Application of a rice plant height negative regulation gene osk8

Abstract

The application discloses a rice plant height negative regulation gene OSK8 and application thereof, and belongs to the field of molecular breeding. The application comprises the following steps: using the rice plant height negative regulation gene OSK8 to reduce the height of a rice plant. The application discloses the application of the rice plant height negative regulation gene OSK8, which comprises the following steps: using the rice plant height negative regulation gene OSK8 to reduce the height of a rice plant, and the height of a receptor plant can be effectively reduced, and the effect is remarkable.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of molecular breeding, and particularly relates to an application of a rice plant height negative regulation gene OSK8. BACKGROUND

[0002] Rice (Oryza sativa) is the first staple crop in China, and more than half of the population relies on rice as their main food. In the 1960s, rice dwarf breeding promoted the occurrence of the first "green revolution", which greatly improved the yield of rice.

[0003] With the development of modern biotechnology, genes and gene loci (Quantitative Trait Locus, QTL) related to rice plant height traits have been discovered. For example, the green revolution gene SD1, which encodes OsGA20ox2, is a key enzyme in the gibberellin synthesis pathway, which positively regulates the plant height of rice by promoting the synthesis of gibberellins (GAs). The mutant allele sd1 leads to dwarf rice, thereby improving the lodging resistance and harvest yield of rice.

[0004] Rice plant height is a complex trait, although multiple related genes have been detected, but due to their own defects, these genes cannot be well applied in rice plant height improvement. Therefore, more rice plant height genes need to be excavated to provide more gene resources for rice research.

[0005] DISCLOSURE

[0006] In order to solve the problems of the prior art, the present disclosure provides an application of a rice plant height negative regulation gene OSK8. The technical solution is as follows:

[0007] The present disclosure provides an application of a rice plant height negative regulation gene OSK8, which comprises: using the rice plant height negative regulation gene OSK8 to reduce the height of rice plants.

[0008] Specifically, the application comprises: knocking out, replacing or modifying the amino acid sequence of the rice plant height negative regulation gene OSK8 to reduce the height of rice plants.

[0009] Specifically, the application comprises:

[0010] selecting a target sequence as a knockout target point in the coding sequence of the rice plant height negative regulation gene OSK8;

[0011] performing first PCR amplification according to the target sequence using a first forward primer and a second reverse primer to obtain a first amplification product;

[0012] performing second PCR amplification according to the target sequence by using the second forward primer and the first reverse primer, to obtain a second amplification product;

[0013] performing, after purification of the first amplification product and the second amplification product, ligation by overlap PCR by using the first forward primer and the first reverse primer, to obtain a ligation product;

[0014] ligating the ligation product to a pCXUN vector to obtain an OSK8 knockout vector;

[0015] transforming the OSK8 knockout vector into a receptor material.

[0016] Further, the target sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0017] Further, the sequence of the first forward primer is shown in SEQ ID NO: 2 in the sequence listing, the sequence of the first reverse primer is shown in SEQ ID NO: 3 in the sequence listing, the sequence of the second forward primer is shown in SEQ ID NO: 4 in the sequence listing, and the sequence of the second reverse primer is shown in SEQ ID NO: 5 in the sequence listing.

[0018] Further, the receptor material is rice ZH11.

[0019] The technical scheme provided by the embodiments of the present disclosure has the beneficial effects that the present disclosure provides an application of a rice plant height negative regulation gene OSK8, which includes using the rice plant height negative regulation gene OSK8 to reduce the height of a rice plant, and the receptor plant can effectively reduce the plant height with a significant effect. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without creative labor.

[0021] Figure 1 is a field seedling phenotype comparison chart of osk8-1, osk8-2 and a control group WT provided by the embodiments of the present disclosure;

[0022] Figure 2 is a plant height difference comparison chart of osk8-1, osk8-2 and a control group WT provided by the embodiments of the present disclosure;

[0023] Figure 3 is a plant height statistical chart of osk8-1, osk8-2 and a control group WT provided by the embodiments of the present disclosure. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0025] Example

[0026] This disclosure provides an application of the rice plant height negative regulatory gene OSK8, which includes using the rice plant height negative regulatory gene OSK8 (LOC_Os11g48030) to reduce the height of rice plants.

