CN106146635B - Corn ZmSTP1 protein and coding gene and application thereof - Google Patents

Abstract

The invention relates to the field of molecular biology, and particularly provides a corn ZmSTP1 protein (shown as SEQ ID No. 1) and application of a coding gene (shown as SEQ ID No. 2) thereof in promoting monosaccharide absorption of a plant root system. The invention obtains a protein with sugar absorption function from important grain crop corn for the first time, the gene is expressed at the root tip of the corn and has the functions of absorbing and transporting various monosaccharides; the gene is transferred into a model plant Arabidopsis thaliana, so that the absorption capacity of a transgenic plant to specific sugar can be improved, the biomass and seed yield of a soil-cultured plant can be obviously improved, and the gene has an important application prospect; meanwhile, from the perspective of improving nitrogen efficiency of carbohydrates, the nitrogen efficiency of plants is expected to be improved, and nitrogen-efficient crops are cultivated.

Description

Corn ZmSTP1 protein and coding gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to application of a corn ZmSTP1 protein and a coding gene thereof in promoting plant root monosaccharide absorption.

Background

Carbohydrates are also called carbohydrates, and are The most abundant and widely distributed important organic compounds in nature, sugars are products of photosynthesis and also substrates of respiration, and provide carbon skeleton and energy for physiological and biochemical reactions in plants, so that sugar supply is important for carbon-nitrogen metabolism, dry matter accumulation and crop yield formation, part of sugars in plants are derived from photosynthesis and starch degradation, and part of sugars are directly absorbed by root systems from soil, neutral sugars such as glucose, fructose and sucrose are important for accumulation of total carbohydrates of plants due to properties such as Molecular weight and hydrophilicity, transmembrane transport of membrane transport proteins is required (Ludwig and Fl ü gge, 2013 Frondiersin Plant Science 4, 231), a series of sugar-transport proteins are currently found on The transmembrane, and biochemical and Molecular characteristics of these proteins are not identical, and more than 50 monosaccharide transport proteins and 20 disaccharide transport proteins (Lande, Annona, mannose, protein, mannose, protein, mannose, glucose, a transport protein, a major protein, a.

Nitrogen is a plant essential mineral element closely related to carbon, the global nitrogen fertilizer dosage in 2014 is about 1.1 hundred million tons, and the requirement for food safety can be met only when the requirement reaches 2.25 hundred million tons in 2050 years (Frink 199)9P Natl Acad SciUSA, 96(4): 1175-; tilman et al, 2011P Natl Acad Sci USA, 108(50): 20260-. A series of environmental problems such as soil acidification, water eutrophication and the like caused by the large amount of nitrogen fertilizer input (Guo et al, 2010Science, 327(5968): 1008-); on the other hand, nitrogen deficiency remains a major limiting factor in agricultural production in developing countries (Diels et al, 2001Agronomy Journal, 93: 1191-. Nitrogen deficiency significantly reduces chlorophyll synthesis and dry matter accumulation, affecting reproductive organ development, ultimately leading to severe yield loss (Marschner 1995Mineral Nutrition of high Plants, 2nd edn). Carbon-nitrogen metabolism is the most important metabolic process in plants, and carbon metabolism is closely related to nitrogen assimilation (Song Jian, 1998plant physiological communication). Spatially, carbon metabolism and NO₂ ^-Assimilation occurs in chloroplasts, and nitrogen metabolism requires a carbon source and an energy source provided by carbon metabolism, and also requires a keto acid synthesized by carbon metabolism as a skeleton to synthesize an amino acid. The activity of Nitrate Reductase (NR) is also affected by carbohydrates (Chenget et al, 1986Metabolism 35, 10-14; Cheng et al, 1992Pacad Sci 89, 1861-1864; Vincentz et al 1993). Studies in tomato have shown that increasing the uptake and utilization of sucrose results in an increase in the expression of nitrate reductase, which in turn accelerates the nitrogen metabolism process and increases the rate of amino acid synthesis (Morcuede et al 1998plant 206, 394-409; Halford et al 2004journal of Experimental Botany55, 35-42). In addition, nitrogen uptake by the root system is also affected by the supply of soluble carbohydrates in the roots (Tolley et al, 1988Journal of Experimental Botany 1988, 39: 613-622; Tolley et al, 1991 cosmetic Gazette 152: 23-33).

As an important food crop, corn plays an important role in global food safety and has great yield-increasing potential relative to other food crops (Chen et al, 2014 Nature). However, the sugar transporters in corn have been reported to date, and this study has cloned the STP1 gene from corn, and it is complementary to yeast and shows strong response to glucose (Glc), fructose (Frc), mannose (Man) and partial response to galactose (Gal), indicating that ZmSTP1 can absorb many kinds of monosaccharides after transferring into yeast system, but has different absorption ability to different monosaccharides. Overexpression into Arabidopsis appears to be responsive to various sugar treatments. The expression level of protein AtSUC2 related to synthesis of chlorophyll a and chlorophyll b and protein AtCAB1 related to synthesis of chlorophyll a and chlorophyll b is obviously increased. The method can provide important candidate genes for the high-efficiency accumulation genetic engineering of corn sugar, has a certain improvement effect on improving the nitrogen absorption and utilization efficiency of plants, particularly grain crops, and has important practical value and direct economic benefit.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a corn ZmSTP1 protein and an application of a coding gene thereof in promoting plant root monosaccharide absorption.

