CN106146635B - Maize ZmSTP1 protein and its encoding gene and application - 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

Maize ZmSTP1 protein and its encoding gene and application

technical field

The invention relates to the field of molecular biology, in particular to the application of maize ZmSTP1 protein and its encoding gene in promoting the absorption of monosaccharide by plant roots.

Background technique

Carbohydrates, also known as carbohydrates, are the most important and widely distributed organic compounds in nature. Sugars are the products of photosynthesis and the substrates of respiration, providing carbon skeleton and energy for physiological and biochemical reactions in plants. Therefore, the supply of sugars is crucial for carbon and nitrogen metabolism, dry matter accumulation, and crop yield formation. Part of the sugar in plants comes from photosynthesis and starch degradation, and part is absorbed directly from the soil by the root system. Among them, the sugar directly absorbed by the root system is of great significance to the accumulation of total plant carbohydrates. Neutral sugars such as glucose, fructose, and sucrose, due to their large molecular weight and hydrophilic properties, require the participation of membrane transport proteins for their transmembrane transport (Ludewig and Flügge, 2013 Frontiersin Plant Science 4, 231). At present, a series of sugar transmembrane transport proteins have been found on the cell membrane, and their biochemical functions and molecular characteristics are different. More than 50 monosaccharide transporters and 20 disaccharide transporters are predicted to exist in Arabidopsis (Lalonde et al., 2004 Annual Review of Plant Biology 55, 341-372). Among them, 14 monosaccharide transporters belong to the STP (Sugar Transporter Subfamily) protein family (Johnson and Thomas, 2007 Molecular Biology and Evolution 24, 2412-2423). The STP proteins that have been functionally studied are mainly expressed in plant reservoir organs, showing high-affinity transport properties (Km 10-100mm) (Buttner 2007 FEBS Letters581, 2318–2324), indicating that they transport and accumulate photosynthetic products in the reservoir. main features. AtSTP1 can reabsorb monosaccharides excreted by roots (Sherson et al., 2000 The Plant Journal 24, 849–857), and Atstp1 mutants have reduced monosaccharide accumulation and are insensitive to galactose and mannose (Sherson et al., 2000 The Plant Journal 24, 849–857); AtSTP13 is mainly expressed in the cortex and endothelium, and is responsible for the reabsorption of monosaccharide substances excreted by the damaged epidermal cells caused by abiotic stress (Nour-Eldin et al., 2006 Plant Methods 2, 17– 25; Yamada et al., 2011 The Journal of Biological Chemistry 286(50), 43577-43586). Arabidopsis plants overexpressing the AtSTP13 gene have increased glucose uptake, significantly increased sugar content, and significantly increased final biomass (Schofield et al., 2009 Plant, Cell and Environment 32, 271–285). Therefore, sugar transporters play a very important role in the absorption and accumulation of sugars. Carbohydrates also regulate the circadian clock, affecting plant hormone synthesis, defense systems and developmental processes (Bolouri Moghaddam and Van den Ende, 2013 Journal of Experimental Botany 64(6), 1439–1449).

Nitrogen is an essential mineral element closely related to carbon. In 2014, the global nitrogen fertilizer consumption was about 110 million tons, and the demand is expected to reach 225 million tons by 2050 to meet the needs of food security (Frink 1999P Natl Acad SciUSA., 96 (4). ): 1175-1180; Tilman et al., 2011 P Natl Acad Sci USA., 108(50):20260-20264). A large number of nitrogen fertilizers have caused a series of environmental problems such as soil acidification and water eutrophication (Guo et al., 2010 Science, 327(5968): 1008-1010); on the other hand, nitrogen deficiency is still the cause of agricultural production in developing countries Major constraints (Diels et al., 2001 Agronomy Journal, 93: 1191-1199; Vitousek 2009). Insufficient nitrogen can significantly reduce chlorophyll synthesis and dry matter accumulation, affect the development of reproductive organs, and ultimately lead to severe yield reduction (Marschner 1995 Mineral Nutrition of Higher Plants, 2nd edn). Carbon and nitrogen metabolism is the most important metabolic process in plants, and carbon metabolism is closely related to nitrogen assimilation (Song Jianmin, 1998 Plant Physiology Communications). From a spatial perspective, carbon metabolism and NO ₂ ^{-assimilation} occur in the chloroplast. Nitrogen metabolism requires carbon source and energy provided by carbon metabolism, and also requires keto acids synthesized by carbon metabolism as the backbone to synthesize amino acids. The activity of nitrate reductase (NR) is also affected by carbohydrates (Chenget al., 1986 Metabolism 35, 10-14; Cheng et al., 1992 PAcad Sci 89, 1861-1864; Vincentz et al. 1993). Studies in tomatoes have shown that by increasing the absorption and utilization of sucrose, the expression of nitrate reductase increases, which in turn accelerates nitrogen metabolism and increases the rate of amino acid synthesis (Morcuende et al. 1998 Planta 206, 394–409; Halford et al. 2004 Journal of Experimental Botany 55, 35-42). In addition, root nitrogen uptake is also affected by the availability of soluble carbohydrates in the root (Tolley et al., 1988 Journal of Experimental Botany 1988, 39:613-622; Tolley et al., 1991 Botanical Gazette 152:23-33).

