Gene ATT1 and application thereof in regulation and control of rice yield and alkali-heat tolerance

Abstract

The invention relates to a gene ATT1 and application of the gene ATT1 in regulation and control of rice yield and alkali-heat tolerance. ATT1 encodes GA20 oxidase and participates in regulating and controlling the alkali-heat resistance of the rice, and NIL-ATT1LTP shows that the single-plant yield and the plot yield of the rice are remarkably increased in a normal field environment or a high-temperature environment. In an alkaline field environment, the NIL-ATT1BART shows that the single-plant yield and the plot yield of the rice are remarkably increased. According to the application, researches find that natural variation sites of ATT1LTP and ATT1BART endow rice with alkali-heat tolerance and yield phenotypes; a promoter region or a coding region of an ATT1 gene is modified by utilizing a CRISPR / Cas9 gene editing technology, or ATT1LTP or ATT1BART gene loci are introduced into a current main cultivated variety in a hybridization manner, so that the alkali resistance and heat resistance of a crop variety are improved, and the yield of the crop variety is increased; therefore, new varieties of high-yield and stress-resistant crops (including but not limited to rice, wheat, corn, sorghum, millet, soybean and the like) are cultivated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene ATT1 and application thereof in regulating rice yield and alkali-heat tolerance. Background Art

[0002] With the rapid increase in the global population, the limited amount of arable land, and climate and environmental changes, improving crop yields under abiotic stress is a challenge that cannot be ignored for modern agriculture. Therefore, there is an urgent need to improve crop yields and improve stress tolerance-related traits to meet future needs. According to statistics from the Food and Agriculture Organization of the United Nations (FAO) in 2021, more than 1.1 billion hectares of arable land in the world are affected by salinization, of which 40% of the soil is alkaline. In addition, it is estimated that the global population is growing rapidly and will reach 11 billion by 2100. The rapid growth of the population and human activities are exacerbating the impact of soil salinization. In addition to alkaline stress, heat stress is also one of the main abiotic stresses threatening crop growth. Since 1900, the global average temperature has risen by 1.2°C and will continue to rise in the next decade, leading to a decline in crop yields and a devastating impact on agricultural production. However, global warming has exacerbated soil salinization, further triggering global food security issues. Therefore, it is urgent to develop new green rice varieties (GRVs) that combine stress resistance and high yield to meet the needs of future population growth and climate change. Rice alkali and heat stress tolerance are quantitative traits controlled by multiple gene loci. Positioning and cloning these potential excellent gene loci and clarifying their inherent molecular mechanisms will help researchers and breeders understand the molecular mechanism of rice alkali-heat tolerance, which is of great significance and value for molecular design and breeding of new alkali-heat resistant crop varieties and new materials. Summary of the invention

[0003] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and to provide a gene ATT1 and its application in regulating rice yield and alkali-heat tolerance.

[0004] The purpose of the present invention can be achieved by the following technical solutions:

[0005] The present invention first provides an ATT1 LTP Gene, the sequence is shown in SEQ ID NO.1. The details are as follows (the yellow highlight and underline represent the start codon and the stop codon (including 5'UTR, 3'UTR)):

[0006]

[0007]

[0008] The present invention further provides ATT1 LTP The promoter, whose sequence is shown in SEQ ID NO.2, is as follows:

[0009]

[0010] The present invention further provides ATT1 LTP The coding region, whose sequence is shown in SEQ ID NO.3, is as follows:

[0011]

[0012] The present invention further provides ATT1 LTP The protein, whose sequence is shown in SEQ ID NO.4, is as follows:

[0013] VAEHPTPPQPHQPPPMDSTAGSGIAAPAAAAVCDLRMEPKIPEPFVWPNGDARPASAAELDMPVVDVGVLRDGDAEGLRRAAAQVAAACATHGFFQVSGHGVDAALARAALDGASDFFRLPLAEKRRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDRAAAPVVADYFSSTLGPDFAPMGRVYQKYCEE MKELSLTIMELLELSLGVERGYYREFFADSSSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGEWRPVSPVPGAMVINIGDTFMALSNGRYKSCLHRAVVNQRRERRSLAFFLCPREDRVVRPPPSAATPRHYPDFTWADLMRFTQRHYRADTRTLDAFTRWLAPPAADAAATAQVEAAS

