Rice HAN2 gene and its application in breeding high-yield and stable-yield rice varieties

Abstract

The present invention relates to the field of plant genetic engineering technology, and in particular to a rice HAN2 gene and its application in cultivating high-yield and stable-yield rice varieties. The nucleotide sequence of the rice HAN2 gene is shown in SEQ ID NO.1 or SEQ ID NO.2. The rice HAN2 gene of the present invention is firstly proved that its encoded protein has IAA transport activity. NPB The discovery of the rice HAN2 gene as a negative regulator of cold tolerance at the seedling and heading stages provides an important theoretical basis for improving rice yield under low temperature stress. NPB Or using gene editing to create mutant alleles to improve the rice's ability to maintain stable yield under low temperature stress has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to rice HAN2 gene and application thereof in cultivating high-yield and stable-yield rice varieties. Background Art

[0002] Rice is the most important food crop in the world, providing staple food for nearly half of the world's population. High yield and stable yield of rice are important factors that determine rice yield. High yield of rice is directly or indirectly determined by plant height, tillering, number of spikelets per panicle, fruit set rate, thousand-grain weight, etc. At the same time, plant height also affects rice lodging resistance. Rice stable yield refers to rice's tolerance or escape from adverse stress and reduce rice yield losses. Improving the cold tolerance of rice seedlings can resist low temperature weather such as late spring cold that early rice often encounters; improving the cold tolerance of the heading stage can resist low temperature weather such as cold dew wind that late rice often encounters; improving the cold tolerance of rice seedlings and heading stages can make rice, especially northern japonica rice, better adapt to the lower temperature growth environment in temperate regions, extend the growth period, and achieve high and stable yields.

[0003] Therefore, discovering genes for high and stable yield of rice and using them to breed high and stable yield rice varieties is an important means to ensure food security in my country and even the world. Summary of the invention

[0004] The purpose of the present invention is to provide a rice HAN2 gene and its application in cultivating high-yield and stable-yield rice varieties, so as to provide an important theoretical basis for high-yield and stable-yield rice under low temperature stress.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In a first aspect, a rice HAN2 gene is provided, the nucleotide sequence of which is shown in any one of the following:

[0007] (1) the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 in the sequence listing;

[0008] (2) a nucleotide sequence that hybridizes with the nucleotide sequence defined in (1) under stringent conditions and encodes a plant auxin transporter;

[0009] (3) a nucleotide sequence having at least 70% identity with the nucleotide sequence in (1);

[0010] (4) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence in (1), but differing in sequence due to the degeneracy of the genetic code;

[0011] (5) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.3, or a nucleotide sequence encoding an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO.3 due to substitution, deletion and / or insertion of one or more amino acid residues, or a nucleotide sequence encoding an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO.3;

[0012] (6) An active fragment of any nucleotide sequence of (1) to (5);

[0013] (7) A nucleotide sequence complementary to any one of the nucleotide sequences (1) to (5).

[0014] In a second aspect, a protein encoded by the rice HAN2 gene is provided, the amino acid sequence of which is shown in any one of the following:

[0015] (1) encoding the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO.3 due to substitution, deletion and / or insertion of one or more amino acid residues, or an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO.3;

[0016] (2) An active fragment of the amino acid sequence in (1).

[0017] In a third aspect, a plant expression vector or a cloning vector is provided, which includes a rice HAN2 gene.

[0018] In a fourth aspect, a host cell is provided, which comprises a rice HAN2 gene.

[0019] Furthermore, the host cell is selected from plant cells or microbial cells.

[0020] In a fifth aspect, a promoter for transcription of rice HAN2 gene is provided, the nucleotide sequence of which is shown in any one of the following:

[0021] (1) the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.5 in the sequence listing;

[0022] (2) a nucleotide sequence having at least 70% identity with the nucleotide sequence of (1);

[0023] (3) An active fragment of any nucleotide sequence from (1) to (2);

[0024] (4) A nucleotide sequence complementary to any one of the nucleotide sequences (1) to (3).