[0027] Specifically, applications include knocking out, replacing, or altering the amino acid sequence of OSK8, a gene that negatively regulates rice plant height, to reduce the height of rice plants. In this embodiment, the coding sequence of the rice plant height negative regulatory gene OSK8 is shown as SEQ ID NO: 6 in the sequence listing, specifically: ATGCTGCGGAAGGGAGAGGCGCCGGGGCACCAAACCCCACCCCACCTGCACAAGGACGACGGCGACGACGACGACGACGCACCGTCCGGCTTCGTCAAGCTCATCAGCGCCGAGGGCTTCGAGTTCGTCGTCGACAAGAAGGCCGCCATGGTCTCCAACACGCTCCGCAACATGCTCACCTCCCCCCCGGCGGCTTCTCCGAGACGCGCGAGGGCGAGGTTAGGTTCCCCGAGATCAGCACCCCCATCCTCGAGAAGATCTGCCAGTACTTCTACTGGTCGCTCCACTACTCCAGTGGGAAGGAGACATCTGAGTTTCAAATTGAACCGGAGATAACTCTGGAGCTGATGATGGCTGCAAACTATCTGGACACCTGA.

[0028] In this embodiment, the amino acid sequence of the rice plant height negative regulatory gene OSK8 is shown in SEQ ID NO: 7 in the sequence listing, specifically: MLRKGEAPGHQTPPHLHKDDGDDDDDAPSGFVK LISAEGFEFVVDKKAAMVSNTLRNMLTSPGGFSETREGEVRFPEISTPILEKIC QYFYWSLHYSSGKETSEFQIEPEITLELMMAANYLDT*. Wherein, * represents a terminator.

[0029] Specifically, the applications include:

[0030] A target sequence is selected from the coding sequence of a rice plant height negative regulation gene OSK8 as a knockout target point;

[0031] The first PCR amplification is performed according to the target sequence by using the first forward primer and the second reverse primer, and a first amplification product is obtained;

[0032] The second PCR amplification is performed according to the target sequence by using the second forward primer and the first reverse primer, and a second amplification product is obtained;

[0033] The first amplification product and the second amplification product are purified, and then connected by overlap PCR by using the first forward primer and the first reverse primer, and a connection product is obtained;

[0034] The connection product is connected to a pCXUN vector, and an OSK8 knockout vector is obtained.

[0035] The OSK8 knockout vector is transformed into a receptor material.

[0036] Further, the target sequence is shown in SEQ ID NO: 1 in the sequence listing, and specifically is: gctgcggaagggagaggcgc.

[0037] Further, the sequence of the first forward primer is shown in SEQ ID NO: 2 in the sequence listing, and specifically is: cccctttcgccaggggtacctatgtacagcattacgtagg.

[0038] The first reverse primer is shown in SEQ ID NO: 3 in the sequence listing, and specifically is: tacgaattcgagctcggtaccgatggtgcttactgtttag.

[0039] The sequence of the second forward primer is shown in SEQ ID NO: 4 in the sequence listing, and specifically is: gctgcggaa gggagaggcgcgttttagagctagaaatagcaagtta.

[0040] The second reverse primer is shown in SEQ ID NO: 5 in the sequence listing, and specifically is: gcgcctctcccttccgcagcaacctgagcctcagcgcagc.

[0041] Further, the receptor material is rice ZH11.

[0042] Further, the reaction system for each 50 μL first PCR amplification comprises: 2x KOD Fx PCR Buffer 25 μL; 2 mM dNTP 10 μL; first forward primer with a concentration of 10 μM 1.5 μL; second reverse primer with a concentration of 10 μM 1.5 μL; U6 template DNA 3 μL; KOD high-fidelity polymerase with a concentration of 5 U / μL 1 μL; ddH2O 8 μL. The template DNA of the first amplification product (422 bp) is obtained. The U6 template DNA and the carrier construction method used in the embodiment can adopt and refer to the method provided in He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR / Cas9 mediated genome editing. J. Genet. Genomics, 2017, 44: 469-472, and is specifically prepared by Wuhan Tianwen Biotechnology Co., Ltd.

[0043] Further, the first PCR amplification program is as follows:

[0044]

[0045] Further, the reaction system for each 50 μL second PCR amplification comprises: 2x KOD Fx PCR Buffer 25 μL; 2 mM dNTP 10 μL; first reverse primer with a concentration of 10 μM 1.5 μL; second forward primer with a concentration of 10 μM 1.5 μL; U6 template DNA 3 μL; KOD high-fidelity polymerase with a concentration of 5 U / μL 1 μL; ddH2O 8 μL. The template DNA of the second amplification product (476 bp) is obtained.

