Corn sucrose transporter ZmSUT3J and coding gene and application thereof
阅读说明：本技术 一种玉米蔗糖转运蛋白ZmSUT3J及其编码基因和应用 (Corn sucrose transporter ZmSUT3J and coding gene and application thereof ) 是由 贺红霞 李楠 朱旭 柳青 郭嘉 孙传波 李传龙 金峰学 李海云 贺红利 杨春明 于 2019-11-26 设计创作，主要内容包括：本发明涉及一种玉米蔗糖转运蛋白ZmSUT3J及其编码基因和应用,属于植物生物工程技术领域。玉米蔗糖转运蛋白ZmSUT3J有氨基酸序列如SEQ ID No.2所述,所述玉米蔗糖转运蛋白ZmSUT3J基因在培育耐逆植物和提高植物产量中的应用,所述植物为拟南芥和玉米。本发明提供的蛋白及其编码基因,可以调控植物生长和发育,受多种胁迫诱导,参与植物对多种胁迫的响应。将本发明的基因重组载体导入植物,可以提高植物的耐盐性。本发明提供的蛋白及其编码基因对于培育耐逆性好的玉米及其他植物新品种具有重要实用价值。本发明在玉米中超表达能够提高玉米的耐逆性及产量,本发明可以应用到玉米育种中,对提高玉米耐逆性和产量有着积极的作用。(The invention relates to a corn sucrose transporter ZmSUT3J, and a coding gene and application thereof, belonging to the technical field of plant bioengineering. The corn sucrose transporter ZmSUT3J has an amino acid sequence shown in SEQ ID No.2, and the corn sucrose transporter ZmSUT3J gene is applied to cultivating stress-tolerant plants and improving the yield of plants, wherein the plants are Arabidopsis thaliana and corn. The protein and the coding gene thereof provided by the invention can regulate and control the growth and development of plants, are induced by various stresses and participate in the response of the plants to the various stresses. The salt tolerance of the plant can be improved by introducing the gene recombinant vector into the plant. The protein and the coding gene thereof provided by the invention have important practical values for cultivating corn with good stress tolerance and other new plant varieties. The overexpression in the corn can improve the stress tolerance and the yield of the corn, and the invention can be applied to corn breeding and has a positive effect on improving the stress tolerance and the yield of the corn.)
1. A corn sucrose transporter zmsout 3J, comprising: the amino acid sequence is shown in SEQ ID No. 2.
2. The corn sucrose transporter ZmSUT3J of claim 1, wherein: the sucrose transporter contains 474 amino acids, and amino acid residues from the 22 nd position to the 495 th position of the amino terminal are conserved structural domains of GPH-sucrose superfamily.
3. A maize sucrose transporter zmsout 3J gene, comprising: the nucleotide sequence is shown in SEQ ID No. 1.
4. A recombinant bacterium or expression cassette comprising the maize sucrose transporter zmsout 3J gene of claim 3.
5. A plant expression vector comprising the maize sucrose transporter zmsout 3J gene of claim 3, having the nucleotide sequence set forth in SEQ ID No. 3.
6. The plant expression vector of claim 5, wherein: the plant expression vector is a recombinant plasmid obtained by inserting a corn sucrose transporter ZmSUT3J gene into a multiple cloning site of pCAMBIA3300 containing ZmSUT4pro promoter.
7. The use of the maize sucrose transporter ZmSUT3J gene of claim 3 for breeding stress tolerant plants, Arabidopsis and maize.
8. The use of the maize sucrose transporter ZmSUT3J gene of claim 7 for breeding stress tolerant plants that are salt and alkali tolerant.
9. The use of the maize sucrose transporter ZmSUT3J gene according to claim 3 for increasing yield in plants selected from but not limited to maize.
10. A method for producing transgenic plants, comprising introducing the target gene of claim 1 into plants to obtain transgenic plants having higher stress tolerance than the plants, wherein the target plants are Arabidopsis thaliana and maize.
Technical Field
The invention relates to the technical field of plant bioengineering, in particular to a corn sucrose transporter ZmSUT3J, and a coding gene and application thereof.
