Rice OsABCB7 gene as well as encoding protein and application thereof

Abstract

The invention relates to a rice OsABCB7 gene as well as an encoding protein and application thereof. The inventor utilizes a CRISPR / Cas9 gene editing system to obtain two homozygous knockout mutants osabcb7-1 and osabcb7-9 of an OsABCB7 gene, wherein the two homozygous knockout mutants osabcb7-1 and osabcb7-9 take japonica rice Nipponbare as a background and have different editing conditions. Many-year and multi-generation agronomic trait investigation shows that under the normal field planting condition, the grain length, grain width, thousand seed weight, single plant yield and the like in the mature period are all remarkably increased compared with those of a wild type contrast, and the maturing rate has no obvious difference; however, under the continuous high-temperature condition, the maturing rate and the yield of a single plant of the mutant are obviously higher than those of a wild type control. Besides, the mutant rice after salt treatment shows enhanced salt tolerance, so that the OsABCB7 gene can be applied to rice crossbreeding and molecular design breeding to improve the rice yield and enhance the salt tolerance and high temperature resistance of the rice.

Description

Technical Field

[0001] The invention belongs to the technical fields of molecular biology, genetic engineering and plant breeding, and particularly relates to the application of rice OsABCB7 gene and its encoded protein in cultivating high-yield crop varieties with enhanced salt and high temperature tolerance. Background Art

[0002] Rice (Oryza sativa L.) is an annual monocotyledonous plant of the Poaceae family. It is one of the three major staple crops in the world and the largest staple crop in my country. Rice is affected by many abiotic stresses during its growth. Saline-alkali land and high temperature largely limit the rice growing area and yield. With the continuous increase in population and food demand at this stage, the available arable land area and natural resources are gradually decreasing, and food supply problems are gradually emerging. Therefore, we urgently need to increase the yield of food crops and the stress tolerance of crops.

[0003] The three components of rice yield include the number of grains per panicle, 1000-grain weight, and the number of effective panicles per plant. The 1000-grain weight is mainly determined by grain shape and grain filling degree. Grain shape, including grain length and grain width, is an important trait that determines yield. Therefore, moderately increasing grain size and weight while maintaining the stability of the number of grains per panicle and the number of effective panicles per plant is a key strategy to increase rice yield.

[0004] The ATP-binding cassette (ABC) transporter family is a large class of transporters widely found in eukaryotes. It has the function of transporting substances using the energy generated by ATP hydrolysis, and transports a wide variety of substrates. In terrestrial plants, the ABC transporter family is divided into eight subfamilies: A, B, C, D, E, F, G, and I, based on their structural characteristics. Most of the subfamilies reported so far are related to important processes such as pollen development, auxin transport, grain yield, and arsenic stress.

[0005] Most of the ABCB subfamily genes reported in Arabidopsis are related to auxin transport. There are 27 genes encoding ABCB subfamily transport proteins in rice, and those reported in rice are related to pollen development and abiotic stress, while studies related to grain shape and yield are rarely reported. The exploration of ABCB protein function is crucial in the process of plant growth and development. By studying the function of ABCB subfamily genes, the role of this gene in regulating rice yield and salt and high temperature tolerance is clarified, providing excellent gene resources for rice molecular design breeding. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a rice OsABCB7 gene and its encoded protein and application. The present invention studies the biological function of the OsABCB7 gene and finds that it plays an important role in regulating rice grain shape and salt and high temperature tolerance. Therefore, the present invention is of great significance for the synergistic improvement of rice yield and multi-resistance traits of important food crops such as rice.

[0007] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0008] The rice OsABCB7 gene has a genomic nucleotide sequence as shown in SEQ ID NO.1, which contains 6201 bases, and a gene coding region (CDS) nucleotide sequence as shown in SEQ ID NO.2, which contains 3705 bases.

[0009] A coded protein is obtained by encoding the rice OsABCB7 gene, and its amino acid sequence is shown in SEQ ID NO.3, containing 1234 amino acids.

[0010] The biological material comprises the rice OsABCB7 gene, wherein the biological material is a recombinant expression vector or an engineered bacterium.

[0011] The use of the above genes, encoded proteins or biological materials in the following (a) or (b): (a) Regulate rice yield and improve salt and high temperature tolerance; (b) Molecular breeding. Specifically, the application includes: using CRISPR / Cas9 technology to knock out the expression of rice OsABCB7 gene and transform target plant cells, tissues or organs, so as to obtain transgenic strains with increased yield and enhanced salt and high temperature tolerance.