[0014] The present invention further provides ATT1 BART The gene, whose sequence is shown in SEQ ID NO.5, is as follows (the red bold mark indicates the natural variation site, and the yellow highlight and underline represent the start codon and the stop codon):

[0015]

[0016]

[0017] The present invention further provides ATT1 BART The promoter gene, whose sequence is shown in SEQ ID NO.6, is as follows (the red bold mark indicates the natural variation site, and the yellow highlight and underline represent the start codon and the stop codon):

[0018]

[0019]

[0020] The present invention further provides ATT1 BART The coding region, whose sequence is shown in SEQ ID NO.7, is as follows:

[0021]

[0022] The present invention further provides ATT1 BART Protein whose sequence is as SEQ ID As shown in NO.8, the details are as follows: MVAEHPTPPQPHQPPPMDSTAGSGIAAPAAAAVCDLRMEPKIPEPFVWPNGDARPASAAAELDMPVVDVGVLRDGDAEGLRRAAAQVAAACATHGFFQVSGHGVDAALARAALDGASDFFRLPLAEKRRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDRAAAPVVADYFSSTLGPDFAPMGRVY QKYCEEMKELSLTIMELLELSLGVERGYYREFFADSSSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGEWRPVSPVPGAMVINIGDTFMALSNGRYKSCLHRAVVNQRRERRSLAFFLCPREDRVVRPPPSAATPRHYPDFTWADLMRFTQRHYRADTRTLDAFTRWLAPPAADAAATAQVEAAS.

[0023] The present invention further provides application of gene ATT1 in regulating rice yield and alkali-heat tolerance.

[0024] Specifically, ATT1 encodes GA20 oxidase, which is involved in regulating rice's alkali-heat resistance. Under normal field conditions or high temperature environments, NIL-ATT1 LTP The rice yield per plant and the yield of the plot were significantly increased. BART The results showed that the yield per plant and the yield in the plot of rice increased significantly. These results indicate that rice yield and alkali-heat stress tolerance can be synergistically regulated by ATT1.

[0025] Furthermore, ATT1 LTP or ATT1 BART Genes are introduced into crops through hybridization or biotechnology to improve the alkali resistance, heat resistance and yield of crop varieties, and to cultivate high-yield and stress-resistant crops.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] This application study discovered the full-length gene sequence of rice alkali-heat stress tolerance and yield control gene ATT1, including coding region and promoter region sequences, as well as the molecular mechanism of ATT1 synergistically regulating rice alkali-heat stress tolerance and yield;

[0028] This study found that ATT1 LTP and ATT1 BART Natural variation loci confer alkali-heat tolerance and yield phenotypes in rice;

[0029] This study found that ATT1 encodes GA20 oxidase 2, which regulates rice alkali-heat stress tolerance and yield by controlling the synthesis of gibberellins;

[0030] Based on this application plan, ATT1 from Longtepu LTP Under normal field planting conditions, the yield increased by about 30%, while in artificially simulated alkaline soil environments, ATT1 BART The gene locus showed less yield loss, while ATT1 LTP The gene locus showed less yield loss. Therefore, the ATT1 gene locus is an important gene resource for breeding high-yield, high-alkali and high-heat resistant rice. CRISPR / Cas9 gene editing technology can be used to modify the promoter region or coding region of the ATT1 gene, or to modify the ATT1 LTP or ATT1 BART Gene loci are introduced into the current main cultivated varieties through hybridization to improve the alkali resistance, heat resistance and yield of crop varieties, and then cultivate new varieties of high-yield and stress-resistant crops (including but not limited to rice, wheat, corn, sorghum, millet, soybeans, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Fine localization of ATT1

[0032] Figure 2 Identification of heat- and alkali-tolerance phenotypes and survival rate of ATT1 near-isogenic lines

[0033] Figure 3 ATT1 promoter sequence analysis

[0034] Figure 4 ATT1 exon variation analysis

[0035] Figure 5 Identification of alkaline heat phenotypes of ATT1 knockout and complement transgenic materials

[0036] Figure 6 NIL-ATT1 LTP and NIL-ATT1 BART Agronomic traits under normal conditions