To achieve the object of the present invention, the present invention firstly provides a maize ZmSTP1 protein derived from maize of the genus Zea (zea mays L.), the amino acid sequence of which is shown in SEQ ID No. 1.

The invention also provides a coding gene ZmSTP1 of the protein, and the nucleotide sequence is shown as SEQ ID No. 2.

The invention also provides application of the ZmSTP1 gene in promoting plant root monosaccharide absorption.

Specifically, the ZmSTP1 gene is transferred into a plant to promote efficient absorption and accumulation of monosaccharide in a plant root system, so that the plant biomass and/or grain yield is improved.

Further, the ZmSTP1 gene is transferred into plant cells by a recombinant expression vector. The existing plant expression vectors can be used to construct recombinant expression vectors containing the ZmSTP1 gene.

When the ZmSTP1 gene is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, such as cauliflower mosaic virus CAMV35S promoter and maize Ubiquitin promoter (Ubiquitin), and the promoters can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

Preferably, the recombinant expression vector is a recombinant plasmid pSuper1300+ -ZmSTP obtained by inserting the ZmSTP1 gene between multiple cloning sites of pPT-HYG.

The invention also provides a recombinant expression vector, a transgenic cell line and a recombinant bacterium containing the ZmSTP1 gene.

The invention also provides a method for promoting efficient absorption and accumulation of plant root system monosaccharide, which is characterized in that the ZmSTP1 gene is transferred into plant cells through a recombinant expression vector.

Preferably, the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between multiple cloning sites of pPT-HYG.

The invention has the beneficial effects that:

the invention clones the maize ZmSTP1 gene, and discovers that the gene is mainly expressed at the root tip of maize seedling by researching the quantitative and qualitative expression characteristics of the gene in maize. The ZmSTP1 gene is further introduced into a model plant Arabidopsis (Columbia) by using a transgenic technology, and the result shows that the ZmSTP1 gene has response to treatment of various monosaccharides, and shows that vegetative growth and reproductive growth are influenced, including biomass, grain yield is remarkably increased, and sugar content is increased; the expression level of sucrose transporter SUC2, chlorophyll a and chlorophyll b synthesis related gene CAB1 is obviously increased. The sugar transport protein gene cloned from corn provides more effective gene resources due to the efficient absorption and accumulation of sugar of main crops, and plays an important role in the research of improving the nutrition and high efficiency performance of plants in genetic engineering. Meanwhile, from the perspective of improving nitrogen efficiency of carbohydrates, the nitrogen efficiency of plants is expected to be improved, and nitrogen-efficient crops are cultivated.

Drawings

FIG. 1 is a phylogenetic tree analysis of the ZmSTP1 gene of the present invention;

FIG. 2 is a quantitative expression analysis of the ZmSTP1 gene in different tissues according to the invention;

FIG. 3a is a tissue expression mapping analysis of the ZmSTP1 gene of the present invention;

FIG. 3b is a subcellular localization analysis of the ZmSTP1 gene of the present invention;

FIG. 4 is the heterology function complementation verification-function complementation phenotype analysis of EBY. VW4000 yeast mutant;

FIG. 5 is a phenotypic analysis of Arabidopsis thaliana plants overexpressing the ZmSTP1 gene (18 days in culture on plates);

FIG. 6 is the rosette leaf phenotype analysis (18 days of plating) of Arabidopsis thaliana over-expressing ZmSTP1 gene;

FIG. 7 is an analysis of Arabidopsis thaliana plants overexpressing ZmSTP1 gene for response to varying concentrations of inorganic nitrogen supply (18 days in culture on plates);

FIG. 8 is a statistical analysis of the phenotype of rosette leaves of Arabidopsis thaliana plants overexpressing ZmSTP1 gene (35 days in soil culture);

FIG. 9 is a statistical analysis of biomass and grain yield of Arabidopsis thaliana over-expressing ZmSTP1 gene (55 days in soil culture);

FIG. 10 is an Arabidopsis thaliana sugar content assay overexpressing ZmSTP1 gene under both plating and soil culture conditions;

FIG. 11 shows quantitative expression analysis of AtSuc2 and AtCAB 1.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified.

Preparation of the Material

1. Strain and tool plasmid

The materials used in the examples of the present invention include E.coli competent cell DH5 α (Code No. CB101, Tiangen Biochemical technology Co., Ltd.), Agrobacterium tumefaciens GV3101 (purchased from Tiangen Biochemical technology Co., Ltd.) and EBY. VW4000 yeast mutant, TA cloning vectorBody:

19-T Vector (Code No. D102, Takara), pSuper1300+ -Kanamycin, pDR195 (the Risk of Ringyuan Ringman for the teacher); pUC-GFP (Takara Bio).