[0046] Further, the second PCR amplification program is as follows:

[0047]

[0048] Furthermore, each 50 μL overlap PCR amplification reaction system includes: 25 μL 2×KOD Fx PCR Buffer; 10 μL 2 mM dNTP; 1.5 μL of the first forward primer at a concentration of 10 μM; 1.5 μL of the first reverse primer at a concentration of 10 μM; 1.5 μL of template DNA for the first amplification product; 1.5 μL of template DNA for the second amplification product; 1 μL of KOD high-fidelity polymerase at a concentration of 5 U / μL; and 8 μL ddH2O.

[0049] Furthermore, the overlap PCR amplification procedure is as follows:

[0050]

[0051] After the overlap PCR amplification reaction, the amplification product was recovered using a 1% agarose gel. The recovered amplification product DNA length was 898 bp, which was the sgRNA expression cassette. Then, the sgRNA expression cassette was ligated to the pCXUN vector using a one-step ligation reaction (Gibson Assembly). The ligation system consisted of 2 μL of the recovered product DNA template, 0.5 μL of pCXUN digested with KpnI, and 7.5 μL of one-step ligase Mixture. After thorough mixing, the mixture was incubated at 50°C for 50 min to obtain the ligation product. The ligation product was transformed into *E. coli* competent cells Trans5α, and single clones were picked and cultured to obtain bacterial culture. After preservation of the bacterial culture, positive single clones were initially identified by PCR, followed by plasmid extraction and sequencing. The unmutated positive single clones were selected as the constructed OSK8 knockout vector. The OSK8 knockout vector was transformed into *Agrobacterium* EHA105 cells using electroporation. Then, the OSK8 knockout vector was transformed into the recipient material rice ZH11 (commercially available) using the Agrobacterium tumefaciens infection method to obtain the OSK8 homozygous knockout mutant.

[0052] The phenotypes of OSK8 homozygous knockout mutants were observed. Specifically, in this embodiment, the OSK8 knockout mutant T0 generation was multiplied and its genes were sequenced to screen out homozygous families with mutations in the OSK8 target sequence from the offspring. Two OSK8 homozygous knockout mutant materials, osk8-1 and osk8-2, were selected from the T1 generation, both of which showed frameshift mutations and premature termination.

[0053] Seedlings of osk8-1, osk8-2, and wild-type rice ZH11 were planted in the field, and their growth during the seedling stage was observed. Wild-type rice ZH11 served as the control group (WT). Specific growth data are as follows: Figure 1 As shown. By Figure 1 It can be seen that during the seedling stage in the field, compared with the control group WT, osk8-1 and osk8-2 had shorter plant heights.

[0054] At the late filling stage of rice, the plant height was investigated again, specifically as shown in Figure 2 At the same time, the plant height was counted, specifically as shown in Figure 3 It can be known from Figure 2 and Figure 3 that, at the late filling stage, the plant height of osk8-1 and osk8-2 is significantly reduced compared with the control group WT.

[0055] The above merely describes optional embodiments of the present disclosure and is not used to limit the present disclosure. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A gene that negatively regulates rice plant height OSK8 The application is characterized by, The application comprises: a rice plant height negative regulation gene OSK8 for reducing the height of a rice plant, a coding sequence of the rice plant height negative regulation gene OSK8 is shown as SEQ ID NO: 6 in the sequence listing, an amino acid sequence of the rice plant height negative regulation gene OSK8 is shown as SEQ ID NO: 7 in the sequence listing, A target sequence is selected as a knockout target in the coding sequence of the rice plant height negative regulation gene OSK8 as shown in SEQ ID NO: 1 in the sequence table; performing first PCR amplification according to the target sequence by using a first forward primer and a second reverse primer, wherein the sequence of the first forward primer is represented by SEQ ID NO: 2 in the Sequence Listing, and the second reverse primer is represented by SEQ ID NO: 3 in the Sequence Listing, to obtain a first amplification product; performing second PCR amplification according to the target sequence by using a second forward primer and a first reverse primer, wherein the sequence of the second forward primer is represented by SEQ ID NO: 4 in the Sequence Listing, and the first reverse primer is represented by SEQ ID NO: 5 in the Sequence Listing, to obtain a second amplification product; performing overlap PCR ligation by using the first forward primer and the first reverse primer after purifying the first amplification product and the second amplification product, to obtain a ligation product; ligating the ligation product to a pCXUN vector to obtain OSK8 knockout vector; The OSK8 Knockout vectors are transformed into recipient material.

2. Use according to claim 1, characterized in that, The receptor material is rice ZH11.

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