[0037] Figure 7 NIL-ATT1 LTP and NIL-ATT1 BARTPhenotypic characteristics of alkali treatment in the field

[0038] Figure 8 High temperature stress in the near-isogenic line NIL-ATT1 LTP and NIL-ATT1 BART Impact on rice yield

[0039] Fig. 9 Conservation analysis of ATT1. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Example 1

[0042] A set of chromosome segment substitution lines (CSSLs) was constructed using African wild rice (O. barthii) as the donor parent and Asian cultivated rice (Oryza Sativa L. ssp. Indica) variety Longtepu (LTP) as the recipient parent. It was used to locate and clone QTLs (quantitative trait loci) (ALKALI-THERMAL TOLERANCE 1) ATT1, which regulates rice alkali stress and heat stress. ATT1 encodes GA20 oxidase and is involved in regulating rice alkali-heat resistance. Under normal field conditions or high temperature conditions, NIL-ATT1 LTP (i.e., rice materials containing the Longtepu ATT1 gene Longtepu as the background material) showed a significant increase in rice yield per plant and plot yield. BART The rice material containing the African wild rice ATT1 gene Long Tepu as the background material showed a significant increase in rice yield per plant and plot yield. These results show that rice yield and alkali-heat stress tolerance can be synergistically regulated by ATT1.

[0043] The linkage analysis combining molecular markers and phenotypes initially located ATT1 between the M1-1 and M1-9 molecular markers (M1-1 and M1-9 distinguished the fragments from Long Te Pu and the fragments from African wild rice). After the preliminary fine positioning and the alkali and heat treatment phenotype identification of the near-isogenic lines, ATT1 was finally located within the range of 174.75kb on chromosome 1 (between the M1-6 and M1-8 molecular markers), as shown in Figure 1. Figure 1 As shown,

[0044] Figure 1(A) Fine mapping of ATT1. The numbers between each molecular marker indicate the number of exchanges obtained, and the n value indicates the individual plants used in the F2 population (a commonly used mapping genetic population, which is formed by hybridizing the replacement line with the recurrent parent to produce the F1 generation, and then self-pollinating the F1 to form the F2 population). The white part in the chromosome pattern diagram indicates the fragment from the recurrent parent Long Tepu, and the black part indicates the fragment from the donor parent wild rice O.barthii. C1 indicates that the fragment is completely derived from the Long Tepu strain, and C2 indicates that the fragment is completely derived from the O.barthii strain. After self-pollination of different F2 populations, molecular markers are used to identify and select the target wild rice fragments in the Long Tepu genetic background (these fragments can be distinguished by molecular markers), and R1-R4 indicates that the fragment is derived from the exchange homozygous strain of O.barthii.

[0045] (B) Phenotypes of recombinant exchange individuals of different target Barthii wild rice fragments under high temperature (42.5°C, RH>90%, 20-30h) and alkali (65mM sodium bicarbonate, 7-14 days) treatments. Scale bar 3cm.

[0046] (C) and (D) show the survival rates of C1-C2 and R1-R4 lines in alkali treatment and heat treatment in ATT1 fine mapping, respectively (n = 8 biological replicates, each replicate consisting of 24 rice seedlings). The data are mean ± standard deviation, and the significant differences were determined by Statix8.0 analysis and post hoc pairwise multiple comparison test (P < 0.05). The data are Mean ± sd; the significant differences were determined by Statix8.0 analysis and post hoc pairwise multiple comparison test (LSD < 0.05).

[0047] based on Figure 1 , the following conclusions can be drawn: 1. A 174.45kb fragment was found from African wild rice and introduced into Longtepu. After the introduction of this fragment, the alkaline tolerance of Longtepu decreased significantly, indicating that the corresponding fragment of cultivated rice Longtepu can enhance the tolerance of rice to alkaline stress.