2. Tool enzyme and biochemical reagent

Various restriction enzymes were purchased from NEB corporation; various Taq enzyme and Trizol RNA miniprep kits were purchased from Takara; dNTP mixtures were purchased from shanghai life; t4DNA ligase was purchased from Promega; a general agarose gel DNA recovery kit (Code No. DP209, Tiangen Biochemical technology Co., Ltd.); plasmid mini-extracts were purchased from (Code No. DP103, Tiangen Biochemical technology Co., Ltd.) ampicillin (Amp), kanamycin (Kan), rifampicin (Rif) from Xinjingke.

3. PCR amplification primer

ZmSTP1-pDR-L：5′-CTCGAGATGGCCGGCGGTGGCATCGTG-3′

ZmSTP1-pDR-R：5′-GGATCCTCACGCGTCGGCCCCCTTGG-3′

ZmSTP1-RT-L：5′-GTCTTCATCGCCTTCTTCCTG-3′

ZmSTP1-RT-R：5′-TTGGTGTCGCTGCCGTTT-3′

ZmUbiquitin-L：5′-GTTGAAGCTGCTGCTGTATCTGG-3′

ZmUbiquitin-R：5′-GCGGTCGCACGATAGTTTTG-3′

ZmSTP1-Situ-F：5′-GATTTAGGTGACACTATAGAATGCTCCA

AAAACGGCAGCGACA-3′

ZmSTP1-Situ-R：5′-TGTAATACGACTCACTATAGGGGTACTAT

TGCTTGGTGGTG-3′

ZmSTP1-GFP-F：5′-TCTAGAATGGCCGGCGGTGGCATCGTG-3′

ZmSTP1-GFP-R：5′-GGATCCCGCGTCGGCCCCCTTGGTG-3′

AtAct2-L：5′-TGATGCACTTGTGTGTGACAA-3′

AtAct2-R：5′-GGGACTAAAACGCAAAACGA-3′

AtSUC2-L：5′-GGATCGCTTGGTTCCCTTTC-3′

AtSUC2-R：5′-GGAGTCAGAGCTGGTGCTTTGG-3′

AtCAB1-L：5′-CCCATTTCTTGGCTTACAACAAC-3′

AtCAB1-R：5′-TCGGGGTCAGCTGAAAGTCCG-3′。

Example 1 cloning of the maize sugar Transporter Gene ZmSTP1 Gene

Amino acid sequences of monosaccharide transporters such as Arabidopsis, rice and wheat were obtained as NCBI, Maizessence and Uniprot, and were aligned using ClustalW1.8 (Thompson et al 1994nucleic acids Research 22, 4673-. The phylogenetic tree analysis showed the closest relationship to AtSTP1, so the gene was named ZmSTP1 (FIG. 1).

1. Total RNA extraction

Grinding 200mg of fresh B73 corn roots in liquid nitrogen; adding Trizol extracting solution provided by 1ml of kit, and shaking for 5 minutes at room temperature; adding 200 mul of trichloromethane, shaking for 30 seconds, centrifuging for 15 minutes at 4 ℃ and 12000 revolutions; taking the supernatant, adding 0.5ml of isopropanol, standing for 1 hour at room temperature, centrifuging for 15 minutes at 4 ℃ at 12000 rpm; taking the precipitate, adding 1ml of 70% ethanol, shaking for 1 minute, centrifuging at 4 ℃ under 10000 rpm for 10 minutes; the supernatant was removed by suction, the precipitate dried in a fume hood, and 50. mu.l of DEPC water was added to dissolve the precipitate. The quality of RNA was checked by electrophoresis on 1% agarose, while the RNA concentration was determined by spectrophotometer.

2. mRNA purification

Adding 500 mu g of total RNA into a new RNA-free enzyme centrifuge tube, and adding binding solution provided by 1ml of kit; placed at 65 ℃ for 10 minutes and then immediately transferred to ice for 1 minute; transferring the liquid into a centrifuge tube containing oligo (dT) resin provided by the kit, gently shaking for 20 minutes at room temperature, centrifuging for 10 minutes at 4000 rpm at room temperature, carefully sucking off the supernatant, and repeating for 2 times; then 0.3ml of binding solution was added to resuspend the resin, transferred to spin-column tubes provided in the kit, washed with 500. mu.l of binding solution, centrifuged at 4000 rpm for 10 seconds at room temperature, and the OD of the eluate was measured₂₆₀If the concentration is more than 0.05, the eluate is washed by adding 500. mu.l of the binding solution again until the OD of the eluate is reached₂₆₀Less than 0.05; adding 200 μ l of eluent, gently suspending the resin, transferring the spin-column tube to a new centrifuge tube, and rotating the tube 4000 rpm at room temperaturemRNA was collected by centrifugation for 10 seconds. Finally, 10. mu.l of 2mg/ml glycoside, 30. mu.l of 2M sodium acetate and 600. mu.l of absolute ethanol were added, the mixture was left at-80 ℃ for 30 minutes, centrifuged at 4 ℃ and 14000 ℃ for 20 minutes, the supernatant was discarded, washed with 70% ethanol once and dissolved in 20. mu.l of TE. 0.5. mu.l of the sample was taken out and OD was measured₂₆₀And the concentration was calculated.

