrhizal fungi and determining the phosphorus content, comprising: the phosphorus content in the overground part, the phosphorus content in the root part and the like.

FIG. 5a and b show the effect of excessive or interference expression GmSWEET6 on phosphorus content of soybean transgenic plants. Wherein the plants are cultivated in sand soil under low phosphorus (50 μ M KH)₂PO₄) Under conditions of no inoculation (NM) or inoculation of arbuscular bacteria (Ri) for 45 days, harvesting, low-phosphorus treated K⁺By K₂SO₄And (5) filling. The results show that: in the case of inoculation Ri, the phosphorus content of the roots decreased significantly after overexpression of gmssweet 6 compared to the control, while the phosphorus content of the roots increased significantly after intervention gmssweet 6.

6. Influence of excessive or interference expression of GmSWEET6 gene on biomass of soybean transgenic plants:

and (3) measuring plant biomass: weighing fresh weights of overground part and root samples by a balance, deactivating enzymes of all samples in a drying oven at 105 ℃ for 30 minutes, drying at 75 ℃ to constant weight, and weighing the dry weights.

FIG. 5c, d is the effect of excessive or interfering expression GmSWEET6 on biomass of soybean transgenic plants. Wherein the plants are cultivated in sand soil under low phosphorus (50 μ M KH)₂PO₄) Under conditions of no inoculation (NM) or inoculation of arbuscular bacteria (Ri) for 45 days, harvesting, low-phosphorus treated K⁺By K₂SO₄And (5) filling. The results show that: in the case of inoculation with Ri, root biomass decreased significantly after overexpression of gmssweet 6 compared to controls, while it increased significantly after intervention gmssweet 6. The method has the advantages that the GmSWEET6 can play a vital role in carbon distribution in the symbiotic process of plants and mycorrhiza, and can influence the mycorrhiza absorption of the plants to phosphorus by regulating and controlling the carbon input of the host plants to a mycorrhiza symbiotic system, so that the growth of the host plants is influenced.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> soybean sucrose transporter important gene GmSWEET6

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tcatcagatc tcggtgacgg g 21

Claims

1. Important gene of soybean sucrose transporterGmSWEET6The application in the aspect of mycorrhizal symbiosis is characterized in that: interferenceGmSWEET6Then, the phosphorus content and the biomass of the root are obviously increased;

the soybean sucrose transporter important geneGmSWEET6The amino acid sequence of the encoded protein is shown in Glyma.04G198600.

2. Use according to claim 1, characterized in that: important gene of soybean sucrose transporterGmSWEET6Application in soybean breeding.

3. Use according to claim 1, characterized in that: important gene of soybean sucrose transporterGmSWEET6Application in breeding transgenic soybean.

4. Use according to claim 1, characterized in that: important gene of soybean sucrose transporterGmSWEET6In regulating formation of beneficial symbiotic relationship between soybean and mycorrhizal fungiApplication is carried out.