Background
Corn is an important grain and feed crop and is also a main raw material in the pharmaceutical industry, the sugar industry, the starch industry, the oil industry, the alcohol industry and the like. Corn belongs to stress-sensitive plants, and is easily interfered and damaged by stress conditions. Soil salinization has become a major environmental factor for corn yield and quality improvement. The plant absorbs water and fertilizer in soil by a root system and generates sugar through photosynthesis of leaves, and the sugar not only serves as an energy source and a structural substance for plant metabolism, but also serves as a signal molecule to participate in a plurality of metabolic processes in the plant body, so that a plurality of physiological and biochemical processes of seed germination, hypocotyl elongation, cotyledon extension, root system growth and development, flowering, senescence and the like of the plant are influenced. Researches of king yufeng and the like find that a large amount of soluble sugar can be accumulated in a corn body under the salt stress to relieve the damage of the salt stress to cells (king yufeng, king celebration et al 2007). Glucose (Glc) and sucrose (Suc) among soluble sugars play an important role in plant growth and development and can be used as signal substances to participate in regulating and controlling plant responses to biotic and abiotic stresses. Sucrose is synthesized in the cytoplasm of plant cells and is the main carbohydrate form, which is also transported from leaves to other parts of the plant body. In addition, Sucrose is an important signal molecule in plants, and is involved in the conduction of plant sugar signals and the regulation of plant growth and development processes, and Sucrose transporters (abbreviated as SUT or SUC) are 12-time transmembrane proteins playing an important role in the transportation and unloading of Sucrose. The sucrose transporter is used as a sucrose transport tool and participates in phloem sucrose unloading, and the plant source-sink relationship and carbon-nitrogen metabolism are regulated to a great extent.
The sucrose transporter is a main regulation factor for adapting to the stress of plants, the regulation and control action mechanism of the sucrose transporter of the plants is known, the relationship between the sucrose transporter and the stress of the plants is revealed, and the sucrose transporter has guiding significance for improving the crop yield and cultivating new varieties with stress tolerance.
Chinese patent application No. CN105255888A discloses that the promoter has cis-acting elements related to specific expression regulation and multiple hormone regulation, GUS histochemical staining proves that the promoter can be used for expressing exogenous genes in roots, leaves and stems of plants, and lays a foundation for the cultivation of transgenic plants.
Disclosure of Invention
The invention provides a corn sucrose transporter ZmSUT3J, and a coding gene and application thereof.
A corn sucrose transporter zmsout 3J, the amino acid sequence of which is set forth in SEQ ID No. 2.
A corn sucrose transporter ZmMUT 3J comprises a sucrose transporter of 474 amino acids, and the 22 nd to 495 th amino acid residues from the amino terminal are GPH-sucrose superfamily conserved structural domains.
The corn sucrose transporter ZmSUT3J gene has a nucleotide sequence shown in SEQ ID No. 1.
The invention provides a primer sequence for homologous cloning of a corn sucrose transporter gene, wherein the names of the primers are respectively ZmS3 BamH: 5 'CGGGATCCCGATGGCTGGTGATGGCATGGAGGTA 3' and ZmS3 SpeRev: 5 'CGAGCTCGTCAGTGCCCTCCTCCCATGGAGACG 3'.
A recombinant strain containing the corn sucrose transporter ZmSUT3J gene.
An expression cassette comprising the maize sucrose transporter ZmSUT3J gene.
The nucleotide sequence of the plant expression vector containing the corn sucrose transporter ZmSUT3J gene is shown in SEQ ID No. 3.
The plant expression vector is a recombinant plasmid obtained by inserting a corn sucrose transporter ZmSUT3J gene into a multiple cloning site of pCAMBIA3300 containing ZmSUT4pro promoter.
The application of the corn sucrose transporter ZmSUT3J gene in cultivating stress-tolerant plants, wherein the plants are arabidopsis thaliana and corn.
The application of the corn sucrose transporter ZmSUT3J gene in cultivating stress tolerant plants, wherein the stress tolerance is salt and alkali tolerance.
The application of the corn sucrose transporter ZmSUT3J gene in improving the yield of plants, wherein the plants are corn, but not limited to the corn.
A method for cultivating transgenic plants is to introduce the target gene into plants to obtain transgenic plants with higher stress tolerance than the plants, wherein the target plants are arabidopsis thaliana and corn.
The protein and the coding gene thereof provided by the invention can regulate and control the growth and development of plants, are induced by various stresses and participate in the response of the plants to the various stresses. The salt tolerance of the plant can be improved by introducing the gene recombinant vector SEQ ID No.3 into the plant. The protein and the coding gene thereof provided by the invention have important practical values for cultivating corn with good stress tolerance and other new plant varieties. Experiments prove that the over-expression of the ZmSUT3J gene in the corn can improve the stress tolerance and yield of the corn, and the ZmSUT3J gene can be applied to corn breeding and has a positive effect on improving the stress tolerance and yield of the corn.