As an important food crop, maize plays an important role in global food security, and has a huge potential to increase production compared with other food crops (Chen et al., 2014Nature). However, so far, the sugar transporter in maize has rarely been reported. In this study, the sugar transporter STP1 gene was cloned from maize, and it was complemented to yeast. ) strong response, with a partial response to galactose (Gal), indicating that ZmSTP1 can absorb a variety of monosaccharides after being transferred into the yeast system, but the absorption capacity of different monosaccharides is different. Overexpression into Arabidopsis showed response to various sugar treatments. Vegetative growth and reproductive growth were affected, including biomass, grain yield increased significantly, sugar content increased, and the expression of sucrose transporter AtSUC2, chlorophyll a and chlorophyll b synthesis-related protein AtCAB1 was also significantly increased. It can provide an important candidate gene for high-efficiency accumulation genetic engineering of corn sugar, and at the same time, it can also improve the nitrogen absorption and utilization efficiency of plants, especially food crops, and has important practical value and direct economic benefits.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the application of a maize ZmSTP1 protein and its encoding gene in promoting the absorption of monosaccharide by plant roots.

In order to achieve the purpose of the present invention, the present invention first provides a maize ZmSTP1 protein, which is derived from maize of the genus Zea (zea mays L.), and the amino acid sequence of the protein is shown in SEQ ID No.1.

The present invention also provides the protein encoding gene ZmSTP1, the nucleotide sequence of which is shown in SEQ ID No.2.

The present invention also provides the application of the ZmSTP1 gene in promoting the absorption of monosaccharide by plant roots.

Specifically, the application is to transfer the ZmSTP1 gene into plants to promote the efficient absorption and accumulation of monosaccharides in the roots of plants, thereby increasing plant biomass and/or grain yield.

Further, the ZmSTP1 gene is transformed into plant cells through a recombinant expression vector. The recombinant expression vector containing the ZmSTP1 gene can be constructed using the existing plant expression vector.

When using ZmSTP1 gene to construct recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus CAMV35S promoter, maize ubiquitin promoter (Ubiquitin), they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can It is the ATG start codon or the adjacent region start codon, etc., but it must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can 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 the multiple cloning sites of pPT-HYG.

The present invention also provides recombinant expression vectors, transgenic cell lines and recombinant bacteria containing the ZmSTP1 gene.

The present invention also provides a method for promoting the efficient absorption and accumulation of monosaccharide in plant roots. The method is to transfer the aforementioned ZmSTP1 gene into plant cells through a recombinant expression vector.

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

The beneficial effects of the present invention are:

The present invention clones the maize ZmSTP1 gene. By studying the quantitative and qualitative expression characteristics of the gene in maize, it is found that the gene is mainly expressed in the root tips of maize seedlings. Then, the ZmSTP1 gene was introduced into the model plant Arabidopsis thaliana (Columbia) by transgenic technology, and the results showed that it responded to a variety of monosaccharide treatments, showing that vegetative growth and reproductive growth were affected, including biomass, grain yield increased significantly, sugar The expression of sucrose transporter SUC2, chlorophyll a and chlorophyll b synthesis related gene CAB1 increased significantly. The sugar transporter gene cloned from maize provided by the present invention provides more effective gene resources for the efficient absorption and accumulation of sugar in main crops, and will play an important role in the research on the nutritionally efficient performance of genetically engineered plants. At the same time, from the perspective of improving the nitrogen efficiency of carbohydrates, it is expected to improve the nitrogen efficiency of plants and cultivate nitrogen-efficient crops.

Description of drawings

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

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

Figure 3a is the tissue expression and localization analysis of the ZmSTP1 gene according to the present invention;

Figure 3b is the subcellular localization analysis of the ZmSTP1 gene according to the present invention;

Figure 4 is the heterologous functional complementation verification - EBY.VW4000 yeast mutant functional complementation phenotype analysis;

Figure 5 is the phenotypic analysis of Arabidopsis plants overexpressing ZmSTP1 gene (plate cultured for 18 days);

Fig. 6 is the phenotype analysis of rosette leaves of Arabidopsis plants overexpressing ZmSTP1 gene (plate cultured for 18 days);

Figure 7 is the response analysis of Arabidopsis thaliana plants overexpressing ZmSTP1 gene to different concentrations of inorganic nitrogen supply (plate culture for 18 days);

Figure 8 is a statistical analysis of the rosette leaf phenotype of Arabidopsis thaliana plants overexpressing the ZmSTP1 gene (35 days of soil culture);

Figure 9 is a statistical analysis of plant biomass and grain yield of Arabidopsis thaliana overexpressing ZmSTP1 gene (55 days of soil culture);

Figure 10 is the determination of sugar content in Arabidopsis thaliana overexpressing ZmSTP1 gene under plate culture and soil culture conditions;

Figure 11 is the quantitative expression analysis of AtSuc2 and AtCAB1.

