Application of rice plant height negative regulation gene OSK8

Abstract

The invention provides application of a rice plant height negative regulation gene OSK8, and belongs to the field of molecular breeding. According to the application, the rice plant height negative regulation gene OSK8 is used for reducing the height of a rice plant. The invention provides application of a rice plant height negative regulation gene OSK8, the application comprises the step that the rice plant height negative regulation gene OSK8 is used for reducing the height of a rice plant, a receptor plant can effectively reduce the plant height, and the effect is remarkable.

Description

Technical Field

[0001] The present disclosure relates to the field of molecular breeding, and in particular to an application of a rice plant height negative regulatory gene OSK8. Background Art

[0002] Rice (Oryza sativa) is the largest staple food crop in my country, with more than half of the population relying on rice as their staple food. In the 1860s, dwarf rice breeding promoted the first "green revolution" and significantly increased rice production.

[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, and positively regulates rice plant height by promoting the synthesis of gibberellins (GAs). The mutant allele sd1 causes the rice plant height to become shorter, thereby improving the rice's resistance to lodging and harvest yield.

[0004] Rice plant height is a complex trait. Although multiple related genes have been detected, these genes cannot be well applied in improving rice plant height due to their own defects. Therefore, it is necessary to explore more rice plant height genes to provide more genetic resources for rice research.

[0005] Public Content

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

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

[0008] Specifically, the application includes: knocking out, replacing or changing the amino acid sequence of the rice plant height negative regulatory gene OSK8 to reduce the height of the rice plant.

[0009] Specifically, the applications include:

[0010] Selecting a target sequence in the coding sequence of the rice plant height negative regulatory gene OSK8 as a knockout target;

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

[0013] The first amplification product and the second amplification product are purified and then connected by overlapping PCR using the first forward primer and the first reverse primer to obtain a connection product;

[0014] The ligated product was connected to the pCXUN vector to obtain an OSK8 knockout vector;

[0015] The OSK8 knockout vector was transformed into recipient material.

[0016] Furthermore, 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 as SEQ ID NO: 2 in the sequence listing, the sequence of the first reverse primer is shown as SEQ ID NO: 3 in the sequence listing, the sequence of the second forward primer is shown as SEQ ID NO: 4 in the sequence listing, and the sequence of the second reverse primer is shown as SEQ ID NO: 5 in the sequence listing.

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

[0019] The beneficial effect brought about by the technical solution provided by the embodiment of the present disclosure is: the present disclosure provides an application of rice plant height negative regulatory gene OSK8, which includes using rice plant height negative regulatory gene OSK8 to reduce the height of rice plants, and the recipient plants can effectively reduce the plant height with significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 It is a comparison diagram of the phenotypes of osk8-1, osk8-2 and the control group WT at the seedling stage in the field provided in the embodiments of the present disclosure;

[0022] Figure 2 It is a comparison diagram of plant height differences among osk8-1, osk8-2 and the control group WT provided in the embodiments of the present disclosure;

[0023] Figure 3 It is a statistical graph of plant height of osk8-1, osk8-2 and control group WT provided in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0025] Example

[0026] The present disclosure provides an application of a rice plant height negative regulatory gene OSK8, and the application includes: using the rice plant height negative regulatory gene OSK8 (LOC_Os11g48030) to reduce the height of rice plants.

[0027] Specifically, the application includes: knocking out, replacing or changing the amino acid sequence of the rice plant height negative regulatory gene OSK8 to reduce the height of the rice plant. In this embodiment, the coding sequence of the rice plant height negative regulatory gene OSK8 is shown in SEQ ID NO: 6 in the sequence listing, specifically: ATGCTGCGGAAGGGAGAGGCGCCGGGGCACCAAACCCCACCCCACCTGCACAAGGACGACGGCGACGACGACGCACCGTCCGGCTTCGTCAAGCTCATCAGCGCCGAGGGCTTCGAGTTCGTCGTCGACAAGAAGGCCGCCATGGTCTCCAACACGCTCCGCAACATGCTCACCTCCCCCGGCGGCTTCTCCGAGACGCGCGAGGGCGAGGTTAGGTTCCCCGAGATCAGCACCCCCATCCTCGAGAAGATCTGCCAGTACTTCTACTGGTCGCTCCACTACTCCAGTGGGAAGGAGACATCTGAGTTTCAAATTGAACCGGAGATAACTCTGGAGCTGATGATGGCTGCAAACTATCTGGACACCTGA.

[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 list, specifically: MLRKGEAPGHQTPPHLHKDDGDDDDDAPSGFVK LISAEGFEFVVDKKAAMVSNTLRNMLTSPGGFSETREGEVRFPEISTPILEKIC QYFYWSLHYSSGKETSEFQIEPEITLELMMAANYLDT*, wherein * represents a terminator.

[0029] Specifically, applications include:

[0030] A target sequence was selected as a knockout target in the coding sequence of OSK8, a negative regulator of rice plant height;

[0031] Performing a first PCR amplification according to the target sequence using a first forward primer and a second reverse primer to obtain a first amplification product;

[0032] Performing a second PCR amplification according to the target sequence using the second forward primer and the first reverse primer to produce a second amplification product;

[0033] The first amplification product and the second amplification product are purified and then connected by overlapping PCR using a first forward primer and a first reverse primer to obtain a connection product;

[0034] The ligation product was connected to the pCXUN vector to obtain the OSK8 knockout vector;

[0035] The OSK8 knockout vector was transformed into recipient material.

