High-yield gene OsXAT8, amplification method thereof and application of high-yield gene OsXAT8 in plant yield improvement

Abstract

The invention discloses a high-yield gene OsXAT8, an amplification method thereof and application of the high-yield gene OsXAT8 in plant yield improvement, and belongs to the technical field of gene engineering. The invention provides a high-yield gene OsXAT8. The nucleotide sequence of the high-yield gene OsXAT8 is as shown in SEQ ID NO. 1. The invention also provides a primer pair for amplifying the high-yield gene OsXAT8, a method for amplifying the high-yield gene OsXAT8 by using the primer pair, and an application of the high-yield gene OsXAT8 in increasing the plant yield. The high-yield gene OsXAT8 and the corresponding protein are applied to genetic engineering genetic breeding of plants to cultivate new varieties of high-yield rice, and the high-yield gene OsXAT8 and the corresponding protein have important reference significance for high-yield related research of other crops.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a high-yield gene OsXAT8 and an amplification method thereof and an application thereof in improving plant yield. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world and plays an irreplaceable role in ensuring national food security and people's basic living needs. Therefore, in-depth research on the high-yield regulation mechanism of rice and breeding and obtaining high-yield varieties have important practical application value in agricultural production.

[0003] In recent years, with the rapid development of molecular biology and genomics technology, research on the high-yield regulation mechanism of rice has made significant progress. Through a variety of modern biotechnology methods such as Agrobacterium-mediated transgenic technology, CRISPR / Cas9 gene editing technology, and RNA interference technology, researchers can overexpress, knock out or modify specific genes in rice, thereby deeply studying the functions of these genes in the growth and development process and their effects on yield traits. For example, by overexpressing genes related to photosynthesis, nutrient absorption, and stress resistance, the yield potential of rice can be significantly improved; and by precisely modifying yield-related genes through gene editing technology, the plant type, panicle type, and grain characteristics of rice can be optimized, thereby further increasing yield. These studies provide an important theoretical basis and technical support for analyzing the molecular mechanism of high crop yield.

[0004] In addition, with the widespread application of high-throughput sequencing technology, researchers can more comprehensively analyze rice genome information and explore important genes and molecular markers related to high yield. Through methods such as genome-wide association analysis (GWAS) and transcriptome analysis, key genes and regulatory networks that control rice yield traits can be identified, providing important candidate gene resources for molecular design breeding. Combining traditional breeding methods with modern molecular breeding technology can accelerate the selection and breeding of new high-yield, high-quality, and stress-resistant rice varieties, providing strong scientific and technological support for achieving sustainable development of rice production.

[0005] In short, the study of the high-yield regulation mechanism of rice not only helps to reveal the genetic basis and molecular mechanism of high-yield crops, but also provides a theoretical basis and technical means for breeding new high-yield, high-quality and stress-resistant rice varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a high-yield gene OsXAT8 and its amplification method and application in plant yield improvement to solve the problems existing in the above-mentioned prior art. The OsXAT8 gene provided by the present invention can improve rice yield and has a good application prospect in cultivating high-yield rice varieties. The present invention lays an important foundation for high-yield rice breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] Technical solution 1: A high-yield gene OsXAT8, the nucleotide sequence of the high-yield gene OsXAT8 is shown in SEQ ID NO.1.

[0009] Technical solution 2: The protein encoded by the high-yield gene OsXAT8.

[0010] Furthermore, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0011] Preferably, the protein includes: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2; 2) a protein derived from 1) with equivalent activity, in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.2.

[0012] Those skilled in the art can replace, delete and / or add one or more amino acids based on the amino acid sequence disclosed in the present invention without affecting its activity to obtain a mutant sequence of the protein.

[0013] Technical solution three: a set of primer pairs for amplifying the high-yield gene OsXAT8, including an upstream primer with a nucleotide sequence such as SEQ ID NO.3 and a downstream primer with a nucleotide sequence such as SEQ ID NO.4.

[0014] Technical solution 4: A method for amplifying the high-yield gene OsXAT8 using the primer pair, comprising the following steps: using the plant nucleic acid molecule as a template, preparing the template and the primer pair into a PCR amplification system for PCR amplification to obtain the high-yield gene OsXAT8.

[0015] Technical Solution 5: A method for increasing plant yield, which increases the plant yield by overexpressing the expression level of the OsXAT8 gene.

[0016] Preferably, the OsXAT8 gene is overexpressed in rice, including using any vector that can guide the overexpression of foreign genes in plants.

[0017] Preferably, it comprises a vector containing the OsXAT8 gene; a host containing the vector, or a transformed plant cell or transgenic plant containing the OsXAT8 gene or a specific fragment thereof.

[0018] In order to facilitate the identification and screening of transgenic plant cells or plants, the vector used can be processed, such as adding plant selectable markers or antibiotic resistance markers.

