Application of OsbZIP1 gene in regulation and control of rice plant type

Abstract

The invention provides application of an OsbZIP1 gene in regulation and control of rice plant types, and relates to the technical field of gene engineering. According to the invention, the OsbZIP1 gene capable of being used for regulating and controlling the plant type of the rice and the encoding protein of the OsbZIP1 gene are obtained through research. Through verification, the OsbZIP1 gene has the function of regulating and controlling the size of the included angle of the rice leaves, namely after the OsbZIP1 gene in the rice is knocked out by the gene, the included angle of the leaves of a rice plant is reduced, and relatively upright leaves can effectively increase the light receiving area of the rice after the included angle of the leaves is reduced, so that the yield of the rice is increased. Therefore, the OsbZIP1 gene provided by the invention has potential application value in improving the plant type of the rice and increasing the yield of the rice.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an application of an OsbZIP1 gene in regulating rice plant type. Background Art

[0002] Rice is one of the most important food crops and the main source of energy for more than half of the world's population. With global warming, food security issues are becoming increasingly prominent, so improving food production capacity is of great significance to ensuring global food security.

[0003] Rice plant type is an important factor affecting rice yield and has long been the focus of breeders. Therefore, the elucidation of the rice plant type regulatory network is of great significance to improving rice yield. Leaf angle, as one of the important factors affecting rice plant type, has a significant impact on rice photosynthesis. Upright leaves can effectively increase the light-receiving area of ​​rice, thereby increasing rice yield. In addition, relatively compact plant leaves are conducive to dense planting of rice, thereby significantly increasing yield.

[0004] Therefore, discovering and identifying genes that regulate rice leaf angle has important guiding significance for further improving rice yield.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide an application of an OsbZIP1 gene in regulating rice plant type. The OsbZIP1 gene has the function of regulating the angle of rice leaves, providing a basis for further studying the molecular mechanism of rice plant type regulation.

[0007] The second object of the present invention is to provide a method for improving rice plant type.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The invention provides an application of the OsbZIP1 gene in regulating rice plant type, wherein the nucleotide sequence of the OsbZIP1 gene is shown as SEQ ID No. 1.

[0010] Furthermore, the amino acid sequence encoded by the OsbZIP1 gene is shown in SEQ ID No.2.

[0011] Furthermore, the application is to inhibit the expression of the OsbZIP1 gene to transform rice plants with reduced leaf angles.

[0012] Furthermore, the transformation includes: constructing a gene knockout vector with OsbZIP1 gene as the target gene, and transforming rice plants with the gene knockout vector to obtain rice plants with reduced leaf angles.

[0013] Furthermore, the gene knockout vector is constructed based on the CRISPR / Cas9 vector;

[0014] Preferably, the knockout is performed using primer sequences shown in SEQ ID No. 3 and SEQ ID No. 4.

[0015] Furthermore, the transformation method includes one or more of Agrobacterium-mediated, Ti plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

[0016] The present invention provides a method for improving rice plant type, characterized in that an OsbZIP1 gene knockout vector is transformed into rice or rice cells to cultivate rice with a reduced leaf angle and a compact plant type.

[0017] Furthermore, the transformation includes: introducing the OsbZIP1 gene knockout vector into rice or rice cells by Agrobacterium-mediated method;

[0018] Preferably, the Agrobacterium is Agrobacterium EHA105.

[0019] Furthermore, the rice variety is japonica rice or indica rice, preferably Zhonghua 11.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides an OsbZIP1 gene and its encoded protein that can be used for regulating rice plant type. Studies have shown that the OsbZIP1 gene has the function of regulating the angle of rice leaves, and is expected to regulate the formation of rice plant type and then design the plant type in a directional manner to increase rice yield. It also provides a basis for further studying the molecular mechanism of rice plant type regulation.