[0012] More specifically, the salt tolerance refers to treating 14-day-old transgenic plant seedlings with 120 mM salt stress for 7 days.

[0013] More specifically, when the mutant plants with high temperature resistance are planted in the field, their fruit setting rate and single plant yield are increased under continuous high temperature conditions of 38°C.

[0014] A molecular design breeding method for improving plant yield and salt and high temperature tolerance comprises the following steps: Step 1, designing target sites at the first exon and the second exon of the OsABCB7 gene, wherein the nucleotide sequence of target site I is shown in SEQ ID NO.6, and the nucleotide sequence of target site II is shown in SEQ ID NO.7; constructing a CRISPR / Cas9 editing vector according to the target site I and target site II; Step 2: The CRISPR / Cas9 editing vector is transferred into the plant to be gene-edited through Agrobacterium-mediated method, and T0 generation positive plants with loss-of-function mutations are cultivated and screened; Step 3: The T0 generation positive plants are self-pollinated for one generation, and the T1 generation homozygous mutant plants are screened.

[0015] Specifically, the homozygous mutant is osabcb7-1, which has a deletion of three bases "TGG" at positions 340-342 of the nucleotide sequence shown in SEQ ID NO.1, and a deletion of 43 bases at positions 659-701, thereby inhibiting the expression of the protein encoded by the OsABCB7 gene, resulting in improved yield and salt and high temperature resistance.

[0016] Specifically, the homozygous mutant is osabcb7-9, which lacks 12 bases at positions 336-347 of the nucleotide sequence shown in SEQ ID NO.1, and a single base "T" is inserted between positions 683 and 684, thereby inhibiting the expression of the protein encoded by the OsABCB7 gene, resulting in improved yield and salt and high temperature resistance.

[0017] Specifically, the plant is any monocotyledon or dicotyledon; monocotyledon is rice, corn, sorghum, wheat, millet, barley, oats, rye, and dicotyledon is cotton, soybean, peanut, tobacco, rapeseed, tomato.

[0018] Beneficial effects: The inventors used the CRISPR / Cas9 gene editing system to obtain two homozygous knockout mutants of the OsABCB7 gene with different editing conditions, osabcb7-1 and osabcb7-9, in the background of japonica rice Nipponbare. After years of multi-generational agronomic trait investigations, it was found that under normal field planting conditions, the grain length, grain width, thousand-grain weight and single-plant yield at maturity were significantly increased compared with the wild-type control, while there was no significant difference in the fruit setting rate; however, under continuous high temperature conditions, the fruit setting rate and single-plant yield of the mutant were significantly higher than those of the wild-type control. In addition, after salt treatment, the mutant rice showed enhanced salt tolerance. Therefore, the OsABCB7 gene can be used in rice hybrid breeding and molecular design breeding to increase rice yield and enhance its salt and high temperature tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the gene editing status of the T1 generation plants of the osabcb7-1 and osabcb7-9 mutants; in the figure, the arrows point to the locations of the two target sites, WT is the gene sequence of the wild-type Nipponbare, and osabcb7-1 and osabcb7-9 are the gene editing status of the two mutant strains and sequencing peak graphs.

[0020] Figure 2 It is a comparison chart of the yield traits of osabcb7-1 and osabcb7-9 mutants; in the figure, the phenotypes of ear length, ear type and grain shape at maturity, as well as plant height, ear length, number of effective ears, fruit setting rate, yield per plant, number of primary branches, grain length, grain width and 1000-grain weight are compared between the wild-type Nipponbare (WT) and the osabcb7 mutant.

[0021] Figure 3 This is a comparison of the phenotypes and yield traits of osabcb7-1 and osabcb7-9 mutants under continuous 38°C high temperature conditions; in the figure, (A) the phenotypes of wild-type Nipponbare (WT) and osabcb7 mutant under continuous 38°C high temperature conditions during maturity; (BD) comparison of plant height, fruit setting rate and single plant yield between WT and osabcb7 mutant under continuous 38°C high temperature conditions.

[0022] Figure 4 The phenotypes of osabcb7-1 and osabcb7-9 mutant 14-d-old plants treated with 120 mM NaCl for 7 d.