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

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

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

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

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

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

[0042] Furthermore, the reaction system for the first PCR amplification per 50 μL includes: 2×KOD Fx PCR Buffer 25 μL; 2 mM dNTP 10 μL; 1.5 μL of the first forward primer with a concentration of 10 μM; 1.5 μL of the second reverse primer with a concentration of 10 μM; 3 μL of U6 template DNA∈; 1 μL of KOD high-fidelity polymerase with a concentration of 5 U / μL; ddH 2 O 8μL. Obtain the template DNA (422bp) of the first amplification product. The U6 template DNA and vector construction methods used in this embodiment can be adopted and referenced 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 / Cas9mediated genome editing. J. Genet. Genomics, 2017, 44: 469-472, and are specifically prepared by Wuhan Tianwen Biotechnology Co., Ltd.

[0043] Further, the first PCR amplification procedure is:

[0044]

[0045] Furthermore, the reaction system for the second PCR amplification per 50 μL includes: 2×KOD Fx PCR Buffer 25 μL; 2 mM dNTP 10 μL; 1.5 μL of the first reverse primer with a concentration of 10 μM; 1.5 μL of the second forward primer with a concentration of 10 μM; 3 μL of U6 template DNA; 1 μL of KOD high-fidelity polymerase with a concentration of 5 U / μL; ddH 2 O 8 μL. Obtain the template DNA (476 bp) of the second amplification product.

[0046] Further, the second PCR amplification procedure is:

[0047]

[0048] Furthermore, each 50 μL of the overlapping PCR amplification reaction system includes: each 50 μL of the recombinant amplification reaction system includes: 25 μL of 2×KOD Fx PCR Buffer; 10 μL of 2 mM dNTP; 1.5 μL of the first forward primer with a concentration of 10 μM; 1.5 μL of the first reverse primer with a concentration of 10 μM; 1.5 μL of the template DNA of the first amplification product; 1.5 μL of the template DNA of the second amplification product; 1 μL of KOD high-fidelity polymerase with a concentration of 5 U / μL; ddH 2 2. 4% dHO: 8 μL.

[0049] Further, the overlapping PCR amplification procedure is:

[0050]

[0051] After the overlapping PCR amplification reaction, the amplified product was recovered by agarose gel with a concentration of 1%. The DNA length of the recovered amplified product was 898bp, which was the sgRNA expression cassette. Then the sgRNA expression cassette was connected to the pCXUN vector by a one-step ligation reaction (Gibson Assembly). The ligation system was: 2μL of the recovered product DNA template, 0.5μL of the KpnⅠ digested vector pCXUN and 7.5μL of the one-step ligase Mixture, mixed evenly and reacted at 50℃ for 50min to obtain the ligation product. The ligation product was transformed into the competent Escherichia coli Trans5α, and a single clone was picked for shaking to obtain a bacterial solution. After the bacterial solution was preserved, the positive single clone was preliminarily determined by the bacterial solution PCR reaction, and then the plasmid was extracted and sequenced. After comparison, the positive single clone without mutation was used as the constructed OSK8 knockout vector. The OSK8 knockout vector was transformed into Agrobacterium EHA105 by electroporation. Then, the OSK8 knockout vector was transformed into the recipient material rice ZH11 (commercially available) by Agrobacterium infection to obtain the OSK8 homozygous knockout mutant.

[0052] The phenotype of the OSK8 homozygous knockout mutant was observed. Specifically, in this embodiment, the OSK8 knockout mutant T0 generation was propagated and sequenced, and homozygous families with mutations in the OSK8 target sequence were screened from the offspring. Two OSK8 homozygous knockout mutant materials, osk8-1 and osk8-2, were selected from the T1 generation, both of which had 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 was observed. The wild-type rice ZH11 served as the control group WT. Figure 1 As shown. Figure 1It can be seen that at the seedling stage in the field, the plant heights of osk8-1 and osk8-2 were shorter than those of the control group WT.

[0054] In the late stage of rice filling, the plant height was examined again. Figure 2 As shown, at the same time, the plant height is counted, as shown in Figure 3 As shown. Combined Figure 2 and Figure 3 It can be seen that in the late grain filling stage, the plant height of osk8-1 and osk8-2 was significantly reduced compared with the control group WT.

[0055] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An application of the rice plant height negative regulatory gene OSK8, characterized in that: The application includes: using the rice plant height negative regulatory gene OSK8 to reduce the height of rice plants.

2. The use according to claim 1, characterized in that The application includes: knocking out, replacing or changing the amino acid sequence of the rice plant height negative regulatory gene OSK8 to reduce the height of the rice plant.

3. The use according to claim 1, characterized in that The applications include: Selecting a target sequence in the coding sequence of the rice plant height negative regulatory gene OSK8 as a knockout target; Performing a first PCR amplification based on the target sequence using a first forward primer and a second reverse primer to obtain a first amplification product; Performing a second PCR amplification based on the target sequence using a second forward primer and the first reverse primer to obtain a second amplification product; The first amplification product and the second amplification product are purified and then connected by overlapping PCR using the first forward primer and the first reverse primer to obtain a connection product; The ligation product was connected to the pCXUN vector to obtain the OSK8 knockout vector; The OSK8 knockout vector was transformed into recipient material.

4. The use according to claim 3, characterized in that The target sequence is shown as SEQ ID NO: 1 in the sequence listing.

5. The use according to claim 3, characterized in that The sequence of the first forward primer is shown as SEQ ID NO: 2 in the sequence listing, the sequence of the first reverse primer is shown as SEQ ID NO: 3 in the sequence listing, the sequence of the second forward primer is shown as SEQ ID NO: 4 in the sequence listing, and the sequence of the second reverse primer is shown as SEQ ID NO: 5 in the sequence listing.

6. The use according to claim 3, characterized in that The receptor material is rice ZH11.

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