Detailed ways

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

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

material preparation

1. Strains and tool plasmids

19-T Vector(Code No.D102，Takara公司)，pSuper1300+-Kanamycin，pDR195(资环院袁力行老师惠赠)；pUC-GFP(Takara Bio)。Materials used in the examples of the present invention include: Escherichia coli competent cell DH5α (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 vector:

19-T Vector (Code No. D102, Takara Company), pSuper1300+-Kanamycin, pDR195 (gifted by Mr. Yuan Lixing, Institute of Information Environment); pUC-GFP (Takara Bio).

2. Tool enzymes and biochemical reagents

Various restriction enzymes were purchased from NEB company; various Taq enzymes and Trizol RNA mini-extraction kits were purchased from Takara company; dNTP mixture was purchased from Shanghai Sangon; T4 DNA ligase was purchased from Promega company; ordinary agarose gel DNA recovery kit (Code No. DP209, Tiangen Biochemical Technology Co., Ltd.); Plasmid Mini Kits were purchased from (Code No. DP103, Tiangen Biochemical Technology Co., Ltd.) Ampicillin (Amp), Kanamycin (Kan) , Rifampicin (Rif) was purchased from Xinjingke Company.

3. PCR amplification primers

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 corn sugar transporter gene ZmSTP1 gene

The amino acid sequences of monosaccharide transporters from Arabidopsis thaliana, rice and wheat were obtained from NCBI, Maizesequence and Uniprot, and were aligned using ClustalW1.8 (Thompson et al. 1994 Nucleic Acids Research 22, 4673-4680). Phylogenetic tree analysis indicated the closest relative to AtSTP1, so the gene was named ZmSTP1 (Fig. 1).

1. Total RNA extraction

Take 200 mg of fresh B73 corn root and grind it in liquid nitrogen; add 1 ml of Trizol extract provided by the kit, shake at room temperature for 5 minutes; then add 200 μl of chloroform, shake for 30 seconds, and centrifuge at 4°C and 12,000 rpm for 15 minutes; Add 0.5 ml of isopropanol, let stand for 1 hour at room temperature, and centrifuge at 4°C and 12,000 rpm for 15 minutes; take the precipitate, add 1ml of 70% ethanol, shake for 1 minute, and centrifuge at 4°C and 10,000 rpm for 10 minutes; aspirate and discard the supernatant , the precipitate was placed in a fume hood to dry, and 50 μl of DEPC water was added to dissolve the precipitate. RNA quality was detected by 1% agarose electrophoresis, and RNA concentration was determined by spectrophotometer.

2. mRNA purification

Take 500 μg of total RNA into a new RNase-free centrifuge tube, add 1 ml of the binding solution provided by the kit; place at 65°C for 10 minutes, then immediately transfer to ice for 1 minute; transfer the liquid to the containing solution provided by the kit. In a centrifuge tube of oligo(dT) resin, shake gently for 20 minutes at room temperature, centrifuge at 4000 rpm for 10 minutes at room temperature, carefully aspirate the supernatant, repeat twice; then add 0.3 ml of binding solution to resuspend the resin, and transfer to the spin provided by the kit -column tube, add 500 μl of binding solution to wash, centrifuge at 4000 rpm for 10 seconds at room temperature, measure the OD ₂₆₀ of the eluate, if it is greater than 0.05, add 500 μl of binding solution again to wash until the OD ₂₆₀ of the eluate is less than 0.05; add 200 μl of washing solution Remove the liquid, suspend the resin gently, transfer the spin-column tube to a new centrifuge tube, and centrifuge at 4000 rpm for 10 seconds at room temperature to collect mRNA. Finally, add 10 μl of 2 mg/ml glycoside, 30 μl of 2M sodium acetate, 600 μl of absolute ethanol, place at -80°C for 30 minutes, centrifuge at 14,000 rpm for 20 minutes at 4°C, discard the supernatant, wash once with 70% ethanol, and dissolve with 20 μl of TE. Remove 0.5 μl to measure the OD ₂₆₀ and calculate the concentration.

3. cDNA first-strand synthesis

Take 5μg of mRNA and use the reverse transcriptase included in the kit for reverse transcription. The details are as follows: add 1 μl Biotion-attB2-oligo(dT) provided by the kit to 10 μL of the mRNA solution purified in the previous step with a concentration of 100 ng/μl, place at 70°C for 5 minutes, quickly transfer to ice for 3 minutes, and then Add the following ingredients:

The following reaction conditions were set according to the kit instructions: 25°C for 10 minutes, 42°C for 60 minutes, 70°C for 10 minutes, and ice bath for 2 minutes.

