High-yield gene osxat8, amplification method thereof and application in plant yield improvement

Abstract

The application discloses a high-yield gene OsXAT8, an amplification method of the high-yield gene OsXAT8 and application of the high-yield gene OsXAT8 in plant yield improvement, and belongs to the technical field of genetic engineering. The application provides the high-yield gene OsXAT8, wherein the nucleotide sequence of the high-yield gene OsXAT8 is shown as SEQ ID NO. 1. The application further 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 application of the high-yield gene OsXAT8 in improving plant yield. The high-yield gene OsXAT8 and the corresponding protein are applied to genetic engineering and breeding of plants, and a new high-yield rice variety is bred, which has important reference significance for high-yield related researches of other crops.

Description

Technical Field

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

[0002] As one of the world's most important food crops, rice (Oryza sativa L.) plays an irreplaceable role in ensuring national food security and meeting the basic needs of the people. Therefore, in-depth research on the mechanisms regulating high-yield rice and the selection and breeding of high-yield varieties has important practical applications in agricultural production.

[0003] In recent years, with the rapid development of molecular biology and genomics technologies, research on the mechanisms regulating high rice yields 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 conducting in-depth research on the functions of these genes in the growth and development process and their impact 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 shape, 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 mechanisms of high crop yield.

[0004] Furthermore, with the widespread application of high-throughput sequencing technology, researchers are able to more comprehensively analyze rice genome information and identify key genes and molecular markers associated with high yield. Through methods such as genome-wide association studies (GWAS) and transcriptome analysis, key genes and regulatory networks controlling rice yield traits can be identified, providing important candidate gene resources for molecular breeding. Combining traditional breeding methods with modern molecular breeding techniques can accelerate the selection and breeding of new high-yield, high-quality, and stress-resistant rice varieties, providing strong scientific and technological support for the 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 present invention aims to provide a high-yield gene, OsXAT8, and its amplification method and application in plant yield improvement, to address the aforementioned problems of the prior art. The OsXAT8 gene provided by the present invention can improve rice yield and has promising application prospects in breeding high-yield rice varieties. This 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 by substituting, deleting or adding one or more amino acids 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 a 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, the application 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 specific fragments thereof.

[0018] In order to facilitate the identification and screening of the transgenic plant cell or plant, the used vector can be processed, such as adding a plant selectable marker or an antibiotic marker with resistance, etc.

[0019] Technical solution six: the application of the high-yield gene OsXAT8, the protein or the high-yield gene OsXAT8 amplified by the method in improving the yield of plants.

[0020] Further, the plant comprises rice.

[0021] The application discloses the following technical effects:

[0022] The application discloses the following technical effects: BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

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

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

[0026] Figure 3The graph shows the yield statistics of OsXAT8-overexpressing strains and wild-type control strains, where OE-1 and OE-2 are OsXAT8-overexpressing 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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 the Agrobacterium GV3101 strain, both of which are commercially available; 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 miniprep kit and agarose gel recovery kit were purchased from Shanghai Jierui Bioengineering Co., Ltd.; MS medium, agar powder, agarose, antibiotics such as spectinomycin (Spe), rifampicin (Rif), hygromycin, and LB medium were purchased from Sigma; various other chemical reagents used in the examples were imported or domestic analytical grade reagents.

[0034] Example 1 Construction of OsXAT8 gene overexpression plants

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

[0036] The gene OsXAT8 encoding plant xylan synthase protein was cloned from Oryza sativa L. spp. japonica. Based on the sequence analysis of the coding region, the upstream and downstream primers designed by the present invention were amplified and ligated to the overexpression vector pCAMBIA1300 with the 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 ligated into the UBi promoter-containing vector pCAMBIA1300. First, OsXAT8 was amplified using rice genomic DNA as a template using the upstream primer shown in SEQ ID NO. 3 and the downstream primer shown in SEQ ID NO. 4. The amplified product was then ligated into the pCAMBIA1300 vector using Kpn I and Bam HI. The ligated 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 confirmed.

[0045] The vector containing pUBI-HZ-OsXAT8 correctly sequenced is transformed into Agrobacterium EH105 strain, and then into rice Nipponbare embryogenic callus. The selection resistance gene of pCAMBIA1300 vector is hygromycin, and the transformed rice callus is screened by hygromycin resistance. Hygromycin-resistant callus is obtained, and seedlings are generated to obtain T0 generation transgenic lines. The T0 generation transgenic lines are normally cultivated in the field to maturity, and T1 generation seeds are harvested. The T1 generation seeds are tested for hygromycin resistance, and 3 / 4 of the lines with resistance and the remaining 1 / 4 without resistance are selected, indicating that the overexpression vector with the target gene is inserted in the form of a single copy in the lines. The hygromycin-resistant plants in these lines are removed, and T2 generation seeds are harvested by single plant seed collection. The T2 generation transgenic lines are screened for hygromycin resistance. If no segregation is observed, the transgenic line is a homozygote, and the homozygous line can be used for breeding and yield analysis experiments. The overexpression line OsXAT8.OE is obtained by segregation.

[0046] 2. Fluorescent quantitative PCR method for detecting the expression level of the target gene of OsXAT8.OE

[0047] The mRNA of wild-type plants (NPB) and OsXAT8.OE transgenic plants is extracted and reverse transcribed into cDNA. The expression level of the OsXAT8 gene in the overexpression plants is detected by fluorescent quantitative PCR technology. The primers, reaction program, and reaction system of the fluorescent quantitative PCR are as follows:

[0048] Primer sequence:

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

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

[0051] 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 6.00 μL; OsXAT8.RT.F 1.00 μL; OsXAT8.RT.R 1.00 μL; 10X Green Mix 10.00μL; ddH2O Up to 20.00μL.

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

[0056] Test results are shown in 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] 100 seeds each of wild-type rice and the OsXAT8 protein-overexpressing strain obtained in Example 1 were soaked in purified water at 28°C for 2 days and germinated in a moist environment at 28°C for 1 day. The seeds, which turned white after germination, were spread onto seedling trays and raised for 30 days to obtain rice seedlings. The rice seedlings were transplanted to a test field in three randomly distributed planting plots. Yields of the wild-type rice and the OsXAT8-overexpressing strain were 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for increasing plant yield, characterized in that: By overexpressing the high-yield gene OsXAT8, the yield of the plant is increased; The nucleotide sequence of the high-yield gene OsXAT8 is shown in SEQ ID NO.1; The plant is rice.

2. The application of the high-yield gene OsXAT8 and its encoded protein in increasing plant yield, characterized in that: The nucleotide sequence of the high-yield gene OsXAT8 is shown in SEQ ID NO.1; The amino acid sequence of the protein is shown in SEQ ID NO.2; The plant is rice.

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