[0023] Figure 5 Comparison of physiological parameters of osabcb7-1 and osabcb7-9 mutant 14-d-old plants treated with 120 mM NaCl for 7 d. (A) Leaf blight rate; (B) Chlorophyll content; (C) Malondialdehyde (MDA) content; (D) Proline content.

[0024] Figure 6 to Figure 12 It is a multiple sequence alignment of rice OsABCB7 protein sequence and homologous genes in monocotyledonous plants. DETAILED DESCRIPTION

[0025] The present invention provides a gene capable of regulating rice yield and salt and high temperature tolerance, and achieves the purpose of increasing rice yield and salt and high temperature tolerance by molecular technology means.

[0026] The present invention confirms that the OsABCB7 gene can simultaneously negatively regulate rice grain shape, yield, salt tolerance and high temperature tolerance. By knocking out the expression of the OsABCB7 gene in rice, the rice grain length, grain width and single plant yield can be significantly increased, while the rice fruit setting rate, single plant yield and salt tolerance of rice under continuous high temperature stress are enhanced. The present invention shows that gene editing of the OsABCB7 gene has important application value in increasing rice yield and increasing salt tolerance and high temperature tolerance.

[0027] The rice OsABCB7 gene and its functionally deleted gene sequence provided by the present invention have the following characteristics:

[0028] 1. The rice OsABCB7 gene of the present invention has a deoxynucleotide sequence as shown in SEQ ID No. 1 in the sequence listing, with a total of 6201 deoxynucleotides, a nucleotide sequence of its gene coding region (CDS) as SEQ ID NO. 2, containing 3705 deoxynucleotides, and an amino acid sequence of its encoded protein as SEQ ID No. 3, with a total of 1234 amino acids.

[0029] 2. The OsABCB7 gene was edited using CRISPR / Cas9 gene editing technology, and two homozygous mutant strains were obtained, both of which showed increased yield and salt and high temperature resistance phenotypes.

[0030] The two homozygous mutants mentioned above comprise the nucleotide sequences shown in SEQ ID No. 4 and SEQ ID No. 5, which are composed of 6155 and 6190 deoxynucleotides respectively.

[0031] 3. The application of the OsABCB7 gene and its gene editing sequence involved in the present invention in molecular breeding markers, transgenic strains, and new transgenic varieties are all protected contents.

[0032] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and drawings.

[0033] The experimental materials used in the following examples mainly include: (a) plant materials: japonica rice variety Nipponbare (Oryza sativa L. japonica); (b) strains and vectors: Escherichia coli strain DH5α, Agrobacterium strain EHA105; related vectors pRGE32, pRGEB32; (c) enzymes and chemical reagents: all are commercially available conventional products.

[0034] In the following examples, PCR primer synthesis and DNA sequencing were performed by Zhejiang Youkang Biotechnology Co., Ltd.

[0035] In the following examples, if no specific experimental methods are specified, all experimental methods can be carried out according to conventional methods or the instructions of the manufacturer.

[0036] Example 1: Construction of CRISPR / Cas9 recombinant vector

[0037] The OsABCB7 gene (Os01g52550) was obtained by scanning the whole genome data of rice. Target sites were designed at the first exon and the second exon of the OsABCB7 gene respectively: the nucleotide sequence of target site I is shown in SEQ ID NO.6, and the nucleotide sequence of target site II is shown in SEQ ID NO.7. Then, primers were designed in combination with the requirements of the CRISPR / Cas9 system (see Table 1), and a CRISPR / Cas9 gene editing recombinant vector was constructed. The constructed recombinant vector was transferred into the Agrobacterium EHA105 strain by heat shock method, and the transformed Agrobacterium was detected by PCR (primers used are shown in Table 1), and the correct single colony was selected for shaking culture, and the strain was preserved with 80% glycerol.

[0038] Example 2: Genetic transformation of CRISPR / Cas9 recombinant vector and identification of editing status

[0039] The japonica rice variety Oryza Sativa L. japonica was used as the background material for genetic transformation. The rice callus was infected with Agrobacterium EHA105 containing the recombinant vector, and after co-culture for 3 days, hygromycin (50 mg / L) and carbenicillin (250 mg / L) were used to screen twice to obtain resistant callus, and then transgenic plants were obtained through differentiation, rooting and seedling hardening, and the T0 generation positive seedlings containing the Cas9 sequence tag were screened for seed propagation.