[0022] The present invention provides a method for improving rice plant type, wherein the method transforms an OsbZIP1 gene knockout vector into rice or rice cells, thereby cultivating rice with a reduced leaf angle and a compact plant type. Therefore, the present application can apply the rice OsbZIP1 gene alone or in combination with other genes to rice cultivation to select rice varieties with specific plant types. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods 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 A detection diagram of the relative expression level of the OsbZIP1 gene in the rice OsbZIP1 knockout strain and overexpression strain provided in Example 4 of the present invention;

[0025] Figure 2 A comparison diagram of the rice OsbZIP1 knockout strain and the wild-type strain provided in Example 4 of the present invention;

[0026] Figure 3 This is a diagram for identifying the sensitivity of rice OsbZIP1 knockout strains to BR provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] According to one aspect of the present invention, the OsbZIP1 gene is used for regulating rice plant type, and the nucleotide sequence of the OsbZIP1 gene is shown as SEQ ID No.1.

[0030]

[0031] In a preferred embodiment of the present invention, the amino acid sequence encoded by the OsbZIP1 gene is shown as SEQ ID No.2.

[0032] The amino acid sequence shown in SEQ ID No. 2 is as follows: MVQGEESSWRMAASTHHERAIPLNQALAYGVQAHASPSVAAAPPASFL DFQPAAAAAAAYFGELEEALIHGANAGGVVDPGMIRADVHSKSAAAAATAGYLAARPPTLEIFPSWPMRQQQQLHSGNSQSVGSTDDSSSAQNTMPQMELVSPASIRASSEHQHQQQQPGQEVM MVTTDDYSYKPGLAAASPSFQQQHQLQHHQQQQLHGGGDHDKRKHGSTRKDGKSVDAKTERRLAQNREAARKSRLRKKAYVQNLETSRVRLQQIEQELQRARSQGLFLGGCRAAGDMSSGAAM FDMEYARWLDDDSKRLTDLRGGLQAHLLDTNLGLIVEECMQHYDELFQLKAALARSDVFHLLTGTWATPAERCFLWMGGFRPSDLLKILIQQLDPLTEQQMLGIYSLQQSSEQAEEALAQGLQ QLHQSLADTVAAGTLNDGPGVPNYMSLMAIALDKLASLESFYQQADNLRQQTLHQLRRILTTRQAARCFLSIGEYYRRLRALSNLWSSRPRENFIGTESVSPTGTELQPMHNQPQQNQYSGF.

[0033] In a preferred embodiment of the present invention, the application is to inhibit the expression of the OsbZIP1 gene to transform a rice plant with a reduced leaf angle.

[0034] The present invention cloned a rice plant type regulating gene (OsbZIP1 gene) through research. After gene knockout, it was found that the gene can change the plant type of rice. After the OsbZIP1 gene in rice was knocked out, the leaf angle of the rice plant became smaller. After the leaf angle became smaller, the relatively upright leaves can effectively increase the light receiving area of ​​the rice, thereby increasing the rice yield. Therefore, the OsbZIP1 gene has potential application value in improving the rice plant type and increasing the rice yield.

[0035] In the above preferred embodiment, the gene knockout vector is constructed based on the CRISPR / Cas9 vector;

[0036] Preferably, the knockout is performed using primer sequences shown in SEQ ID No. 3 and SEQ ID No. 4.

[0037] The nucleotide sequence shown in SEQ ID No.3 is as follows: TGTGTGACCATCATGACCTCCTGCCC.

[0038] The nucleotide sequence shown in SEQ ID No. 4 is as follows: AAACGGGCAGGAGGTCATGATGGTCA.

[0039] In the above preferred embodiment, the transformation method includes one or more of Agrobacterium-mediated, Ti plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

[0040] According to one aspect of the present invention, a method for improving rice plant type is provided, wherein an OsbZIP1 gene knockout vector is transformed into rice or rice cells to cultivate rice with a reduced leaf angle and a compact plant type.

[0041] The present invention provides a method for improving rice plant type, wherein the method transforms an OsbZIP1 gene knockout vector into rice or rice cells, thereby cultivating rice with a reduced leaf angle and a compact plant type. Therefore, the present application can apply the rice OsbZIP1 gene alone or in combination with other genes to rice cultivation to select rice varieties with specific plant types.

[0042] In a preferred embodiment of the present invention, the transformation comprises: introducing the OsbZIP1 gene knockout vector into rice or rice cells by Agrobacterium-mediated method;

[0043] In the above preferred embodiment, the Agrobacterium is Agrobacterium EHA105.