3. First Strand cDNA Synthesis

Mu.g of mRNA was collected and reverse transcribed using reverse transcriptase carried in the kit. The method comprises the following specific steps: mu.l of Biotion-attB2-oligo (dT) provided in the kit was added to 10. mu.L of the mRNA solution purified in the previous step at a concentration of 100 ng/. mu.l, left at 70 ℃ for 5 minutes, quickly transferred to ice for 3 minutes, and then the following components were added:

the following reaction conditions were set at 25 ℃ for 10 minutes, 42 ℃ for 60 minutes, 70 ℃ for 10 minutes, and ice bath for 2 minutes, with reference to the kit instructions.

4. Clone ZmSTP1

The STP1 is cloned by taking specific cloning primers (ZmSTP1-OE-F, ZmSTP1-OE-R) and cDNA as templates, and the specific operation process comprises the following steps:

the following ingredients were added to a 200 μ l centrifuge tube:

after centrifugal mixing, PCR is carried out, and the PCR reaction program is as follows:

and (5) detecting and recovering agarose electrophoresis gel, and performing column type kit operation according to the tiangen centrifugation.

Add tail a and add the following ingredients to a 200 μ l centrifuge tube:

purified DNA product 7. mu.l

After shaking up, incubation was carried out for 30min at 72 ℃. Use of

And (3) cloning the recovered amplified fragment to a T Vector by using a 19-T TA cloning kit to construct a recombinant plasmid.

Ligation was performed overnight at 16 ℃ and heat shock transformed into 50. mu.L of competent cells. Activating and oscillating the shaking table at 37 ℃ and 150rpm for 60 min; 100. mu.l of the cell suspension was applied to a LB + Amp solid medium plate and incubated overnight (12 to 16 hours) in a 37 ℃ incubator. And (4) picking a single clone, and carrying out verification sequencing. Taking a plasmid with correct sequencing as a template, adding different enzyme cutting sites according to subsequent experiments, repeating the operation 4, and connecting ZmSTP1 containing the enzyme cutting sites

And (3) carrying out sequencing on the 19-T vector, and preserving bacteria for later use after the sequencing is correct.

Example 2 Real-Time PCR analysis of maize ZmSPT1 Gene expression profiles in various tissues.

The tested materials were planted in Shanzhuang laboratory of China agricultural university, corn samples of different tissues were harvested one week after spinning, rapidly placed in liquid nitrogen, taken back to the laboratory, and placed in a-80 deg.C refrigerator for use.

Quantitative results indicated that ZmSTP1 was expressed in the highest amount in roots (FIG. 2).

The attached Real-Time PCR operation step:

1. extracting total RNA from roots of different treatment samples (same method as example 1);

2. 50. mu.g of total RNA was used to remove genomic DNA by DNase I (TaKaRa, Cat. No.: D2215) as follows:

reaction system (50 μ l):

reacting at 37 ℃ for 30 minutes;

adding 150. mu.l DEPC water, adding 200. mu.l phenol/chloroform/isoamyl alcohol (25:24:1), and mixing well;

centrifuging at 4 ℃ and 12000rpm for 10 minutes, and transferring the upper layer into a new centrifuge tube;

adding 200 μ l chloroform/isoamyl alcohol (24: 1), mixing well;

centrifuging at 12000rpm at 4 deg.C for 10 min, and transferring the upper layer into a new centrifuge tube;

add 20. mu.l of 3M NaAc (pH 5.2), add 500. mu.l of precooled absolute ethanol and leave at-20 ℃ for 60 minutes;

centrifuging at 4 deg.C and 12000rpm for 15 min, recovering precipitate, washing with 70% precooled ethanol for 2 times; centrifuging at 7500rpm for 5 minutes at 4 ℃ each time;

blow-drying, and dissolving DEPC in water again.

3. The first strand cDNA was synthesized by reverse transcription in the conventional manner (the same procedure as in example 1).

4. The Real-time PCR was performed to detect gene abundance by selecting SYBR Green RealtimePCR Master Mix (catalog number 91620F3) from TOYOBO, quantitative PCR instrument model Bio-Rad iCycler iQ5system (BIO-RAD), diluting the reverse product 10 times as the Real-time PCR template

Reaction system:

PCR reaction procedure: 2 minutes at 50 ℃, 10 minutes at 95 ℃, 45 cycles (15 seconds at 95 ℃, 30 seconds at 61 ℃, 1 minute at 72 ℃);

a curve melting step: 95 ℃ for 15 seconds, circulating for 10 seconds, increasing the temperature from 60 ℃ to 95 ℃ at a speed of 0.5 ℃ per circulation, and performing 70 cycles;

and (3) calculating the relative expression amount of ZmSTP1 in different tissues by using ZmUbi as an internal reference and adopting a relative quantitative algorithm.

Example 3 localization of ZmSPT1 expression using in situ hybridization, Green fluorescent protein technology.