4. ZmSTP1 clone

Using specific cloning primers (ZmSTP1-OE-F, ZmSTP1-OE-R) and the above cDNA as templates to clone STP1, the specific operation process:

Add the following components to a 200 μl centrifuge tube:

After centrifugation and mixing, PCR was performed. The PCR reaction procedure was as follows:

Agarose electrophoresis gel detection and recovery, according to the operation steps of Tiangen spin column kit.

To add A tail, add the following to a 200 μl centrifuge tube:

7 μl of purified DNA product

19-T TA克隆试剂盒，将回收的扩增片断克隆到T Vector上，构建重组质粒。After shaking, incubate at 72°C for 30min. use

19-T TA cloning kit, clone the recovered amplified fragments into T Vector to construct recombinant plasmids.

19-T载体上，进行测序，测序正确后保菌备用。The cells were ligated overnight at 16°C and transformed into 50 μL of competent cells by heat shock. 37°C, 150rpm shaker for activation and shaking for 60min; draw 100 μl of bacterial liquid and spread it on LB+Amp solid medium plate, and place it in a 37°C constant temperature incubator for overnight (12-16 hours). Single clones were picked for verification sequencing. Using the correctly sequenced plasmid as a template, add different restriction sites according to the subsequent experiments, repeat the operation 4, and ligate the ZmSTP1 containing restriction sites in

19-T carrier, sequenced, and kept the bacteria for later use after the sequencing was correct.

Example 2. Real-Time PCR analysis of the expression characteristics of the maize ZmSPT1 gene in each tissue.

The tested materials were planted at the Shangzhuang Experimental Station of China Agricultural University, and corn samples of different tissues were harvested one week after spinning, quickly placed in liquid nitrogen, and brought back to the laboratory to be placed in a -80 degree refrigerator for use.

Quantitative results showed that ZmSTP1 was expressed at the highest level in roots (Fig. 2).

Attached Real-Time PCR operation steps:

1. Extract the total RNA in the roots of the samples with different treatments (the method is the same as that in Example 1);

2. Take 50 μg of total RNA and remove genomic DNA with DNase I (TaKaRa company, catalog number: D2215), the method is as follows:

Reaction system (50μl):

37°C for 30 minutes;

Add 150 μl DEPC water, add 200 μl phenol/chloroform/isoamyl alcohol (25:24:1), and mix well;

Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the upper layer to a new centrifuge tube;

Add 200 μl chloroform/isoamyl alcohol (24:1) and mix well;

Centrifuge at 12000rpm at 4°C for 10 minutes, take the upper layer and transfer it to a new centrifuge tube;

Add 20 μl of 3M NaAc (pH=5.2), add 500 μl of pre-cooled absolute ethanol, and place at -20°C for 60 minutes;

Centrifuge at 12,000rpm for 15 minutes at 4°C, recover the precipitate, and wash the precipitate twice with 70% pre-cooled ethanol; centrifuge at 7,500rpm for 5 minutes at 4°C each time;

Dry and redissolve in DEPC water.

3. Synthesize the first strand of cDNA by reverse transcription in a conventional method (the method is the same as that in Example 1).

4. Real-time PCR was used to detect gene abundance. The reagents were SYBR Green RealtimePCR Master Mix (catalog number 91620F3) from TOYOBO Company, and the quantitative PCR instrument model was Bio-Rad iCycler iQ5system (BIO-RAD Company). The inversion products were diluted 10 times as Real-time PCR template

reaction system:

PCR reaction program: 50°C for 2 minutes, 95°C for 10 minutes, 45 cycles (95°C for 15 seconds, 61°C for 30 seconds, 72°C for 1 minute);

Melting curve steps: 95°C for 15 seconds, a cycle of 10 seconds, each cycle increases the rate of 0.5°C from 60°C to 95°C for 70 cycles;

Using ZmUbi as the internal reference, the relative expression of ZmSTP1 in different tissues was calculated by the relative quantification algorithm.

Example 3. Using in situ hybridization and green fluorescent protein technology to analyze the expression and localization of ZmSPT1.

1. Preparation of plant material

Hoagland medium was used for plant culture, and the root tips of maize seedlings at the three-leaf stage were taken. Cut the root tip 0.5-1cm and put it into FAA fixative solution (each 100ml of fixative solution contains: 50% ethanol 90ml, glacial acetic acid 5ml, formaldehyde 5ml);

Then the plant material is dehydrated, transparent, and wax-dipped, as follows:

Discard FAA fixative and wash twice with DEPC;

50% ethanol, 50% ethanol+10% tert-butanol, 50% ethanol+20% tert-butanol, 50% ethanol+35% tert-butanol, 50% ethanol+50% tert-butanol, 25% ethanol+75% tert-butanol, 25% ethanol+75% tert-butanol+0.1% eosin Y), and 100% tert-butanol were treated for 2 hours in turn;

Transfer to 2/3 tert-butanol + 1/3 paraffin oil for 4 hours;

Pour out 1/3 of the tert-butanol paraffin oil mixture, add the same volume of paraffin wax melted at 60°C to the upper layer to form a solidified wax cover, and place it at 60°C for 12 hours (repeat 3 times);

Pour out all the liquid, add pure melted paraffin, 60 ℃, 8 hours, (repeated 2 times);

It was then embedded on a 65°C ironing plate.