[0048] Among them, the molecular marker primers used for positioning are as follows:

[0049] The 5' oligonucleotide primer sequence of M1-1 is:

[0050] 5'-ACTAACAAAACAAGCTCAAGG-3'

[0051] The 3' primer sequence is:

[0052] 5'-ACCATTTTCTAGAGTTCAAAGT-3'

[0053] The 5' oligonucleotide primer sequence of M1-2 is:

[0054] 5'-GACTACTTGAACATAAAAGCCA-3'

[0055] The 3' primer sequence is:

[0056] 5'-AAAATCGTTGGCAGTGTAGG-3'

[0057] The 5' oligonucleotide primer sequence of M1-3 is:

[0058] 5'-TGCAAGATTCCTACTCTGAG-3'

[0059] The 3' primer sequence is:

[0060] 5'-GACCAATCAAACTCAACTAGC-3'

[0061] The 5' oligonucleotide primer sequence of M1-4 is:

[0062] 5'-TGATAATAGGTGGCTGAATGG-3'

[0063] The 3' primer sequence is:

[0064] 5'-TGACCAAAAGGTGATAGAGAG-3'

[0065] The 5' oligonucleotide primer sequence of M1-5 is:

[0066] 5'-GCAGCTTGGGTCCTAAAA-3'

[0067] The 3' primer sequence is:

[0068] 5'-TCCACACCTATCATAATGGC-3'

[0069] The 5' oligonucleotide primer sequence of M1-6 is:

[0070] 5'-GGCAGCAAGTTCTACGATA-3'

[0071] The 3' primer sequence is:

[0072] 5'-GTACTTTACTCCTAACTTATGT-3'

[0073] The 5' oligonucleotide primer sequence of M1-7 is:

[0074] 5'-TGATGATCATGGTCGCAACT-3'

[0075] The 3' primer sequence is:

[0076] 5'-CAGCGGAGATGAAAACCCTA-3'

[0077] The 5' oligonucleotide primer sequence of M1-8 is:

[0078] 5'-GTTTGGTGGCATAGTTGGGG-3'

[0079] The 3' primer sequence is:

[0080] 5'-CGAGAAGAAGGTTTGGTGCA-3'

[0081] The 5' oligonucleotide primer sequence of M1-9 is:

[0082] 5'-CCATGAGGGTAGGAGACAT-3'

[0083] The 3' primer sequence is:

[0084] 5'-ATACCTCCACTAACCTTCCA-3'

[0085] In this example, by investigating the alkali and heat resistance phenotypes, combined with genotypes for linkage analysis and molecular marker-assisted selection, ATT1 was precisely located in 10,870 plants, and the candidate segment of ATT1 was narrowed down to the 174.75 kb region of chromosome 1, and near-isogenic lines of ATT1 were constructed (through large-scale PCR identification of rice materials, combined with PCR-based molecular marker genotyping identification, rice lines with 174.45 KB African wild rice fragments introduced into the Long Te Pu background were found). The near-isogenic lines were investigated for alkali-heat resistance phenotypes.

[0086] 2. Identification of rice alkali and heat stress tolerance phenotypes

[0087] Identification of alkaline stress phenotypes: In rice seedlings (near isogenic line NIL-ATT1 LTP , NIL-ATT1 BART and gene-edited att1 (NIL-ATT1 BART background) and transgenic gATT1 BART (NIL-ATT1 LTPAfter 12 days of cultivation (when the seedlings have one leaf and one heart), add sodium bicarbonate to 1× Yoshida nutrient solution to ensure that the final concentration of sodium bicarbonate in the nutrient solution is 65 mM, and treat for 7-14 days (the rice seedlings are grown in a nutrient solution containing 65 mM sodium bicarbonate for 14 days, until the leaves begin to turn yellow, curl, and wilt, then they are replaced with a nutrient solution without sodium bicarbonate. Since the growth of rice seedlings is affected by many factors, including light, temperature, and the quality of germinating seeds, the duration of each alkali treatment will be adjusted accordingly according to the situation), replace the alkali treatment solution every four days, until the rice leaves begin to turn yellow, curl, and wilt, stop the alkali treatment, and recover for 7-14 days (due to the different resistances of different materials, the treatment time is determined according to the specific situation), take pictures, and calculate the survival rate.