[0040] The TPS method was used to extract T1 rice genomic DNA, and plants without Cas9 sequence tags were screened. Then primers were designed at both ends of the two target sites of the OsABCB7 gene sequence (see Table 1). The extracted genomic DNA was used as a template for PCR amplification using this primer. The resulting product was cut from the gel and sent to the company for sequencing. The sequencing results were compared with the OsABCB7 genome sequence to clarify the specific editing conditions of different edited strains ( Figure 1 ).

[0041] T1 generation plants were sequenced to obtain two homozygous mutants, osabcb7-1 and osabcb7-9, with two editing conditions. In osabcb7-1, three bases "TGG" were deleted at positions 340-342 of the nucleotide sequence shown in SEQ ID NO.1, and 43 bases were deleted at positions 659-701; while in osabcb7-9, 12 bases were deleted at positions 336-347 of the nucleotide sequence shown in SEQ ID NO.1, and a single base "T" was inserted between positions 683 and 684. Both allelic mutants will eventually lead to premature termination of translation, thereby inhibiting the expression of the protein encoded by the OsABCB7 gene, and obtaining rice mutant plants.

[0042] Example 3: Phenotypic analysis of rice osabcb7-1 and osabcb7-9 mutants at maturity

[0043] 3.1 Cultivation of transgenic plants

[0044] Select a number of healthy and full test material seeds, disinfect them with 0.5% sodium hypochlorite solution for 15 minutes, rinse them with distilled water 3-5 times, soak them in distilled water, and place them in a 37°C incubator until the rice seeds turn white. Pick them out and spread them on a nylon net floating on the nutrient solution to grow. When the rice grows to about 4-5 cm high, fix them in a plastic container containing 1.6L nutrient solution and continue to cultivate. Place 48 seedlings in each container. Change the nutrient solution every 3 days. The above experiments were carried out in an intelligent incubator with 16 hours of light (26°C), 8 hours of darkness (24°C) and 60% humidity.

[0045] Nutrient solution formula: 0.274mM MgSO4, 0.091mM KNO3, 0.5μM MnCl2, 0.183mM Ca(NO3)2, 0.1mM KH2PO4, 0.4μM ZnSO4, 0.183mM (NH4)2SO4, 0.2μM CuSO4, 3μM H3BO3, 0.1μM (NH4)6Mo7O 24 , 40 μM NaFe(III)-EDTA, 2 mM 2-morpholinesulfonic acid (MES). Use NaOH and HCl to adjust the pH value of the nutrient solution to about 5.8-6.0.

[0046] After the experimental materials have grown for one month, they are transferred to the experimental fields for planting. When the rice grows to maturity, the agronomic traits of each strain are examined in the field.

[0047] 3.2 Statistical analysis of yield-related agronomic traits

[0048] After years of multi-generational agronomic trait investigation, under normal field planting conditions, the plant height, ear length, and grain size of the osabcb7-1 and osabcb7-9 knockout mutants were significantly increased compared with the wild-type Nipponbare (WT) ( Figure 2 ). The grain length, grain width, 1000-grain weight and yield per plant of each mutant strain at maturity were significantly higher than those of WT, while the number of primary branches and fruiting rate were not significantly different from those of the wild type ( Figure 2 ).

[0049] However, when planted in the field and exposed to continuous high temperatures above 38°C, the mutant strains showed significantly higher fruit set rate and yield per plant than the wild-type control ( Figure 3 ), indicating that the OsABCB7 gene significantly negatively regulates the fruit setting rate and yield of rice under high temperature.

[0050] Example 4: Analysis of salt tolerance of rice osabcb7-1 and osabcb7-9 homozygous mutants at the seedling stage

[0051] The method of nutrient solution culture at the seedling stage is the same as 3.1. When the seedlings of the test materials reach 14 days old (the first day of seedling age is counted when they are laid on the seed-laying net), 120mM NaCl is added to the nutrient solution for salt stress treatment for 7 days. After the treatment, take photos in time to record the phenotype and harvest the leaves of the test materials, and place them in a -80℃ refrigerator for later use.