[0025] In the sixth aspect, the application of rice HAN2 gene in breeding high-yield and stable-yield rice varieties is provided. Rice HAN2 gene can positively regulate rice plant height, negatively regulate rice tiller number, and negatively regulate rice cold resistance at seedling and heading stages.

[0026] A seventh aspect provides a method for cultivating a high-yield and stable-yield rice variety, comprising the following steps:

[0027] The transgenic rice plants are obtained by transfecting rice plants with recombinant Agrobacterium containing the rice HAN2 gene, or by combining the indica rice allele 9311 containing the rice HAN2 gene with the japonica rice allele 9311-HAN2 NPB The offspring rice plants were obtained by hybridizing two varieties of rice, from which rice materials with enhanced cold resistance and increased yield were screened.

[0028] The beneficial effects of the present invention are:

[0029] (1) The rice HAN2 gene of the present invention is the first to demonstrate that the protein it encodes has IAA transport activity.

[0030] (2) In the present invention, whether 9311-HAN2 NPB Both the han2 mutant and the han2 mutant can significantly reduce plant height, increase the number of tillers and single plant yield, and have important application value in production practice.

[0031] (3) The discovery of the rice HAN2 gene as a negative regulator of cold tolerance at the seedling and heading stages provides an important theoretical basis for improving rice yield under low temperature stress. NPB Or using gene editing to create mutant alleles to improve the rice's ability to maintain stable yield under low temperature stress has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Plant type diagram;

[0033] Figure 2 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Plant height statistics results;

[0034] Figure 3 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Statistical results of tillering number per plant;

[0035] Figure 4 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Statistical results of single plant yield;

[0036] Figure 5 The plant type diagrams of ZH11 and han2 mutants;

[0037] Figure 6 The statistical results of plant height of ZH11 and han2 mutants;

[0038] Figure 7 The tillering statistics of individual plants of ZH11 and han2 mutants are shown;

[0039] Figure 8 The statistical results of single plant yield of ZH11 and han2 mutants;

[0040] Fig. 9 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Seedling performance under normal conditions and after 10°C low temperature treatment;

[0041] Fig.10 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Statistical results of seedling survival rate after 10℃ low temperature treatment;

[0042] Fig.11 Seedling performance of ZH11 and han2 mutants under normal conditions and after 4℃ low temperature treatment;

[0043] Fig.12 The statistical results of the survival rate of ZH11 and han2 mutants at the seedling stage after low temperature treatment at 4℃;

[0044] Fig.13 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Ear shape diagram under normal conditions and after 10℃ low temperature treatment;

[0045] Fig.14 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Statistical results of fruit set rate under normal conditions;

[0046] Fig.15 It is the near isogenic line of 9311 and HAN2, 9311-HAN2 NPB Statistical results of fruit setting rate after 19℃ low temperature treatment;

[0047] Fig.16 The ear shape diagrams of ZH11 and han2 mutants under normal conditions and after 16℃ low temperature treatment;

[0048] Fig.17 The statistical results of fruiting rate of ZH11 and han2 mutants under normal conditions;

[0049] Fig.18 The statistical results of the fruiting rate of ZH11 and han2 mutants after low temperature treatment at 16℃;

[0050] Fig.19 is the atomic percentage excess of deuterium atoms remaining in GFP and HAN2-GFP yeast cells;

[0051] Fig. 20 is the atomic percentage excess of deuterium atoms remaining in GFP and HAN2-GFP Xenopus egg cells;

[0052] Fig.21 is the relative content of D5-IAA effluxed in protoplasts of WT and han2 mutant;

[0053] Fig. 22 The relative transfer rates of D5-IAA in different leaves and sheaths of WT and han2 mutants. DETAILED DESCRIPTION

[0054] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0055] Example 1: Cloning of the rice high-yield and stable-yield gene HAN2