[0088] Identification of heat stress phenotypes: In rice seedlings (near isogenic line NIL-ATT1 LTP , NIL-ATT1 BART and gene-edited att1 (NIL-ATT1 BART background) and transgenic gATT1 BART (NIL-ATT1 LTP After culturing for 12 days (when the seedlings have one leaf and one heart), the rice seedlings were placed in a heat treatment box at 42°C and 90% humidity for 20-30 hours (the rice seedlings were placed in a high temperature environment (temperature 42°C, humidity 90%, due to the different heat resistance of different materials, the treatment time was adjusted according to the specific conditions of the treatment), recovered for 7 days, photographed and the survival rate was calculated. During each stress treatment, ensure

[0089] One biological group was sown with 24 seeds and three biological replicates were set.

[0090] Figure 2 Middle, (AC) near isogenic line NIL-ATT1 LTP and NIL-ATT1 BART Phenotype (A) and survival rate (B and C) under alkaline stress and high temperature stress. Alkaline treatment, 65 mM sodium bicarbonate treatment for 14 days; high temperature treatment, 42 ° C, RH>90% treatment for 28 hours, scale bar, 10 cm. Data are mean ± SD. n = 3 replicates, each replicate consisting of 24 rice seedlings, using two-tailed t-test for significant difference test, ***P < 0.001.

[0091] The results showed that NIL-ATT1 from indica rice LTP The survival rate under alkali-heat stress was significantly higher than that of NIL-ATT1 from African wild rice BART This indicates that the candidate interval identified does contain the gene ATT1 that is both alkali-resistant and heat-resistant. LTP(ATT1 gene from Long Tepu) has stronger resistance to stress.

[0092] 3. CRISPR / Cas9 gene editing and genetic complementation

[0093] To further validate the candidate gene, NIL-ATT1 was constructed LTP Background contains NIL-ATT1 BART The ATT1 genomic DNA was used to construct the transgenic complementation strain, and the NIL-ATT1 was constructed using CRISPR / Cas9 technology. BART The att1 mutant was genetically transformed by Agrobacterium tumefaciens EHA105-mediated rice mature embryo transformation method, and the transgenic positive lines were screened and planted in the field. 2 Generation examination phenotype.

[0094] The specific method is as follows:

[0095] 1. Constructed NIL-ATT1 LTP Background contains NIL-ATT1 BART Transgenic complementation lines of ATT1 genomic DNA:

[0096] Using high-fidelity DNA polymerase (KOD-one Taq enzyme), amplify the DNA fragment from NIL-ATT1 BART The gDNA (gene fragment) of ATT1 (i.e. from African wild rice) is then recovered, and then the amplified fragment is amplified with the adapter primer, the fragment is recovered by gel, and the tailed fragment is recovered. The pCAMBIA1300 empty vector is double-digested and the main electrophoresis band is recovered; the pCAMBIA1300 enzyme-digested empty vector is ligated under the action of homologous recombinase, and the ligation product is transformed into T1 competent cells, coated with Kan-resistant plates, placed in a 37°C incubator, and then the clone is selected and shaken in Kan-resistant LB liquid to extract the plasmid. The final recombinant plasmid can be sequenced and identified using public primers. The correctly sequenced recombinant plasmid is transformed into Agrobacterium competent cells and used to transfect plants to obtain transgenic rice strains.

[0097] 2. Construction of NIL-ATT1 using CRISPR / Cas9 technology BART Background of att1 mutant

[0098] Construction of knockout vector CRISPR / Cas9-ATT1:

[0099] Knockout target selection and adapter primer design: ATT1 knockout target and adapter primer design were designed using the target gene target primer design online software crispr-p2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR). For adapter primer design, select a conventional target 19 bases upstream of the NGG PAM sequence, or select an unconventional target 20 bases upstream. The steps for constructing the CRISPR / Cas9-ATT1 vector are as follows:

[0100] (1) Digest the pRGEB31 vector with Bsa I: pRGEB31, 2 μg; 10x NEB Buffer 4, 2 μl; 10x BSA, 2 μl; Bsa I (NEB) 1 μl; Add H2O to 20 μl; Purify the digested vector using a PCR purification kit.

[0101] (2) Prepare DNA oligonucleotide duplex: Forward oligo (100uM), 1μl; Reverse oligo, (100uM), 1μl; 10x T4 DNA ligase Buffer, 1μl; T4 PNK (NEB), 0.5μl; H2O, 6.5μl; PCR program: 37℃, 60min; then 95℃ 10min. 1:200 diluted oligonucleotide duplex.