1. Preparation of plant material

Hoagland culture solution is used for plant culture, and root tip samples are taken from maize seedlings in the trefoil stage. Cutting 0.5-1cm of root tip, and adding into FAA fixative (containing 50% ethanol 90ml, glacial acetic acid 5ml, and formaldehyde 5ml per 100ml fixative);

then dehydrating, transparentizing and waxing the plant material, wherein the method comprises the following steps:

removing FAA stationary liquid, and washing with DEPC water twice;

50% ethanol, 50% ethanol + 10% tert-butyl alcohol, 50% ethanol + 20% tert-butyl alcohol, 50% ethanol + 35% tert-butyl alcohol, 50% ethanol + 50% tert-butyl alcohol, 25% ethanol + 75% tert-butyl alcohol + 0.1% eosin Y), 100% tert-butyl alcohol were treated sequentially for 2 hours each;

transferring into 2/3 tert-butyl alcohol and 1/3 paraffin oil for 4 hours;

pouring out 1/3 tert-butyl alcohol and paraffin oil mixed solution, supplementing the same volume of paraffin melted at 60 ℃ to the upper layer to form a solidified wax cover, and standing at 60 ℃ for 12 hours (repeating for 3 times);

pouring out all the liquid, adding pure molten paraffin, and carrying out heating at 60 ℃ for 8 hours (repeating for 2 times);

then embedded on a 65 ℃ hot plate.

2. Synthesis and purification of probes

RNA probe synthesis was carried out while preparing plant materials, and the following procedure was followed with reference to the method of using T7-RNA polymerase (catalog No.: P2075) by Takara:

huada Gene Corp synthesized a forward primer containing the T7 promoter and ZmSTP 1:

5′-GATTTAGGTGACACTATAGAATGCTCCAAAAACGGCAGCGACA-3′

reverse primer:

5-TGTAATACGACTCACTATAGGGGTACTATTGCTTGGTGGTG-3′；

a DNA template containing a T7 promoter is amplified from a plasmid by using KOD enzyme and transcribed into an RNA probe in vitro, and the reaction system is as follows:

20 mul in total, mixed evenly and reacted for 2 hours at 37 ℃;

adding 4. mu.l of DNase I (TaKaRa Co., Cat. No. D2215) and reacting at 37 ℃ for 15 minutes to remove the genomic DNA;

after completion of the reaction, the reaction mixture was placed on ice and 0.8. mu.l of 0.5M EDTA (pH 8.0) was added to terminate the reaction;

adding 2 μ l of 5M LiCl and 75 μ l of anhydrous ethanol pre-cooled at-20 ℃, uniformly mixing, and placing at-20 ℃ for 2 hours;

13000rpm, centrifugation at 4 ℃ for 15 minutes;

discarding the supernatant, adding 50 μ l of 70% precooled ethanol to wash the precipitate, and centrifuging at 13000rpm at 4 ℃ for 5 minutes;

removing the supernatant, and drying the precipitate;

add 24. mu.l DEPC-H2O to dissolve, and store at-80 ℃ for further use.

3. Tabletting:

the embedded wax blocks were cut into 8-10 μm pieces using a Shanghai Hongyu model QP-4 microtome. The wax tape (containing the plant sample) in the proper position is cut and stuck on a slide glass (Sigma company, catalog number P0425-72EA) of polylysine, the wax tape is put into DEPC water and is spread on a baking table at 45 ℃, excessive water is absorbed after the spreading is sufficient, and the piece is baked in an oven at 40 ℃ for 24-48 hours, so that the piece is fully dried.

Slicing and dewaxing: washing the slices with xylene for 3 times and 5 minutes respectively, washing with anhydrous ethanol for 2 times and 2 minutes respectively, sequentially washing with 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, and 30% ethanol for 1 minute respectively, and washing with DEPC for 2 times and 1 minute each.

And (3) protease K treatment: adding proteinase K reaction buffer (100mM Tris-HCl pH7.5, 50mM EDTA) and proteinase K to a final concentration of 1 μ g/ml in a staining jar, placing the pretreated slide, and keeping the temperature at 37 ℃ for 20 minutes;

washing the slide with DEPC water for 2 times, each for 1 minute;

acetylation treatment: the slide glass was placed in a staining jar, 40ml of a 0.1mol/L triethanolamine solution (pH 8.0) in which 100 μ L acetic anhydride was dissolved was added, and the mixture was left at room temperature for 10 minutes; the solution was decanted and washed twice with 2 × SSC solution for 7 minutes each;

the tissue sections were dehydrated by washing the slices with different dilutions of aqueous ethanol (30%, 50%, 70%, 85% and 95%) sequentially at room temperature for 1 minute per stage. Then washed 2 times with fresh absolute ethanol for 2 minutes each time. And (5) drying at room temperature.

4. Hybridization of

The composition of the hybridization solution is as follows: each slide was run with 200. mu.l of hybridization solution containing: mu.l of deionized formamide, 20. mu.l of 10 Xhybridization buffer (100mM Tris pH7.5, 10mM EDTA, 3M NaCl), 24. mu.l of 50% dextran sulfate, 20. mu.l of 10 Xblocking Solution, 250. mu.g salmon sperm DNA, 5. mu.l of probe, 36.5. mu.l of 50 XDenhardt's Solution. Hybridization was performed for 16-30 hours in the absence of light.

Washing: immersing the slices in 2 XSSC to make the cover sheets fall off, and then placing the slices at room temperature for 30 minutes; replacing the new 2 XSSC at 65 ℃ for 1 hour; 0.1 XSSC, 65 ℃ for 1 hour.