[0056] Positional cloning: 1) Using chromosome segment substitution lines (CSSLs) of japonica rice Nipponbare (Nip) and indica rice 9311, a QTL-qHAN2 affecting rice cold tolerance was located on rice chromosome 1 through multi-year multi-point measurement; 2) Based on the chromosome segment substitution line population, a chromosome segment substitution line CSSL10 containing the NPB qHAN2 locus was screened out; 3) CSSL10 was backcrossed with 9311 to construct a BC4F1 progeny population. Through the screening of recombinant plants and the determination of the low-temperature phenotype of homozygous recombinant plants (BC3F2), qHAN2 was located in the 70-kb interval of rice chromosome 1; 4) This interval contains 8 predicted genes, including HAN2 (LOC_Os01g50100); 5) There are polymorphic differences in the promoter region of this gene between NPB and 9311, and there are also retrotransposon differences in the 3′UTR region, and the expression level in the 9311 allele is significantly higher than that in the NPB allele; 6) By comparing the near-isogenic lines 9311 and 9311-HAN2 NPBThe cold tolerance test at the seedling stage found that the high expression level 9311 was higher than 9311-HAN2. NPB , the latter has stronger cold tolerance, indicating that LOC_Os01g50100 is the candidate gene HAN2 of qHAN2-QTL; 7) Through the identification of cold tolerance of HAN2 mutants at seedling stage and the detection of trehalose, it was found that while the cold tolerance of mutants was enhanced, their trehalose content was also increased, further indicating that the mutation of HAN2 can improve the cold tolerance of rice seedlings, indicating that HAN2 is the target gene. The cDNA sequence of the gene is shown in SEQ ID NO.1, its genomic nucleotide sequence is shown in SEQ ID NO.2, its encoded protein amino acid sequence is shown in SEQ ID NO.3, and its promoter nucleotide sequence is shown in SEQ ID NO.4 and SEQ ID NO.5.

[0057] Example 2: HAN2 positively regulates rice plant height and negatively regulates rice tillering number

[0058] Field observation 9311, 9311-HAN2 NPB , wild-type ZH11 and han2 mutant materials ( Figure 1 , Figure 5 ) found that the plant height of 9311 was significantly higher than that of the near isogenic line 9311-HAN2 NPB ( Figure 2 ), the plant height of ZH11 was significantly higher than that of mutant han2 ( Figure 6 ), and the number of tillers 9311-HAN2 NPB Higher than 9311( Figure 3 ), the tiller number of the han2 mutant was higher than that of the wild type ZH11 ( Figure 7 ), indicating that under normal growth conditions, HAN2 has the function of positively regulating rice plant height (i.e., the japonica rice allele or mutant allele of HAN2 can reduce rice plant height; the indica rice allele can increase rice plant height) and negatively regulating rice tillering (i.e., the japonica rice allele or mutant allele of HAN2 can increase the number of rice tillers).

[0059] 9311-HAN2 NPB The yield per plant was significantly higher than that of 9311( Figure 4 ), the yield per plant of the han2 mutant was also significantly higher than that of the wild-type control ZH11 ( Figure 8 ), indicating that the japonica rice allele or mutant allele of HAN2 has the potential to increase yield.

[0060] Example 3: HAN2 negatively regulates cold tolerance of rice at seedling and heading stages

[0061] Low temperature treatment at seedling stage: about 15 days old, in artificial climate box, 10h / 14h, 10℃ treatment for 3-6 days; recovery: intelligent greenhouse room 1, 11h / 13h, 30℃ / 25℃; NIL-HAN2, a near-isogenic line with low HAN2 expression NPB The survival rate of seedlings was significantly higher than that of 9311 ( Figure 9-10 In addition, the seedlings were treated in an artificial climate box at 10h / 14h, 4℃ for 3-6 days; recovery: Room 1 of the smart greenhouse, 11h / 13h, 30℃ / 25℃; the survival rate of the mutant strain of han2 was significantly better than that of the wild type ZH11 ( Figure 11-12 ), further indicating that after HAN2 mutation, its cold tolerance is enhanced under low temperature conditions at the seedling stage. The results show that HAN2 negatively regulates the cold tolerance of rice seedlings.