[0102] (3) Ligation reaction: 1μl (~50ng) of pRGEB31 vector was digested with Bsa I. 1μl of oligonucleotide double-stranded DNA diluted 1:200; 10x T4 DNA ligase Buffer, 0.5μl T4 ligase (NEB); 1μl; Add H2O to 5μl. Incubate at room temperature (25°C) for 2-4 hours. Transform the ligation product into T1 competent cells, apply Kan-resistant plates, and place them in a 37°C incubator. Then, select clones and shake them in Kan-resistant LB liquid to extract plasmids. The final recombinant plasmid can be sequenced and identified using public primers. The correctly sequenced recombinant plasmid is transformed into Agrobacterium competent cells and used to transfect plants to obtain transgenic rice lines.

[0103] 3. Genetic transformation and positive detection

[0104] The above plasmid was transformed into EHA105 Agrobacterium, and the constructed pCAMBIA1300-NIL-ATT1 was transformed into rice transgenic by Agrobacterium-mediated transformation. BART The ATT1 genomic fragment vector was transferred into NIL-ATT1 LTP strains, and CRISPR / Cas9-ATT1 vectors were transformed into NIL-ATT1BART The corresponding transgenic seedlings were obtained through callus induction, infection, hygromycin resistance screening, differentiation induction and rooting.

[0105] 4. Positive detection of transgenic seedlings:

[0106] Amplification primers were designed based on a special gene sequence of hygromycin protein on the vector. After PCR amplification and agarose gel electrophoresis detection, if there was a band, it indicated that the plasmid vector had been inserted into the rice genome, which was a positive material and could be retained; if there was no band, it indicated that the transformation was unsuccessful and it was discarded. In addition to PCR detection, the mutation of the knockout target gene was also detected by first-generation sequencing. A pair of PCR primers were designed at a position near 500bp upstream and downstream of the knockout target (at a position near 500bp upstream and downstream of the knockout target), and first-generation sequencing was performed after PCR reaction amplification, and compared with the target gene sequence to determine the mutation type of the ATT1 gene. After field cultivation, T1 generation seeds were obtained. After field planting, T1 generation seeds were further tested to ensure that the mutant strain was a homozygous material, and self-pollination was retained as seeds, that is, T2 generation. T2 generation seeds were phenotypically identified.

[0107] Among them, the primers for complement vector construction are:

[0108] The 5' oligonucleotide primer sequence is:

[0109] 5'-ACGAATTCGAGCTCGGTACC TATATTTACAAGTTAACGACGT-3'

[0110] The 3' primer sequence is:

[0111] 5'-GCAGGTCGACTCTAGAGGATCC TGAGCCGAATTCGAATTCAAT-3'

[0112] Primers for CRISPR / Cas9 knockout vector construction:

[0113] The 5' oligonucleotide primer sequence of the target is:

[0114] 5'-GGTGTACCAGAAGTACTGCG-3'

[0115] The 3' primer sequence is:

[0116] 5'-CGCAGTACTTCTGGTACACC-3'

[0117] The rice gene prediction website (http: / / rice.plantbiology.msu.edu / ) was used to predict candidate genes in the ATT1 candidate interval. There are a total of 21 candidate genes in the ATT1 candidate interval, of which 15 have functional annotation genes, three of which are retrotransposons and transposons, as shown in Table 1. In the candidate interval of ATT1, there is a GA20 oxidase 2 (gibberellin 20oxidase 2; LOC_Os01g66100). This enzyme plays a key role in the synthesis of gibberellin. Many studies have shown that the anabolism and signal transduction of gibberellin are closely related to the abiotic stress response of rice. Sequence alignment identified that compared with NIL-ATT1 LTP , LOC_Os01g66100 in NIL-ATT1 BART There are a large number of mutations in the promoter and four base substitutions in the gene coding region ( Figure 3 , Figure 4 ).

[0118] Figure 3 , Promoter sequence analysis of ATT1 in near-isogenic lines. Red boxes indicate regulatory element variations caused by genetic polymorphisms. Promoter alignment was performed by Clustal Omega software, and regulatory elements were predicted by the PlantCARE database. Figure 4 Analysis of the coding region of ATT1 in the near-isogenic line. The green part represents the coding region of ATT1.