2. Probe synthesis and purification

Synthesize RNA probes while preparing plant materials. Refer to T7-RNA polymerase (catalog number: P2075) from Takara Company. The specific operations are as follows:

BGI synthesizes forward primers containing T7 promoter and ZmSTP1:

5′-GATTTAGGTGACACTATAGAATGCTCCAAAAACGGCAGCGACA-3′

Reverse primer:

5-TGTAATACGACTCACTATAGGGGTACTATTGCTTGGTGGTG-3';

The DNA template containing the T7 promoter was amplified from the plasmid with KOD enzyme, and transcribed into an RNA probe in vitro. The reaction system is as follows:

A total of 20 μl, mixed, and reacted at 37°C for 2 hours;

Add 4 μl of DNase I (TaKaRa company, catalog number D2215) and react at 37°C for 15 minutes to remove genomic DNA;

After the reaction, put it on ice, and add 0.8 μl of 0.5M EDTA (pH=8.0) to stop the reaction;

Add 2μl 5M LiCl, 75μl -20℃ pre-cooled absolute ethanol, mix well and place at -20℃ for 2 hours;

Centrifuge at 13000rpm, 4°C for 15 minutes;

Discard the supernatant, add 50 μl of 70% pre-cooled ethanol to wash the pellet, and centrifuge at 13,000 rpm at 4°C for 5 minutes;

Discard the supernatant and dry the pellet;

Add 24 μl DEPC-H2O to dissolve, and store at -80°C for later use.

3. Production:

The embedded wax blocks were cut into 8-10 μm slices with a Shanghai Hongyu QP-4 microtome. Cut the wax tape (containing plant samples) at the appropriate position and stick it on the slide of polylysine (Sigma company, catalog number P0425-72EA), put the wax tape into DEPC water at 45°C and put it on the baking stage to display the slide. After the slices are full, absorb the excess water, and bake the slices in a 40°C oven for 24-48 hours to fully dry the slices.

Section dewaxing: the slices were washed 3 times in xylene for 5 minutes each, 2 times in absolute ethanol for 2 minutes each, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol for 1 minute each, DEPC Wash 2 times, 1 minute each time.

Proteinase K treatment: Add proteinase K reaction buffer (100mM Tris-HCl pH7.5, 50mM EDTA) and proteinase K to a final concentration of 1 μg/ml in a staining jar, put the pretreated slides, and incubate at 37°C for 20 minutes ;

Wash the slides twice with DEPC water, 1 minute each time;

Acetylation treatment: place the glass slide in a staining jar, add 40 ml of 0.1 mol/L triethanolamine solution (pH=8.0) dissolved in 100 μl of acetic anhydride, and place at room temperature for 10 minutes; pour off the solution, and wash with 2×SSC solution for two times. times, 7 minutes each time;

Tissue sections were dehydrated by sequentially washing the slides with different dilutions of aqueous ethanol (30%, 50%, 70%, 85% and 95%) for 1 min at room temperature. Then wash twice with fresh absolute ethanol for 2 minutes each. Dry at room temperature.

4. Hybrid

Hybridization solution composition: 200 μl hybridization solution per slide, including: 100 μl deionized formamide, 20 μl 10× hybridization buffer (100 mM Tris pH 7.5, 10 mM EDTA, 3M NaCl), 24 μl 50% dextran sulfate, 20 μl 10× Blocking Solution, 250 μg salmon sperm DNA, 5 μl probe, 36.5 μl 50× Denhardt’s solution. Hybridize in the dark for 16-30 hours.

Rinsing: The slides were immersed in 2×SSC to make the coverslip fall off, and then placed at room temperature for 30 minutes; replaced with new 2×SSC, 65°C for 1 hour; 0.1×SSC, 65°C for 1 hour.

Blocking: Dry the back of the slide with absorbent paper, place it in a wet box, add 2ml of 1% blocking solution to each slide (each 400ml contains 2g of Boehringer Block reagent, 0.1M Tris-HCl, pH=7.5; and 0.15M NaCl ) at room temperature for 1 hour.

Equilibration: Remove the blocking solution, add 1 ml of washing solution (100 mM Tris-HCl pH=7.5, 150 mM NaCl, 0.3% Triton X-100, 1% BSA) to each piece and equilibrate for 15 minutes.

Antibody adsorption: remove the equilibration solution, add 400 μl antibody solution (each 400 μl antibody solution contains: 399 μl washing buffer, 1.32 μl Anti-DIG-AP provided by the kit), hybridize for 2 hours at room temperature or overnight at 4°C.

Washing: The slides were washed 3 times in washing solution for 10 minutes each time.

Equilibration before color development: immerse in color development buffer for 5 minutes (1ml color development buffer formula: 100 μl 1M Tris-HCl pH9.5, 20 μl 5M NaCl, 860 μl ddH2O, 0.1 g polyhexenol).