[0044] In a preferred embodiment of the present invention, the rice variety is japonica rice or indica rice, preferably Zhonghua 11.

[0045] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0047] Example 1 Cloning of Rice Leaf Angle Regulatory Gene OsbZIP1

[0048] The rice variety Zhonghua 11 was used as the experimental material. The total RNA of leaf tissue was extracted by TRIzol method, and then cDNA was synthesized. The obtained cDNA was used as a template to amplify the OsbZIP1 gene using primer pair F / R, and then sequenced and analyzed after electrophoresis recovery and purification.

[0049] F: 5'-ATGGTGCAAGGTGAGGAG-3' (SEQ ID No. 5).

[0050] R: 5'-TCAAAATCCTGAGTACTGATT-3' (SEQ ID No. 6).

[0051] Here are the steps:

[0052] 1. RNA extraction: Put the tissue frozen with liquid nitrogen into a pre-cooled mortar and grind it into powder. Transfer it into a 1.5mL centrifuge tube (RNA-free) (about 100-150μL) before the tissue is thawed; add 1mL TRIZOL solution, mix by inverting, and place at room temperature for 5 minutes; transfer the centrifuge tube to a high-speed centrifuge, centrifuge at 12000rpm for 5 minutes; take 800μL of supernatant to a new 1.5mL centrifuge tube (RNA-free); add 200μL of chloroform to the centrifuge tube, mix by inverting, and place at room temperature for 15 minutes; transfer the centrifuge tube to a 4℃ centrifuge, centrifuge at 12000rpm for 15 minutes; aspirate about 400μL of the upper aqueous phase to a new 1.5mL centrifuge tube, and be sure not to aspirate the middle interface; add 400μL of isopropanol to the centrifuge tube, gently shake and mix, and place at room temperature for 5-10 minutes; centrifuge at 4℃, 15000rpm for 10 minutes, pour off the supernatant, and a white precipitate can be seen at the bottom of the centrifuge tube; add 1mL to the tube 75% ethanol, gently shake the centrifuge tube until the precipitate is suspended; centrifuge at 80,000 rpm for 3 minutes at 4°C, remove the supernatant, and let the centrifuge tube air-dry at room temperature for 3-6 minutes; add 20 mL RNA-free H2O to the tube, and tap the tube wall with your hand to promote the dissolution of the RNA precipitate.

[0053] 2. RNA digestion (removal of DNA contamination):

[0054]

[0055]

[0056] Digestion procedure: 37℃ for 30min, 75℃ for 5min, use immediately or store in -80℃ freezer.

[0057] 3. Synthesis of cDNA:

[0058] The cDNA was obtained by reverse transcription using the First Strand cDNA Synthesis Kit from TOYOBO.

[0059] 4. (a) Removal of DNase I contamination:

[0060]

[0061] Treat with PCR instrument at 75°C for 5 min, then place on ice for 2 min;

[0062] (b) Synthesis of first-strand cDNA:

[0063]

[0064] Treat with PCR instrument at 42°C for 60 min, 75°C for 5 min, and then store at -20°C.

[0065] 5. Cloning results:

[0066] The nucleotide sequence of the OsbZIP1 gene is shown in SEQ ID No.1.

[0067] The amino acid sequence encoded by the OsbZIP1 gene is shown in SEQ ID No.2.

[0068] Example 2 Obtaining OsbZIP1 gene knockout transgenic strains

[0069] 1. Construction of knockout vector

[0070] The target site for knocking out OsbZIP1 was designed using the website ( https: / / crispr.dbcls.jp / ), and then the primer pair cr-OsbZIP1-F / R was used to generate dimers, which were then constructed into the CRISPR / Cas9 vector;

[0071] cr-OsbZIP1-F:5'-TGTGTGACCATCATGACCTCCTGCCC-3' (SEQ ID No. 3).

[0072] cr-OsbZIP1-R: 5'-AAACGGGCAGGAGGTCATGATGGTCA-3' (SEQ ID No. 4).