Drawings
FIG. 1A is the electrophoresis chart of total RNA of the inbred line of Ji 853 maize, in which 1-4 are root, stem, pistil and tassel;
FIG. 1B is a graph of RT-PCR amplification of the ZmSUT3J gene, where M: DL2000 molecular weight Marker, 1-4 are cDNA of root, stem, pistil and tassel as template;
FIG. 2 is a vector map of plant expression vector pCAM-S4P-SUT 3J;
FIG. 3A is the result of RT-PCR of T3 plant of transgenic pCAM-S4P-SUT3J Arabidopsis thaliana; taking Marker as the center, the left side 1-12 is SUT3 amplification result, and the right side 1-12 is internal reference TUB2 amplification result; the samples were: 1-3: s8-2-3; 4-6: s8-2-4; 7-8: s8-2-5; 9-10, S4P-GUS; 11-12: wild type, marker: gene ruler mix (Thermoscientific);
FIG. 3B is the T2 phenotype of ZmSUT3OE transgenic Arabidopsis thaliana, 10d post-transplant, with wild type Arabidopsis thaliana on the left and SUT3J OE on the right (line 8-2);
FIG. 3C is the T2 phenotype of ZmSUT3OE transgenic Arabidopsis, 15d post-transplant, wild type Arabidopsis on the left and SUT3J OE on the right (line 8-2);
FIG. 3D is the T2 phenotype of ZmMUT 3OE transgenic Arabidopsis thaliana grown 14 days after addition of 140mM NaCl in MS0 solid medium, wild type Arabidopsis thaliana on the left and SUT3J OE on the right (strain 8-2);
FIG. 4A is an example of PCR detection of the bar gene in ZmSUT3J maize, 1 and 14: Marker 2K; 2, a plasmid; 3, water; 4-13,15-26, ZmSUT3J corn;
FIG. 4B is an example of PCR detection of the ZmSTT 3J gene using ZmSTT 3J maize, 1, 26, 27 and 52 Marker 2K; 2, a plasmid; 3, water; 4-25 and 28-51 ZmSUT3OE maize
FIG. 5 is a field phenotype of ZmSTT 3J transformed maize homozygous positive material AA (right side) and homozygous negative material AA (left side);
FIG. 6A is a PCR assay of ZmSUT3J transformed maize; 1, 2K Marker; 2, a plasmid; 3, water; 4, ZmSUT3 transgenic maize; 5, non-gene corn;
FIG. 6B is a RT-PCR assay of ZmSUT3J transformed maize;
FIG. 7 is a Southern hybridization of ZmSUT 3J-transgenic maize; m is 15 kbMarker; 1, a plasmid; 2, non-transgenic corn; 3, ZmSUT3 transgenic corn;
FIG. 8A is a graph of ear row number differences for ZmSUT3J corn;
FIG. 8B is a graph of ear thickness variation for ZmSUT3J transformed maize;
FIG. 8C is a graph of ear weight variation for ZmSUT3J transformed maize;
FIG. 8D is a graph of the difference in hundred kernel weight for ZmSUT3J transformed corn;
FIG. 8E is a graph of ear phenotype differences for ZmSTT 3J transgenic maize, wherein A is a
FIG. 9A is a graph of plant height variation under ZmSUT3J transgenic corn salt stress conditions;
FIG. 9B is a graph of plant height variation under ZmSUT3J transgenic corn alkali stress conditions;
FIG. 9C is a graph of stalk thickness differences under ZmSUT3J maize salt stress conditions;
FIG. 9D is a graph of stalk thickness differences under ZmSUT3J maize alkali stress conditions;
FIG. 9E is a graph of the difference in ear bearing rates under ZmSTT 3J maize salt stress conditions;
FIG. 9F is a graph of the difference in ear bearing rates under ZmSTT 3J maize alkali stress conditions.
Detailed Description
The invention is further illustrated by the following specific examples.
Experimental materials:
plasmid: pBACKZERO-T (Kyoto King Ltd.), pCAMBIA3300(Cambia Labs);
the strain is as follows: coli TransT1 (beijing holotype gold), agrobacterium EHA105 (commercially available);
plant material: the maize receptor material Hi-II, the Ji 853 maize inbred line and the tetra 287 maize inbred line are all planted in the test fields of Jilin province agricultural science institute.
The methods used in the following examples are all the methods described in general molecular biology, tissue culture techniques and genetic engineering, unless otherwise specified. The specific steps can be seen in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory)ry Manual,3rdedition, 2001, NY, Cold Spring Harbor). Various reagents and endonucleases: purchased from TaKaRa, Shanghai Czeri Biotechnology, Inc., the gene synthesis was performed by Czeri Biotechnology, Inc., and the sequencing analysis was performed by Huada gene.