[0019] Technical Solution 6: Application of the high-yield gene OsXAT8, the protein or the high-yield gene OsXAT8 amplified by the method in increasing plant yield.

[0020] Furthermore, the plant includes rice.

[0021] The present invention discloses the following technical effects:

[0022] The present invention studies the gene OsXAT8 encoding the plant xylan synthase protein OsXAT8, and finds that the transgenic plant overexpressing the gene exhibits a phenotype of increased yield. The OsXAT8 gene has a nucleotide sequence shown in SEQ ID NO.1, and is composed of 1449 bases. The reading frame of the gene is from the 1st to the 1449th bases from the 5' end. The OsXAT8 gene is derived from Nipponbare rice, and is numbered as LOC_Os01g02930.1 in the rice genome database. After the OsXAT8 gene of the present invention is overexpressed, the rice exhibits a phenotype of increased yield. The OsXAT8 gene of the present invention provides a gene resource for breeding new varieties of high-yield crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Comparison of gene expression levels between the overexpression strain OsXAT8.OE and the control wild-type strain NPB;

[0025] Figure 2 The phenotypes of OsXAT8 overexpression strains and control wild-type strains are compared; OsXAT8.OE1 (a) and OsXAT8.OE2 (b) are OsXAT8 overexpression strains, and NPB (c) is a wild-type strain;

[0026] Figure 3The yield statistics of OsXAT8 overexpression strains and control wild-type strains, among which OE-1 and OE-2 are OsXAT8 overexpression strains, and NPB is a wild-type strain. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The pCAMBIA1300 vector in the following examples is a commonly used cloning vector and a commonly used cloning vector for Agrobacterium GV3101 strain, both of which can be purchased commercially; the rice variety is Nipponbare.

[0033] The main reagents in the following examples are: various restriction endonucleases, Taq enzyme, T4 ligase, Pyrobest Taq enzyme, KOD, etc. were purchased from biological companies such as TAKARA, Promega, NEB, and Toyobo; dNTPs were purchased from Genestar; plasmid mini-extraction kit and agarose gel recovery kit were purchased from Shanghai Jierui Bioengineering Co., Ltd.; MS medium, agar powder, agarose, spectinomycin (Spe), rifampicin (Rif), hygromycin and other antibiotics and LB Medium were purchased from Sigma; various other chemical reagents used in the examples were imported or domestic analytical reagents.

[0034] Example 1 Construction of OsXAT8 gene overexpression plants

[0035] 1. Construction of overexpression strain OsXAT8.OE

[0036] The gene OsXAT8 encoding plant xylan synthase protein was cloned from Oryza sativa L. spp. japonica. According to the sequence analysis of the coding region, the upstream and downstream primers were designed to amplify the coding region of the gene and connected to the overexpression vector pCAMBIA1300 with UBi promoter.

[0037] Nucleotide sequence of OsXAT8:

[0038]

[0039] Amino acid sequence of the protein encoded by OsXAT8:

[0040] MGSEVKPAKLGLRRHLNAGFFAGFLLVLLTYVIVSQQFAMETPTAVTSRAPRIDENESVTKARVETEKKREQEWQRPKDTSGAVSAEEFSKRDSTNAKPIENGKVVCGSNGFYSDTCDVDG DVRINGTALSVTLVPASRRSERRREWKIQPYPRRTVSGIAEVTVTRQQDRAAAPACTVTHGVPGVVFALGGLTGNYWHDFSDVLVPLFVASRRYGGEVQFLVSNIQPWWLGKYEAVVRRLSR YDAVDLDRDTEVRCFRRVAVGLRMHKEFSVKPELAPGGQRLTMADFAAFLRDTYALPRAAAAGARRPRLVVIRRAHYRKIVNMDEVVRAEAAAGFEAAVMSPRFDEPVEEVARKVNAFDAM VGVHGAGLTNAVFLPAGAVVIQVVPYGRLERMARADFGEPVADMGLRYMEYSVAADESTLLEMLGPEHQVVKDPEAVHRSGWDKVAEYYLGKQDVRINVARFAATLAAAFDHLRPSHS(SEQ ID NO.2).

[0041] The upstream and downstream primers used were:

[0042] Upstream primer: 5'-ATGGGCAGCGAGGTGAAGC-3' (SEQ ID NO. 3);

[0043] Downstream primer: 5'-CTATGTGATGGTCGAAGGT-3' (SEQ ID NO. 4).

[0044] The OsXAT8 gene was connected to the vector pCAMBIA1300 with the UBi promoter: First, rice genomic DNA was used as a template, and OsXAT8 was amplified using the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4, and the amplified product was connected to the pCAMBIA1300 vector using Kpn I and BamHI. The connection product was named pUBI-HZ-OsXAT8. The plasmid obtained in the previous step was sequenced, and the next step of the experiment was performed after the sequencing was correct.

