Use of HT2 gene / protein in increasing rice yield under high temperature stress

Abstract

The application discloses a use of an HT2 gene / protein in improving the yield of rice under high-temperature stress, and belongs to the technical field of genetic engineering. The HT2 gene / protein can improve the seed setting rate of rice panicles under high-temperature stress, the nucleic acid sequence of the HT2 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The application finds a related gene HT2 (Heat tolerance 2) which can improve the seed setting rate of rice panicles under high-temperature stress, and can significantly improve the yield of rice under high temperature.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to the use of an HT2 gene / protein in increasing the yield of rice under high temperature stress. Background Art

[0002] High temperature stress has the most serious impact on the heading and flowering period of rice. Exposure to high temperatures during this period can lead to abnormal fertilization or inability to normally fill nutrients, changes in the structure of rice starch granules, a decrease in the fruit set rate and an increase in the empty grain rate, ultimately leading to reduced yield or even total crop failure.

[0003] Over the past two decades, researchers have mapped multiple QTL loci associated with heat tolerance across 12 rice chromosomes, using various traits as evaluation indicators. They have also cloned and characterized numerous genes that respond to high temperatures and regulate rice growth and development, including TT1, TT2, and TT3, discovered by Academician Lin Hongxuan's team (Li et al., 2015; Kan et al., 2022; Zhang et al., 2022). However, these studies have primarily focused on regulating heat resistance during the seedling stage. The cloning and molecular mechanism of heat tolerance genes during heading and flowering are still in their early stages, and there are no reports demonstrating simultaneous effects on heat tolerance during the heading stage and rice yield. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of an HT2 gene / protein in increasing rice yield under high temperature stress, which can effectively increase rice yield under high temperature.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] Use of the HT2 gene / protein in increasing rice yield under high temperature stress.

[0007] Furthermore, the nucleic acid sequence of the HT2 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the HT2 gene is shown in SEQ ID NO.2.

[0008] Furthermore, the HT2 gene can increase the seed setting rate of rice under high temperature stress during the panicle stage.

[0009] A method for cultivating high-yield rice under high temperature comprises introducing HT2 gene into the genome of rice and overexpressing the HT2 gene in rice.

[0010] Furthermore, the introduction method is selected from a genetic transformation method, a genome editing method or a gene mutation method.

[0011] The use of the HT2 gene in cultivating high-yield rice varieties under high temperature stress.

[0012] Use of the HT2 gene in screening / identifying rice varieties with high-yield characteristics under high temperature stress.

[0013] The use of the above-mentioned HT2 gene in improving high-yield rice germplasm resources.

[0014] Beneficial effects of the present invention:

[0015] The present invention discovered a gene in rice called HT2 (Heat tolerance 2) that can improve the seed setting rate of rice at the panicle stage under high temperature, which can significantly increase the yield of rice under high temperature stress at the panicle stage. In addition, an excellent natural allele HT2 that is beneficial for improving the seed setting rate of rice at the panicle stage under high temperature stress was also discovered. 9311 , providing new genetic resources and genetic materials for analyzing the high-yield mechanism of rice and cultivating new high-yield varieties under high temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 1 shows the expression changes of HT2 under high temperature. (A) Transcriptome analysis of HT2 expression in spikelets under normal and high temperature conditions. (B, C) qPCR analysis of the expression trends of HT2 in stamens and pistils under high temperature at the heading stage. The horizontal axis represents the high temperature treatment time. Data in the figure are mean ± SD. P values ​​were calculated using the t-test. * indicates P < 0.05, and ** indicates P < 0.01.

[0017] Figure 2 Effects of HT2 on rice seed setting at the panicle stage under high temperature; (A) Phenotypes of wild-type ZH11 and HT2 knockout mutants at the panicle stage treated with or without high temperature; Control: Phenotype of untreated field control; Heat tress: Phenotype of indoor high temperature treatment at the panicle stage. Scale bar, 2 cm; (B) Statistical results of single panicle seed setting rate. Data in the figure are mean ± SD, P values ​​were calculated using t-test, ** indicates P < 0.01; (C) Phenotypes of wild-type ZH11 and HT2 overexpressing materials at the panicle stage treated with or without high temperature. Scale bar, 2 cm; (D) Statistical results of single panicle seed setting rate;

[0018] Figure 3 HT2 9311 Haplotype detection; (A) HT2 haplotype analysis; (B) HT2 sequence differences between Nip and 9311;

[0019] Figure 4 HT2 9311 Yield testing at high temperature; (A) 9311 and SSSL-HT2 NipHigh-temperature treated / untreated phenotypes at the heading stage; Control: field control phenotype, Heat tress: phenotype after indoor high-temperature treatment at the heading stage; scale, 2 cm; (B) Statistical results of single-ear seed setting rate; Data in the figure are mean ± SD, P values ​​were calculated using t-test, ** indicates P < 0.01. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0021] Example 1 Effect of HT2 on Rice Yield