Blocking the slide was dried on the back with absorbent paper and placed in a wet box, 2ml of 1% blocking solution (2 g of Boehringer Block reagent, 0.1M Tris-HCl, pH 7.5; and 0.15M NaCl) was added to each slide, and the slide was left at room temperature for 1 hour.

Equilibration the blocking solution was removed and 1ml of wash solution (100mM Tris-HCl pH7.5, 150mM NaCl, 0.3% Triton X-100, 1% BSA) was added to each slide and equilibrated for 15 minutes.

Antibody adsorption: the equilibration solution was removed and 400. mu.l of antibody solution (per 400. mu.l antibody solution: 399. mu.l wash, 1.32. mu.l Anti-DIG-AP supplied from the kit) was added and hybridized for 2 hours at room temperature or overnight at 4 ℃.

And (3) film washing: the slides were washed 3 times in wash solution for 10 minutes each time.

Balancing before color development: the mixture was immersed in a developing buffer for 5 minutes (1ml developing buffer formulation: 100. mu.l of 1M Tris-HClpH9.5, 20. mu.l of 5M NaCl, 860. mu.l of ddH2O, 0.1g of polyhexenol).

Color development: adding 20 mu l of NBT/BCIP into the color development buffer solution, dripping 500 mu l of color development solution into each piece, developing for 0.5-4h at the dark place at room temperature in a wet box, stopping the reaction when a positive signal is light red or reddish brown in microscopic examination and the background is not clearly developed, and washing for 3 times with water for 5 minutes each time. After mounting with neutral gum, the reddish brown positive signal turns blue or bluish purple.

As shown in FIG. 3a, the results of probe hybridization are shown in the upper and lower graphs, respectively, as a longitudinal and transverse root tip cut graph. The results indicate that ZmSTP1 is expressed throughout the maize root tip, consistent with its biological function of mediating sugar uptake from the environment by the root.

An expression vector of the recombinant plasmid fused with ZmSTP1 is constructed by using a PUC-GFP vector and a 35S promoter, and is injected into tobacco leaves to transiently express ZmSTP1, so that the ZmSTP1 is positioned, and the result shows that the ZmSTP is expressed on cell membranes and cell nuclei (figure 3 b).

Example 4 verification of Yeast heterologous functional complementation of ZmSTP1

The pDR195 vector contains XhoI and BamHI cleavage sites at both ends. Taking 1-1.5ml of bacterial liquid, extracting plasmid according to the specification of the Tiangen plasmid miniextraction kit, selecting XhoI and BamHI to extract ZmSTP1 gene from

19-T is cut off, recovered by electrophoresis, and is connected between XhoI and BamHI enzyme cutting sites on a pDR195 vector, and a forward connected clone is selected by sequencing.

Vw4000 yeast mutants, which lack all hexose and galactose sugar transporters, are rendered non-viable in an environment where monosaccharides are the sole carbon source (Wieczorke et al, 1999), but can grow in an environment where maltose is the carbon source, and we used this property to examine the function of ZmSTP 1. The recombinant plasmid of the yeast pDR195 constructed by the operation is introduced into the EBY.VW4000 yeast mutant, and the activity of the yeast is analyzed under a certain carbon source environment.

Yeast expression vector transferred into EBY.VW4000 yeast mutant

Yeast to be transformed was inoculated into 5ml of liquid YPD medium and cultured overnight at 30 ℃ with shaking at 200 rpm. Determination of concentration to OD₆₀₀0.5 in 50ml YPD medium, and cultured at 30 ℃ and 200rpm with shaking to OD₆₀₀Centrifuging at 3000 Xg for 5min to collect cells 2, discarding the supernatant, and suspending the cells in 25mlCentrifuging again in bacteria water to collect cells, discarding water, suspending the cells in 1ml of sterile water, transferring to a sterile 1.5ml centrifuge tube, centrifuging, removing supernatant, adding 1ml of sterile water to suspend the cells,

the cell suspension was aliquoted according to the amount of transformation (about 200. mu.l), centrifuged again for 1-2min to pellet the cells, the supernatant carefully aspirated with a pipette, and then the premixed transformation mixture was added:

after vigorous shaking to completely mix the cells, the cells were placed in a 42 ℃ water bath and heat shocked for at least 40min (Suga and Hatakeyama, 2005), centrifuged at high speed for 30sec to remove the conversion mixture, 0.2-1.0ml of sterile water was added, and the suspension pellet was gently extracted up and down with a pipette.

pDR-EBY.VW4000 transferred into the empty vector is used as a negative control, and wild type pDR-23344c is used as a positive control. After transformation, selection culture was carried out on a solid agar medium containing 6.7g/L YNB (yeast reagent base) in which uracil was replaced with ammonium sulfate (0.5g/L) maltose (20 g/L). Growth medium 2% glucose, fructose, galactose or mannose was added as the sole carbon source and ammonium sulfate (0.5g/L) as the nitrogen source on the basis of the selection medium. Taking OD₆₀₀1.0 yeast liquid, diluted to 10^-1，10^-2，10^-3，10^-4Four gradients, 10. mu.l of the drop were pipetted onto the previous medium and cultured upside down at 30 ℃ for 3 days to observe the phenotype. Each treatment was repeated three times with different monoclonals. In sharp contrast to the mutants, the yeast with ZmSTP1 was introduced to grow significantly on glucose (Glc), fructose (Frc) and mannose (Man), and the growth was improved to some extent under galactose (Gal) supply conditions (FIG. 4), indicating that ZmSTP1 could absorb multiple monosaccharides but different monosaccharides after transfer into the yeast system.