Color development: add 20μl of NBT/BCIP to the color development buffer, add 200-500μl of color development solution dropwise to each piece, and develop color in a dark place at room temperature for 0.5-4h in a wet box. The reaction can be stopped when the background has no obvious color, and washed with water 3 times for 5 minutes each time. After mounting with neutral gum, the reddish-brown positive signal turned blue or blue-purple.

The results are shown in Figure 3a, which are the results of probe hybridization. The upper side of the two figures is a longitudinal section of the root apex, and the lower side is a cross-section of the root apex. The results showed that ZmSTP1 was expressed in the entire maize root tip, which is consistent with its biological function of mediating root uptake of sugar from the environment.

Using the PUC-GFP vector and the 35S promoter, the expression vector of the recombinant plasmid fused with ZmSTP1 was constructed, and ZmSTP1 was transiently expressed by injecting tobacco leaves. The results showed that it was expressed on the cell membrane and nucleus (Figure 3b).

Example 4. ZmSTP1 yeast heterologous functional complementation verification

19-T上切下来，电泳回收，连入pDR195载体上的XhoI和BamHI酶切位点之间，经测序选取正向连入的克隆。Both ends of the pDR195 vector contain XhoI and BamHI restriction sites. Take 1-1.5ml of bacterial liquid, refer to the instructions of Tiangen Plasmid Extraction Kit, extract the plasmid, select XhoI and BamHI to extract the ZmSTP1 gene from ZmSTP1.

19-T was excised, recovered by electrophoresis, and ligated between the restriction sites of XhoI and BamHI on the pDR195 vector. The clones that were connected in the forward direction were selected by sequencing.

The EBY.VW4000 yeast mutant, lacking all hexose and galactose sugar transporters, cannot survive in environments where monosaccharides are the sole carbon source (Wieczorke et al., 1999), but can survive in environments where maltose is the carbon source. environment, and using this feature we examined the function of ZmSTP1. The yeast pDR195 recombinant plasmid was constructed and introduced into the EBY.VW4000 yeast mutant through the above operations, and the yeast activity was analyzed under a certain carbon source environment.

Yeast expression vector into EBY.VW4000 yeast mutant

The yeast to be transformed was inoculated into 5 ml of liquid YPD medium, and cultured overnight at 30°C with shaking at 200 rpm. Determine the concentration to OD ₆₀₀ = 0.5 and inoculate it into 50 ml of YPD medium, shake and culture at 30 ° C, 200 rpm to OD ₆₀₀ = 2, centrifuge at 3000 × g for 5 min to collect cells, discard the supernatant culture medium, and suspend the cells in 25 ml of sterile water. Then centrifuge to collect the cells, discard the water, suspend the cells in 1ml sterile water, transfer to a sterile 1.5ml centrifuge tube, centrifuge, remove the supernatant, and add 1ml sterile water to suspend the cells,

Aliquot the cell suspension according to the transformation volume (about 200 μl), centrifuge again for 1-2 min to pellet the cells, carefully aspirate the supernatant with a pipette, and then add the premixed transformation mixture:

Vigorously shake until the cells are completely mixed, heat-shock for at least 40 min in a 42°C water bath (Suga and Hatakeyama, 2005), centrifuge at high speed for 30 sec, remove the transformation mixture, add 0.2-1.0 ml of sterile water, and gently pipette up and down. Lightly extract the suspended pellet.

The pDR-EBY.VW4000 transformed into the empty vector was the negative control, and the wild-type pDR-23344c was the positive control. After transformation, screening culture was carried out on solid agar medium with 6.7 g/L YNB (yeast nitrogen base) in which uracil was replaced by ammonium sulfate (0.5 g/L) maltose (20 g/L). The growth medium was supplemented with 2% glucose, fructose, galactose or mannose as the sole carbon source and ammonium sulfate (0.5 g/L) as the nitrogen source on the basis of the screening medium. Take the yeast liquid with OD ₆₀₀ = 1.0, dilute it into four gradients of 10 ^-1 , 10 ^-2 , 10 ^-3 , and 10 ^-4 , pipette 10 μl onto the previous medium, and invert at 30°C for 3 days Observe the phenotype. Each treatment was repeated three times with a different monoclonal. In sharp contrast to the mutants, ZmSTP1-introduced yeast grew significantly on glucose (Glc), fructose (Frc), and mannose (Man), and the growth condition was also improved to some extent under the condition of galactose (Gal) supply (Figure 4). , indicating that ZmSTP1 can absorb a variety of monosaccharides after being transferred into the yeast system, but the absorption capacity of different monosaccharides is different.