[0052] like Figure 4 and Figure 5 As shown in Figure 2, after salt treatment, the leaves of both WT and mutant plants dried up and curled, but compared with WT, the degree of leaf drying in mutant plants was weaker ( Figure 4 ), and the leaf blight rate was significantly lower than that of WT ( Figure 5 A). Before treatment, there were no significant differences in total chlorophyll, malondialdehyde (MDA) and proline content between WT and mutant strains. However, after salt treatment, the total chlorophyll content of the mutant strain was significantly higher than that of WT ( Figure 5 B), while the MDA content was significantly lower than that of WT ( Figure 5 C); In addition, the proline accumulation in the mutant plants was significantly higher than that in the WT ( Figure 5 D) These results indicate that the osabcb7 mutant line is more salt tolerant than the WT.

[0053] Example 5. Conservation analysis of rice OsABCB7 in monocots

[0054] Using the protein sequence encoded by the rice OsABCB7 gene as shown in SEQ ID No. 3, the protein sequence database of NCBI in the United States was searched by sequence alignment and it was found that the OsABCB7 gene has one or more orthologous genes in both monocotyledonous and dicotyledonous plants. The results of multiple sequence alignment showed that the OsABCB7 protein sequence is highly conserved in monocotyledonous plants such as African rice, corn, sorghum, wheat, and Brachypodium distichum.

[0055] Table 1 Primers related to CRISPR-Cas9 gene editing and PCR amplification

[0056] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. Rice OsABCB7 The gene, whose genomic nucleotide sequence is shown in SEQ ID NO.1, contains 6201 bases, and whose gene coding region nucleotide sequence is shown in SEQ ID NO.2, contains 3705 bases.

2. A protein encoding a protein from rice OsABCB7 The gene encoding is obtained, and its amino acid sequence is shown in SEQ ID NO.3, which contains 1234 amino acids.

3. The rice as claimed in claim 1 OsABCB7 The biological material of the gene is a recombinant expression vector or an engineered bacterium.

4. The rice according to claim 1 OsABCB7 Use of the gene, the encoded protein according to claim 2 or the biological material according to claim 3 in the following (a) or (b): (a) Regulating rice yield and improving salt and high temperature tolerance; (b) Molecular breeding.

5. The use according to claim 4, characterized in that: The application includes: using CRISPR / Cas9 technology to knock out rice OsABCB7 Genes are introduced and transformed into target plant cells, tissues or organs to obtain transgenic strains with increased yield and enhanced salt and high temperature tolerance.

6. The use according to claim 5, characterized in that: The salt tolerance refers to the treatment of 14-day-old transgenic plant seedlings with 120 mM salt stress for 7 days.

7. The use according to claim 5, characterized in that: The mutant plants with high temperature resistance are planted in the field, and under continuous high temperature conditions of 38° C., their fruit setting rate and single plant yield are increased.

8. A molecular design breeding method for improving plant yield and salt and high temperature tolerance, characterized in that: The following steps are involved: Step 1: OsABCB7 Target sites are designed at the first exon and the second exon of the gene, wherein the nucleotide sequence of target site I is shown in SEQ ID NO.6, and the nucleotide sequence of target site II is shown in SEQ ID NO.7; Constructing a CRISPR / Cas9 editing vector according to the target site I and target site II; Step 2: The CRISPR / Cas9 editing vector is transferred into the plant to be gene-edited through Agrobacterium-mediated method, and T0 generation positive plants with loss-of-function mutations are cultivated and screened; Step 3: The T0 generation positive plants are self-pollinated for one generation, and the T1 generation homozygous mutant plants are screened.

9. The molecular design breeding method according to claim 8, characterized in that: The homozygous mutant is osabcb7- 1 , osabcb7-1 There is a deletion of three bases "TGG" at positions 340-342 of the nucleotide sequence shown in SEQ ID NO.1, and a deletion of 43 bases at positions 659-701, thereby inhibiting OsABCB7 The expression of the gene-encoded protein resulted in improved yield and salt and high temperature tolerance.

10. The molecular design breeding method according to claim 8, characterized in that: The homozygous mutant is osabcb7-9 , osabcb7-9 In the nucleotide sequence shown in SEQ ID NO.1, 12 bases are missing from positions 336 to 347, and a single base "T" is inserted between positions 683 and 684, thereby inhibiting OsABCB7 The expression of the gene-encoded protein resulted in improved yield and salt and high temperature tolerance.

Images