The extraction method of the plasmid DNA of the subsidiary yeast comprises the following steps:

selecting the growing bacterial plaque, inoculating the growing bacterial plaque into 0.5ml YNB liquid culture medium containing 1mM Arg, and carrying out shaking culture at 30 ℃ and 230rpm for overnight; centrifuging at 4,000 rpm for 5min to collect thallus;

pouring out the supernatant, suspending the thallus by using fresh liquid culture medium (the total volume is about 50 mu l), adding 10 mu l of 10mg/ml lysozyme solution into each tube, and fully shaking to ensure that the solution and the thallus are completely mixed;

the tube was incubated at 30 ℃ for 60 minutes at 230rpm with shaking;

adding 10 mul of 20% SDS into each tube, and shaking vigorously for 1 minute to mix thoroughly;

putting the sample at-20 ℃ for 2 hours, taking out the sample to be thawed, and oscillating violently to crack fully;

each tube volume was made up to 200 μ l with TE buffer (pH 7.0);

add 200. mu.l phenol: carrying out simulation: isoamyl alcohol (25:24:1), shaking vigorously for 5 minutes; centrifuging at 14,000 rpm for 10 minutes, and transferring the supernatant into a new centrifuge tube;

add 8. mu.l 10M NH₄Ac and 500. mu.l absolute ethanol;

placing in a refrigerator at the temperature of-80 ℃ for 1 hour, and centrifuging at 14,000 rpm for 10 minutes;

the supernatant was discarded, the precipitate was dried by blowing, and the residue was washed with 20. mu. l H₂Dissolving and precipitating O;

transferring 0.5. mu.l of plasmid into E.coli competent cells (DH5 α strain), shaking at 37 ℃, extracting plasmid, carrying out enzyme digestion identification, and then sending to the company for sequencing.

Example 5 construction, transformation and phenotypic analysis of ZmSTP1 Arabidopsis overexpression vector.

Wild type Arabidopsis thaliana col was transformed with the recombinant expression vector pSuper1300+ -ZmSTP 1. The specific method comprises the following steps: inoculating 0.5ml of positive Agrobacterium strain liquid into 500ml of YEB liquid culture medium, and shake culturing at 28 deg.C to OD₆₀₀To 0.5. The cells were collected by centrifugation at 5000rpm and 4 ℃ for 15 minutes. The cells were resuspended in 200ml of infiltration buffer (1 XMS macroelements, 5% sucrose) and silwet L-77(GE, cat # S5505) was added to a final concentration of 0.2%. 1 × macroelement contains 1.65g/LNH₄NO₃，1.9g/L KNO₃，0.44g CaCl₂∙2H₂O，0.37g/L MgSO₄∙7H₂O and 0.17g/L KH₂PO₄. Soaking the flower of Arabidopsis immediately after bolting in a resuspension for 30 seconds. Wrapping the plant with a fresh-keeping bag, keeping away from light at 16 deg.C for 24 hr, and vertically growing until T is harvested₀And (5) seed generation.

1. Screening of transgenic Positive plants

As the Arabidopsis plants transformed with the pSuper1300+ -ZmSTP1 vector have hygromycin resistance, they grew normally on MS solid medium containing hygromycin, while wild-type seeds not transformed with the gene did not grow normally and died. Transforming the current transgenic plant into T₀Instead, from the T₀The seeds produced by selfing the generation plants and the plants grown from them are T₁And (4) generation. Mixed collection T₁Seeding on MS solid culture medium containing hygromycin 50 microgram/ml, screening plant capable of normally growing, transplanting in pot to continue growing, and harvesting the single plant. T is₂After the generation seeds are screened for 1 time of hygromycin resistance, the T is harvested from a single plant₃And (5) seed generation. Also after a resistance selection, all individuals can grow homozygote plants which are trans-pSuper 1300+ -ZmSTP1, and are left for later use.

2. Molecular detection of transgenic Arabidopsis

And (3) PCR detection: separately extracting T₃Transferring total RNA of pSuper1300+ -ZmSTP1 gene Arabidopsis homozygous plant, reverse transcribing into first strand cDNA under the guide of oligo (dT), and detecting the expression level of ZmSTP1 gene in transgenic Arabidopsis by RT-PCR with ZmSTP1-RT-L and ZmSTP1-RT-R as primers.