[0073] 2. Transfer the constructed vector into Agrobacterium EHA105, the steps are as follows: add 1-3 μL plasmid to 100 μL Agrobacterium competent medium, place on ice for 5 minutes; freeze the Agrobacterium competent medium with the added plasmid in liquid nitrogen for 5 minutes; after the liquid nitrogen treatment, transfer the competent medium to a 37°C water bath for 5 minutes; add 400 μL YEP medium (without antibiotics) to the competent medium, and culture it in a shaking incubator at 28°C for 2-4 hours; spread the cultured Agrobacterium on a plate containing kanamycin and rifampicin antibiotics, and culture it at 28°C for 2-3 days.

[0074] 3. Genetic transformation

[0075] The knockout vector was transferred into the japonica rice variety Zhonghua 11 using Agrobacterium-mediated transformation, and then cultured, differentiated, and rooted.

[0076] 4. Acquisition of transgenic positive seedlings and genotype identification

[0077] The primer pair HYG-F / R was used to perform PCR amplification on the T0 generation transgenic plants, and the plants that could amplify the bands were positive plants; then the primer pair OsbZIP1-JC-F / R was used to amplify the target fragment and sequence it to analyze its genotype.

[0078] HYG-F: 5'-GTGCTTTCAGCTTCGATG-3' (SEQ ID No. 7);

[0079] HYG-R: 5'-AACCAAGCTCTGATAGAG-3' (SEQ ID No. 8);

[0080] OsbZIP1-JC-F: 5'-CGCTGTTGTTCTTGGCTCTT-3' (SEQ ID No. 9);

[0081] OsbZIP1-JC-R: 5'-AGTCGTCGTGGTCCTGCTCT-3' (SEQ ID No. 10).

[0082] Example 3 Obtaining OsbZIP1 gene overexpression transgenic strain

[0083] 1. Construction of overexpression vector:

[0084] Using Zhonghua11 cDNA as a template, the CDS fragment of OsbZIP1 was amplified by primers OX-F / R and constructed into the vector pCAMBIA1305-GFP, and then introduced into the japonica rice variety Zhonghua11 by Agrobacterium-mediated transformation.

[0085] OX-F: 5'-GCCCAGATCAACTAGTATGGTGCAAGGTGAGGAG-3' (SEQ ID No. 11);

[0086] OX-R: 5'-TGCTCACCATGGATCCAAATCCTGAGTACTGATTCT-3' (SEQ ID No. 12).

[0087] 2. Detection of overexpression positive plants:

[0088] The primer pair HYG-F / R was used to perform PCR amplification detection on the OsbZIP1 overexpressing T0 generation transgenic plants, and the plants that could amplify the bands were positive plants.

[0089] Example 4 Detection of OsbZIP1 gene expression

[0090] RNA was extracted from leaves of Zhonghua 11, OsbZIP1 knockout strain (T1, two strains were numbered OsbZIP-1, OsbZIP-2) and overexpression strain (T1 two strains were numbered OX-OsbZIP-1, OX-OsbZIP-2) respectively, and reverse transcribed to obtain cDNA for real-time fluorescence quantitative PCR experiments. The primers used to detect the expression of OsbZIP1 gene were:

[0091] OsbZIP1-RT-F: 5'-ACCCACCATGAGAGAGCAAT-3' (SEQ ID No. 13);

[0092] OsbZIP1-RT-R: 5'-GCTGGAAGTCCAAGAAGCTG-3' (SEQ ID No. 14).

[0093] This experiment used THUNDERBIRD™ SYBR qPCR Mix without ROX (TOYOBO) kit was used.

[0094] Real-time fluorescence quantitative PCR reaction system:

[0095]

[0096] Real-time fluorescence quantitative PCR reaction program: 95°C for 30 s; amplification for 40 cycles (95°C for 5 s, 60°C for 30 s); 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

[0097] Calculation of real-time fluorescence quantitative PCR results: The rice Actin gene (Os03g0718150) was used as an internal reference, and the 2-ΔΔCT method was used to calculate and analyze the experimental results.

[0098] Figure 1 This is a graph showing the relative expression level of the OsbZIP1 gene in the rice OsbZIP1 knockout strain and overexpression strain provided in this example. Figure 1 A in the middle is a schematic diagram of the OsbZIP1 knockout target and mutation method. Figure 1 Middle B is the relative expression detection diagram; among them: ZH11 is the wild type; OsbZIP-1 and OsbZIP-2 are OsbZIP1 gene knockout strains; OX-OsbZIP-1 and OX-OsbZIP-2 are overexpression strains.