[0022] To identify genes that regulate high temperature tolerance and yield at the panicle stage of rice, we sampled spikelets at normal and high temperatures and sequenced their transcriptomes. Data screening revealed a gene that was upregulated by high temperature ( Figure 1 A), which was speculated to be involved in regulating high temperature tolerance of rice at the heading stage, was named HT2 (Heat tolerance 2). Its nucleic acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0023] To further determine whether HT2 responds to high temperature during the heading stage, we measured the expression of HT2 in stamens and pistils under high temperature during the heading stage and found that its expression was upregulated in both stamens and pistils ( Figure 1 To verify whether HT2 is directly involved in regulating rice yield at the heading stage under high temperature, HT2 knockout mutant lines (HT2-KO1, 1 bp deletion, premature termination; HT2-KO2, 5 bp deletion, premature termination) and HT2 overexpression transgenic lines (HT2-OE1, HT2-OE2) were constructed in the japonica rice Zhonghua 11 (ZH11) background. These lines were subjected to high temperature treatment at the heading stage indoors, and the seed set rate under high temperature was measured. The treatment process is as follows:

[0024] 1) Select rice seedlings with the same growth and at the eighth stage of panicle development in the field and transplant them into plastic buckets;

[0025] 2) Cultivate in the field for 3 days and mark the ears that have flowered before treatment and those to be treated;

[0026] 3) High temperature treatment at the heading stage was performed in an artificial greenhouse (42-32°C temperature gradient, 60% humidity). The treatment duration was selected according to the different background materials (2 days for japonica rice and about 3 days for indica rice).

[0027] 4) The seeds are then transplanted to the field, and after the grains mature, the marked ears are classified and counted.

[0028] The results of high temperature treatment of HT2 knockout mutants showed that the fruit set rate of wild type ZH11 and HT2 knockout lines decreased significantly after high temperature treatment, of which ZH11 decreased by 40.14%, and the fruit set rate of the two HT2-KO lines decreased by 68.68% and 69.56%, respectively. The decrease in KO was greater than that in ZH11. Figure 2 A, B). The results of high temperature treatment of HT2 overexpression lines at the heading stage showed that the seed setting rates of ZH11 and HT2 overexpression lines decreased significantly after high temperature treatment. The seed setting rate of wild type ZH11 decreased by 40.23%, while the seed setting rates of overexpression lines decreased by 26.06% and 25.15%, respectively. The decrease in seed setting rate of ZH11 was greater than that of overexpression ( Figure 2 C, D). This indicates that HT2 can increase the seed setting rate of rice at the panicle stage under high temperature and improve rice yield.

[0029] Example 2HT2 9311 Has breeding value

[0030] 1. HT2 haplotype analysis

[0031] In order to identify the superior natural alleles of HT2, genotyping of HT2 was performed using 405 sequenced resource materials in our laboratory. The results showed that HT2 was divided into five major haplotypes, among which Hap2, Hap4, and Hap5 were mainly distributed in indica rice, while Hap1 ( Figure 3 A). Analysis revealed that the indica rice 93-11 belongs to Hap2, while the japonica rice Nip belongs to Hap1, representing two typical indica and japonica rice types, respectively. Sequence alignment revealed that the HT2 promoter has multiple SNPs and indels between 93-11 and Nip, with significant differences in the promoter region. A large 29bp deletion exists in the Nip promoter ( Figure 3 B).

[0032] 2. 93-11 and SSSL-HT2 Nip High temperature treatment

[0033] For 93-11 and SSSL-HT2 Nip High temperature treatment was carried out at the ear stage. The results of high temperature treatment at the ear stage showed that after high temperature treatment, 93-11 and SSSL-HT2 Nip The fruit setting rate decreased significantly, while SSSL-HT2Nip The decrease in fruit setting rate was greater than that of 93-11( Figure 4 A, B).

[0034] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. HT2 Use of a gene or its encoded protein in increasing rice yield under high temperature stress, the HT2 The nucleic acid sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that described HT2 Genetically improve the seed setting rate of rice at the panicle stage under high temperature stress.

3. A method for cultivating high-yield rice under high temperature, characterized in that: Including introducing the nucleic acid sequence shown in SEQ ID NO.1 into the genome of rice HT2 gene and overexpressed it in rice.

4. The method according to claim 3, characterized in that The method of introduction is selected from a genetic transformation method or a genome editing method.

5. HT2 Use of the gene in cultivating high-yield rice varieties under high temperature stress, the HT2 The nucleic acid sequence of the gene is shown in SEQ ID NO.1.

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