3. Analysis of monosaccharide absorption capacity of transgenic plants

For T₃The seeds were vernalized, surface sterilized, and cultured for germination in ATS sugar-free solid agar medium (Schofield et al, 2009Plant, Cell and Environment 32, 271-285). After 4 days, the seedlings of ZmSTP1-OE 3 lines and Col-0 with consistent growth were selected and transferred to squares (13X 13 cm)²) On a screening medium. The screening culture medium is the previous seedling culture medium, and sugars with different concentrations are added as carbon sources, and the method comprises the following steps: glucose (Glc), fructose (Frc), sucrose (Suc), ribose (Rib), galactose (Gal), inositol (MI), xylose (Xyl), mannose (Man). The sugar-free ATS culture medium is used as negative control, and trimethylglucose (glucose) is addedGlucose analog, unable to be phosphorylated by hexokinase) was used as a positive control. The number of rosette leaves is obviously increased under the supply of Glc (2, 5, 10mM), Frc (5, 10mM), Suc (5mM) or Rib (5, 55 mM); the number of rosette leaves at high concentrations of Glc, Frc, Gal, Suc, Xyl, Rib, Man, MI was reduced (data not in full). Rosette leaf diameter increased at low concentrations of Glc, Frc, Rib carbon source and decreased at high concentrations of Glc, Frc, Gal, Suc, Xyl, Rib, Man or MI, with the final biomass also being significantly different (FIG. 5, FIG. 6). Addition of 9 or 1mM NO at 55mM different carbon sources simultaneously₃ ^-Under nitrate nitrogen, growth was significantly inhibited (fig. 7), indicating that the over-expressed plants were more sensitive to nitrogen.

In the soil culture experiment, transgenic Arabidopsis thaliana and wild type were grown under short-day conditions (8/16 day/night, 84. mu. mol m)^-2s^-122 ℃ C.) for up to 35 days. After 35 days, the short day was changed to long day (16/8h day/night, 56. mu. mol m)^-2s^-122 ℃ C.) until 55 days of maturation. Statistics were made for the first inflorescence height, fresh weight, dry weight of the plants, and weight of each seed. Soluble sugars were extracted by chloroform/methanol method (Antonio et al, 2008Rapid Communications in Mass Spectrometry22, 1399-. Under short-day conditions, vegetative growth of ZmSTP1 overexpression plants was promoted, which was manifested by increased leaf blade, increased petiole length, increased number, and significantly increased rosette leaf diameter (FIG. 8). Under long-day conditions, the over-expressed plants showed increased inflorescence number, increased dry weight and darker leaf color (FIG. 9). Upregulated expression of AtSUC2(sucrose transporter 2) in overexpressed plants (fig. 10), indicating that ZmSTP1 affects transport of other sugars; ZmSTP1 may also be associated with the light and light pathways, and the chlorophyll a and chlorophyll B synthesis related protein AtCAB1 was upregulated in overexpressing plants (fig. 11). Taken together, ZmSTP1 may ultimately increase yield by affecting the transport of other sugars and regulating photosynthetic pathways.

Attached: arabidopsis culture and transgenic seedling screening

Taking out proper amount of the mixtureThe Arabidopsis seeds were placed in a 1.5mL centrifuge tube and deionized water was added. The seeds were allowed to sink in water as much as possible and were vernalized in a refrigerator at 4 ℃ for 2 days. Sterilizing seeds in a super clean bench, adding 75% ethanol for sterilization for 1 minute, washing with sterile water once, adding 2% sodium hypochlorite for sterilization for 2 minutes, and washing with sterile water for 5-7 times. The seeds were spotted evenly on 1/2MS (or 1/2MS +50mg/ml hygromycin) medium using a 10. mu.L pipette, and the seeded medium was placed vertically in a culture chamber (light: 100. mu. E. m)^-2s^-1And photoperiod: 16h day/8 h night, temperature 22/20 ℃, humidity 100%). Transplanting the seedlings with good growth vigor into a flowerpot filled with wet nutrient soil (m vermiculite/m nutrient soil is 1: 1). 4 plants are placed in each pot, 12 pots are placed in each tray, and the surfaces of the pots are covered with a layer of preservative film to prevent excessive evaporation of water from affecting the growth of seedlings. Pouring 400ml of water every 5 days, removing the preservative film after 10 days, and pouring 400ml of water every 3 days.

Get T₀Vernalization and sterilization of Arabidopsis seeds, and uniform spreading of the seeds on 1/2MS screening medium (containing 50. mu. mol/L hygromycin). Wrapping a layer of black plastic film, placing in a laboratory, culturing in the dark for 5-7 days, if the stem of the seedling is very high, selecting 12 seedlings from each transgenic plant, numbering, transferring to 1/2MS culture medium, culturing for 3-5 days, transferring to nutrient soil after 4 young leaves grow, placing in a culture room, culturing, and completing the whole growth period. Repeating the above operations until obtaining homozygous T₃And (5) seed generation.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (6)

1. The application of the maize ZmSTP1 gene in regulating and controlling a plant photosynthetic pathway; the nucleotide sequence of the ZmSTP1 gene is shown as SEQ ID No. 2.

2. The use of claim 1, wherein the gene is transferred into a plant to promote expression of a plant photosynthetic pathway gene, thereby increasing plant biomass and/or seed yield.

3. The use according to claim 2, wherein the gene is transferred into a plant cell by means of a recombinant expression vector.

4. The use according to claim 3, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between the multiple cloning sites of pPT-HYG.

5. A method for regulating plant photosynthetic pathway is characterized in that corn ZmSTP1 gene is transferred into plant cells through a recombinant expression vector; the nucleotide sequence of the ZmSTP1 gene is shown as SEQ ID No. 2.

6. The method according to claim 5, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between multiple cloning sites of pPT-HYG.

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