[0062] Low temperature treatment at the heading stage: NIL-HAN2 under normal conditions NPB There was no significant difference in fruiting rate between 9311, wild type ZH11 and mutant han2 ( Fig.14 , Fig.17 ). At the heading stage, in the walk-in artificial climate box, the indica rice background was 12h / 12h, 19℃ for 7 days; the japonica rice background was 12h / 12h, 16℃ for 7 days; recovery: intelligent greenhouse room 1, 11h / 13h, 30℃ / 25℃. After low temperature treatment at the heading stage, NIL-HAN2 NPB The fruiting rate was significantly higher than that of 9311( Fig.13 , Fig.15 ); The fruiting rate of the han2 mutant was significantly increased compared with the wild type ZH11 ( Fig.16 , Fig.18 The results showed that HAN2 negatively regulated cold tolerance in rice at the heading stage.

[0063] Example 4: HAN2 has IAA transport function

[0064] First, the yeast 3-Indoleacetic acid-d5 (d5-IAA) stable isotope transport experiment showed that ( Fig.19 ), after 3 hours of incubation with 150μM d5-IAA, the d5-IAA contained in yeast cells heterologously expressing HAN2-GFP was not significantly different from that in GFP empty cells. After 12 hours of efflux, the d5-IAA contained in yeast cells heterologously expressing HAN2-GFP was significantly reduced compared with that in GFP empty cells. Secondly, the stable isotope transport experiment of d5-IAA in Xenopus oocytes showed that ( Fig. 20), after 4 hours of incubation with 150 μM d5-IAA, the d5-IAA contained in yeast cells heterologously expressing GFP-HAN2 was not significantly different from that in GFP empty cells. After 4 hours of efflux, the d5-IAA contained in yeast cells heterologously expressing GFP-HAN2 was significantly lower than that in GFP empty cells. Finally, the d5-IAA transport experiment in rice protoplasts showed that ( Fig.21 ), the relative transfer amount of d5-IAA in wild-type protoplasts was significantly higher than that in han2 mutant protoplasts after 0.5, 1, and 2 hours of efflux. The above results prove that HAN2 has the function of transporting auxin to the extracellular space. The experiment of feeding d5-IAA to rice plant leaves showed that ( Fig. 22 ), the relative transfer rate of d5-IAA in wild-type plants was significantly higher than that in han2 mutants, confirming that HAN2 can transfer d5-IAA from the relatively mature leaves of the source to other leaves and roots.

[0065] Example 5: Application of HAN2 in Indica-Japonica subspecies hybridization

[0066] In the process of breeding new rice varieties, the han2 mutant allele can be created by gene editing, thereby improving the cold tolerance of rice at the seedling and heading stages. 93-11 Rice and japonica allele NIL-HAN2 Nip The offspring rice plants are obtained by hybridizing rice, from which rice materials with high yield (plant height, tillering, single plant yield) and stable yield (cold resistance) are screened.

[0067] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0068] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of rice HAN2 gene in breeding high-yield and stable-yield rice varieties, characterized in that: The nucleotide sequence of the rice HAN2 gene is shown in SEQ ID NO.1 or SEQ ID NO.2; the rice HAN2 gene can positively regulate rice plant height, negatively regulate rice tiller number, and negatively regulate rice cold tolerance at seedling and heading stages.

2. A method for cultivating high-yield and stable-yield rice varieties, characterized in that: The following steps are involved: The invention relates to obtaining a transgenic rice plant by transfecting a rice plant with a recombinant Agrobacterium containing the rice HAN2 gene according to claim 1, or by combining an indica rice allele 9311 containing the rice HAN2 gene according to claim 1 with a japonica rice allele 9311-HAN2 NPB The offspring rice plants were obtained by hybridizing two varieties of rice, from which rice materials with enhanced cold resistance and increased yield were screened.

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