Attached yeast plasmid DNA extraction method:

The growing plaques were picked and placed in 0.5 ml of YNB liquid medium containing 1 mM Arg, and incubated overnight at 30°C with shaking at 230 rpm; the cells were collected by centrifugation at 4,000 rpm for 5 minutes;

Pour off the supernatant, resuspend the cells with fresh liquid medium (total volume is about 50 μl), then add 10 μl of lysozyme solution with a concentration of 10 mg/ml to each tube, and shake well to mix the solution and the cells completely;

Incubate the test tube at 30°C, shaking at 230rpm for 60 minutes;

Add 10 μl 20% SDS to each tube, shake vigorously for 1 minute to mix well;

Put the sample at -20°C for 2 hours, take it out and thaw, and shake vigorously to fully lyse;

Make up the volume of each tube to 200 μl with TE buffer (pH=7.0);

Add 200 μl phenol: imitation: isoamyl alcohol (25:24:1), shake vigorously for 5 minutes; centrifuge at 14,000 rpm for 10 minutes, transfer the supernatant to a new centrifuge tube;

Add 8 μl 10M NH ₄ Ac and 500 μl absolute ethanol;

Place in -80°C refrigerator for 1 hour, centrifuge at 14,000rpm for 10 minutes;

Discard the supernatant, blow dry the pellet, and dissolve the pellet with 20 μl H ₂ O;

Take 0.5 μl of the plasmid and transfer it to E.coli competent cells (DH5α strain), shake the bacteria at 37°C, extract the plasmid, identify it by enzyme digestion, and send it to the company for sequencing.

Example 5. Construction, transformation and phenotype analysis of ZmSTP1 Arabidopsis thaliana overexpression vector.

Wild-type Arabidopsis col was transformed with the recombinant expression vector pSuper1300+-ZmSTP1. Specific method: take 0.5ml of Agrobacterium liquid that has been identified as positive and inoculate it into 500ml YEB liquid medium, shake and culture at 28°C to an OD of ₆₀₀ to 0.5. Centrifuge at 5000rpm for 15 minutes at 4°C to collect bacteria. The cells were resuspended with 200 ml of infiltration buffer (1×MS macroelement, 5% sucrose), and silwet L-77 (GE Company, product number: S5505) was added to the final concentration of 0.2%. 1×major elements contain 1.65g/LNH ₄ NO ₃ , 1.9g/L KNO ₃ , 0.44g CaCl ₂ ﹒ 2H ₂ O, 0.37g/L MgSO ₄ ﹒ _7H2O and 0.17 g/L _{KH2PO4 .} The flowers of Arabidopsis thaliana that have just bloomed after bolting were immersed in the resuspension for 30 seconds to infect. The plants were wrapped in a fresh-keeping bag, placed in the dark at 16°C for 24 hours, and then upright and normal growth until the T ₀ generation seeds were harvested.

1. Screening of transgenic positive plants

Since the Arabidopsis plants transformed into the pSuper1300+-ZmSTP1 vector are resistant to hygromycin, they can grow normally on MS solid medium containing hygromycin, while the wild-type seeds without the transgene cannot grow normally and die. The transformed contemporary transgenic plant is the T ₀ generation, and the seeds produced by the self-crossing of the T ₀ generation plant and the plants grown therefrom are the T ₁ generation. The seeds of the T ₁ generation were mixed and collected, sown on MS solid medium containing 50 μg/ml hygromycin, and the plants that could grow normally were screened and transplanted into pots to continue to grow, and each plant was harvested. The T ₂ generation seeds were screened for hygromycin resistance once again, and the T ₃ generation seeds were harvested from a single plant. After another resistance screening, all individuals can grow as homozygous plants transfected with pSuper1300+-ZmSTP1, which are reserved for future use.

2. Molecular detection of transgenic Arabidopsis

PCR detection: Extract the total RNA of Arabidopsis thaliana homozygous plants transfected with pSuper1300+-ZmSTP1 gene of T ₃ generation, reverse transcribed into first-strand cDNA under the guidance of oligo(dT), and use ZmSTP1-RT-L and ZmSTP1-RT- R is the primer for RT-PCR to detect the expression level of ZmSTP1 gene in transgenic Arabidopsis.

3. Analysis of monosaccharide absorption capacity of transgenic plants

The T ₃ generation seeds were vernalized and surface sterilized, and seedlings were cultured on ATS sugar-free solid agar medium (Schofield et al., 2009 Plant, Cell and Environment 32, 271-285). After 4 days of treatment, 3 ZmSTP1-OE lines and Col-0 seedlings with the same growth vigor were selected and transferred to a square (13×13 cm ² ) screening medium. The screening medium was the previous seedling medium, and different concentrations of sugar were added as carbon sources, namely: glucose (Glc), fructose (Frc), sucrose (Suc), ribose (Rib), galactose (Gal), Inositol (MI), Xylose (Xyl), Mannose (Man). Sugar-free ATS medium was a negative control, and ATS medium supplemented with trimethyl glucose (a glucose analog, which cannot be phosphorylated by hexokinase) was a positive control. Under the supply of Glc (2, 5, 10 mM), Frc (5, 10 mM), Suc (5 mM) or Rib (5, 55 mM), the number of rosette leaves increased significantly; high concentrations of Glc, Frc, Gal, Suc, Xyl, Rib, Man, MI decreased the number of rosette leaves (data not listed). The rosette leaf diameter increased at low concentrations of Glc, Frc, Rib carbon sources and decreased at high concentrations of Glc, Frc, Gal, Suc, Xyl, Rib, Man or MI, and the final biomass was also significantly different (Fig. 5, Fig. 6) . Under the simultaneous addition of 9 or 1 mM _NO3 ^- nitrate at 55 mM different carbon sources, growth was significantly inhibited (Fig. 7), indicating that the overexpressed plants were more sensitive to nitrogen.