[0045] The sequenced vector containing pUBI-HZ-OsXAT8 was transformed into Agrobacterium tumefaciens EH105 strain, and then transferred into rice Nipponbare embryonic callus. The selection resistance gene carried by the pCAMBIA1300 vector was hygromycin. The transformed rice callus was screened with hygromycin resistance to obtain callus with hygromycin resistance, and seedlings were regenerated to obtain T0 transgenic strains. The T0 transgenic strains were cultivated in the normal field until maturity, and T1 seeds were harvested. The T1 seeds were then tested for hygromycin resistance. The test of resistance was carried out, and strains with 3 / 4 resistance and the remaining 1 / 4 without resistance were selected, indicating that the overexpression vector with the target gene was inserted in the strain in a single copy form; the plants with hygromycin resistance in these strains were removed, and then the T2 generation seeds were harvested by individual planting; the T2 generation transgenic strains were screened for hygromycin resistance, and if there was no separation, it indicated that the transgenic strain was homozygous, and the homozygous strain could be used for seed breeding and yield analysis experiments; the overexpression strain OsXAT8.OE was isolated.

[0046] 2. Detection of target gene expression level of OsXAT8.OE by fluorescence quantitative PCR

[0047] The mRNA of wild-type plants (NPB) and OsXAT8.OE transgenic plants was extracted and reverse transcribed into cDNA. The expression level of OsXAT8 gene in overexpressed plants was detected by fluorescence quantitative PCR technology. Among them, the primers, reaction procedures and reaction systems of fluorescence quantitative PCR were:

[0048] Primer sequences:

[0049] OsXAT8.RT.F: 5'-CACAAGGAGTTCAGCGTGAAG-3' (SEQ ID NO.5);

[0050] OsXAT8.RT.R: 5'-GTTCACGATCTTCCGGTAGTG-3' (SEQ ID NO.6);

[0051] Internal reference sequence:

[0052] Ubiquitin.F: 5'-GAGGCCAAGCTTCGCCGGCT-3' (SEQ ID NO.7);

[0053] Ubiquitin.R: 5'-AAGAAGCTGAAGCATCCAGC-3' (SEQ ID NO. 8).

[0054] Reaction system: cDNA 6.00μL; OsXAT8.RT.F 1.00μL; OsXAT8.RT.R 1.00μL; 10X Green Mix 10.00μL; ddH 2 O Up to 20.00μL.

[0055] Reaction procedure: 95℃5min, 95℃30sec, 60℃(Tm)15sec, 72℃25sec, 55℃-85℃81 cycles with temperature rising gradually, 25℃10sec. The gene expression calculation formula is:

[0056] Test results see Figure 1 , it can be seen that the expression level of OsXAT8 gene was increased in the overexpression line (OsXAT8.OE transgenic plants).

[0057] Example 2 Yield detection of rice lines overexpressing OsXAT8 protein

[0058] Take 100 seeds of wild-type rice and OsXAT8 protein overexpression seeds obtained in Example 1, soak them in pure water at 28°C for 2 days, germinate them at 28°C for 1 day in a humid environment, spread the seeds that turn white after germination on seedling trays, raise seedlings for 30 days, obtain rice seedlings, transplant the rice seedlings to the experimental field, set up 3 planting plots, and randomly distribute them. The yield of wild-type rice and OsXAT8 overexpression lines was measured at maturity.

[0059] The phenotype photos of OsXAT8 overexpression lines and wild-type control lines are shown in Figure 2 , the production statistics are shown in Figure 3 The results showed that the yield of OsXAT8 overexpressing lines increased by 13.8%-16.7% compared with the wild-type control.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-yield gene OsXAT8, characterized in that: The nucleotide sequence of the high-yield gene OsXAT8 is shown in SEQ ID NO.

1.

2. The protein encoded by the high-yield gene OsXAT8 according to claim 1.

3. The protein according to claim 2, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. A set of primer pairs for amplifying the high-yield gene OsXAT8 according to claim 1, characterized in that: It includes an upstream primer having a nucleotide sequence such as SEQ ID NO.3 and a downstream primer having a nucleotide sequence such as SEQ ID NO.

4.

5. A method for amplifying the high-yield gene OsXAT8 according to claim 1 using the primer pair according to claim 4, characterized in that: The following steps are involved: The nucleic acid molecule of the plant is used as a template, and the template and a primer pair are prepared into a PCR amplification system for PCR amplification to obtain the high-yield gene OsXAT8.

6. A method for increasing plant yield, characterized in that: By overexpressing the expression level of the OsXAT8 gene according to claim 1, the plant yield is increased.

7. Use of the high-yield gene OsXAT8 according to claim 1, the protein according to claim 2 or 3, or the high-yield gene OsXAT8 amplified by the method according to claim 5 in increasing plant yield.

8. The use according to claim 7, characterized in that: The plants include rice.

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