[0099] like Figure 1 As shown, compared with the wild type, the expression level of OsbZIP1 in the knockout transgenic line was significantly decreased, while it was significantly increased in the overexpression positive line.

[0100] Figure 2 This is a comparison diagram of the rice OsbZIP1 knockout strain and the wild-type strain provided in this example.

[0101] Figure 2 A in the middle is a comparison of plant morphology. Figure 2 Middle B is a comparison of the sword leaf morphology of a single plant. Figure 2 Middle C is a statistical chart of leaf angles. Figure 2 ZH11 is the wild type; OsbZIP-1 and OsbZIP-2 are strains with OsbZIP1 gene knocked out.

[0102] like Figure 2 As shown in Figure 2, knocking out OsbZIP1 significantly reduced the leaf angle of rice, while overexpressing OsbZIP1 did not change the morphology of the plant.

[0103] Example 5: BR sensitivity identification

[0104] Brassinosteroid (BR) is an important sterol plant hormone that plays an important role in the growth and development of plants. In rice, BR regulates many important agronomic traits, including rice leaf angle and grain size, which means that BR has great potential in improving rice productivity. As one of the most BR-sensitive tissues in rice, BR-insensitive mutants generally lead to a smaller rice leaf angle, while BR-sensitive mutants generally lead to an increase in rice leaf angle. Therefore, studying the mechanism of action of brassinosteroid signaling in rice is of great significance to the improvement of rice plant type and the increase of yield.

[0105] The present invention identified the BR sensitivity of rice OsbZIP1 knockout strains, and the specific method is as follows:

[0106] The sterilized seeds of Zhonghua 11 and OsbZIP1 knockout strains were germinated and grown in a growth chamber for 8 days. Then, fragments of about 2 cm from the pulvinus were cut and placed in a solution containing 1 μM BL (24-epibrassinolide, the active form of BR). After incubation at 28°C for 2 days, the leaf angles were observed and measured.

[0107] Figure 3 This is a diagram for identifying the sensitivity of the rice OsbZIP1 knockout strain to BR provided by the present invention. Figure 3 ZH11 is the wild type; OsbZIP-1 and OsbZIP-2 are strains with OsbZIP1 gene knocked out.

[0108] like Figure 3 As shown, after BR sensitivity testing of seedlings of Zhonghua11 and OsbZIP1 knockout lines, it was found that the sensitivity of OsbZIP1 knockout lines to BR was weakened, which preliminarily indicated that the OsbZIP1 gene could positively regulate the transduction of BR signals.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of OsbZIP1 gene in regulating rice plant type, the nucleotide sequence of the OsbZIP1 gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by the OsbZIP1 gene is shown in SEQ ID No.

2.

3. The use according to claim 1, characterized in that: The application is to inhibit the expression of the OsbZIP1 gene to transform rice plants with reduced leaf angles.

4. The use according to claim 3, characterized in that: The transformation comprises: constructing a gene knockout vector with OsbZIP1 gene as the target gene, transforming rice plants with the gene knockout vector, and obtaining rice plants with reduced leaf angles.

5. The use according to claim 4, characterized in that: The gene knockout vector is constructed based on the CRISPR / Cas9 vector; Preferably, the knockout is performed using primer sequences shown in SEQ ID No. 3 and SEQ ID No.

4.

6. The use according to claim 4, characterized in that: The transformation method includes one or more of Agrobacterium-mediated, Ti plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

7. A method for improving rice plant type, characterized in that: The OsbZIP1 gene knockout vector is transformed into rice or rice cells to cultivate rice with a smaller leaf angle and a compact plant shape.

8. The method for improving rice plant type according to claim 7, characterized in that: The transformation comprises: introducing the OsbZIP1 gene knockout vector into rice or rice cells by Agrobacterium-mediated method; Preferably, the Agrobacterium is Agrobacterium EHA105.

9. The method for improving rice plant type according to claim 7, characterized in that: The variety of the rice is japonica rice or indica rice.

10. The method for improving rice plant type according to claim 9, characterized in that: The rice variety is Zhonghua No. 11.

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