In soil culture experiments, transgenic Arabidopsis and wild type were grown under short-day conditions (8/16 day/night, 84 μmol m ⁻² s ⁻¹ , 22° C.) until 35 days. Short-day light was changed to long-day light after 35 days (16/8h day/night, 56 μmol m ^-2 s ^-1 , 22°C) until maturity at 55 days. The height of the first inflorescence, the fresh weight of the plant, the dry weight, and the weight of each seed were counted. Soluble sugars were extracted by the chloroform/methanol method (Antonio et al., 2008 Rapid Communications in Mass Spectrometry 22, 1399-1407) and determined by liquid chromatography spectrophotometry (HP 1100, Agilent Technologies, Palo Alto, CA, USA) according to Focks et al. Human method Starch content was determined using a kit (Sigma-Aldrich, St. Louis, USA). Under short-day conditions, the vegetative growth of ZmSTP1-overexpressing plants was promoted, manifested as enlarged leaves, increased petiole length, increased number, and significantly increased rosette leaf diameter (Fig. 8). Under long-day conditions, overexpressing plants exhibited increased inflorescence number, increased dry weight, and darker leaf color (Fig. 9). The expression of AtSUC2 (sucrose transporter 2) was up-regulated in the overexpressed plants (Fig. 10), indicating that ZmSTP1 affects the transport of other carbohydrates; ZmSTP1 may also be related to the light and pathway, and AtCAB1, a protein related to the synthesis of chlorophyll a and chlorophyll B, was upregulated in the overexpressed plants (Figure 11). Taken together, ZmSTP1 may ultimately increase production by affecting the transport of other carbohydrates and regulating photosynthetic pathways.

Attachment: Arabidopsis culture and screening of transgenic seedlings

Take an appropriate amount of Arabidopsis seeds into a 1.5mL centrifuge tube and add deionized water. Make the water cover all the seeds as much as possible, and place them in the refrigerator at 4°C for vernalization for 2 days. Disinfect the seeds in an ultra-clean bench, add 75% ethanol to sterilize for 1 minute, rinse with sterilized water, then add 2% sodium hypochlorite for 2 minutes, and rinse with sterilized water for 5-7 times. Use a 10μL pipette to evenly spot the seeds on the 1/2MS (or 1/2MS+50mg/ml hygromycin) medium, and place the seeded medium vertically in the culture room (light: 100 μE m ^-2 s ^-1 , photoperiod: 16h day/8h night, temperature 22/20°C, humidity 100%). The seedlings with better growth were transplanted into flowerpots containing moist nutrient soil (m vermiculite/m nutrient soil=1:1). 4 plants per pot, 12 pots per plate, and the surface of the flower pot is covered with a layer of plastic wrap to prevent excessive evaporation of water from affecting the growth of seedlings. Pour 400ml of water every 5 days, remove the plastic wrap after 10 days, and pour 400ml of water every 3 days.

The seeds of T ₀ generation Arabidopsis were vernalized and sterilized, and the seeds were evenly spread on 1/2 MS selection medium (containing 50 μmol/L hygromycin). Wrap a layer of black plastic film, place it in the laboratory, and cultivate in the dark for 5-7 days. If the seedling stems are very high, it may be a resistant transgenic seedling. Select 12 seedlings from each transgenic plant, number them, and move them to 1/2MS The medium is cultivated for 3-5 days, and after 4 young leaves are grown, it is moved to nutrient soil and placed in a culture room for cultivation to complete its entire growth period. The above operation was repeated until homozygous T ₃ generation seeds were obtained.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (6)

1. Application of the maize ZmSTP1 gene in regulating the photosynthetic pathway of plants; the nucleotide sequence of the ZmSTP1 gene is shown in SEQ ID No.2. 2 . The application according to claim 1 , wherein the gene is transferred into a plant to promote gene expression of plant photosynthetic pathway, thereby increasing plant biomass and/or grain yield. 3 . 3. The application according to claim 2, wherein the gene is transferred into a plant cell through a recombinant expression vector. The application 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, characterized in that the maize ZmSTP1 gene is transferred into plant cells through a recombinant expression vector; the nucleotide sequence of the ZmSTP1 gene is shown in SEQ ID No.2. The method according to claim 5, wherein the recombinant expression vector is a recombinant plasmid obtained by inserting the gene between the multiple cloning sites of pPT-HYG.

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