[0119] These results indicate that the changes in the ATT1 promoter are the main reason for the differences in the alkali-resistance and heat-resistance phenotypes of near-isogenic lines. Therefore, LOC_Os01g66100 may be a candidate gene for ATT1.

[0120] Table 1 ATT1 candidate interval genes

[0121]

[0122] To further confirm that LOC_Os01g66100 is a candidate gene for ATT1, NIL-ATT1 was constructed. LTP Background contains NIL-ATT1 BART The transgenic complementation line of ATT1 genomic DNA was obtained by CRISPR / Cas9 technology. BART The mutant strains of att1 in the background (see the method above for details). Among them, LOC_Os01g66100 has two types of mutations, including 25bp deletion and 2bp deletion. The complementary strains and mutant strains were identified for alkali-heat resistance ( Figure 5 ).

[0123] Figure 5 Medium, (A)NIL-ATT1 LTP and NIL-ATT1 LTP Background contains NIL-ATT1 BART Relative expression of ATT1 in transgenic complementation lines containing ATT1 genomic DNA; (B) att1(NIL-ATT1 BART Background) Gene mutation site and type; (CE) NIL-ATT1 LTP Background contains NIL-ATT1 BART Phenotype (C) and survival rate (D, E) of transgenic complementation lines expressing ATT1 genomic DNA under alkaline stress and high temperature stress; (FG) att1 (NIL-ATT1 BART Phenotype (F) and survival rate (G, H) of mutants under alkaline stress and high temperature stress. Alkaline treatment, 65mM sodium bicarbonate treatment for 14 days; high temperature treatment, 42℃, RH>90% treatment for 28 hours, scale bar, 10cm. Data are mean ± standard deviation. (n=3 replicates), two-tailed t-test was used for significant difference test, **p<0.01, ***p<0.001.

[0124] The results showed that att1(NIL-ATT1 BART ) showed significantly higher tolerance to alkaline stress and heat stress than NIL-ATT1 BART . NIL-ATT1 LTP Background contains NIL-ATT1 BART The tolerance of transgenic lines complemented with ATT1 genomic DNA was significantly lower than that of NIL-ATT1 LTP ( Figure 5 ). This indicates that the LOC_Os01g66100 gene is a candidate gene for ATT1, which is involved in regulating the alkali-heat stress response of rice.

[0125] 4. Field investigation of agronomic traits of near-isogenic line materials

[0126] Planting the near-isogenic line NIL-ATT1 in a farm in Songjiang District, Shanghai LTP and NIL-ATT1 BART Three replicates were set for each material, with about 300 plants in each replicate. The plant and panicle types of the individual plants were photographed at maturity. After all the rice grains turned yellow, 20 individual plants were collected from the near-isogenic lines to examine the individual plant yield, plant height, number of tillers, panicle length, number of panicles, fruit setting rate, 1000-grain weight, grain length, and grain width. 180 individual plants were selected in the field to form a yield measurement plot, and the plot area and total plot yield were examined to calculate the yield per unit area.

[0127] 5. Field alkali treatment experiment

[0128] The experiment was conducted in an alkaline soil cement pond in Songjiang District, Shanghai. The soil in the alkaline pond was the soil that had been subjected to rice alkaline stress (sodium bicarbonate was applied) the previous year. Before transplanting, the soil was plowed and then irrigated to a water layer above 5 cm. Rice seedlings (near gene line NIL-ATT1 LTP and NIL-ATT1 BART ) Transplantation. The fertilizer management mode was consistent with that of normal rice fields. Alkaline soil sampling began at the tillering stage of rice. The electrical conductivity (EC), pH value and redox potential of alkaline soil were measured. Sodium bicarbonate was added when the pH value was lower than 9.0, and the soil pH value was always kept above 9.0. Alkaline stress treatment was continued until the rice matured.

[0129] 6 Field heat treatment experiment

[0130] The rice planted in Shanghai Songjiang Farm has grown to the heading stage and is about to enter the grain filling stage. It is covered with film and the steel frame structure is spliced ​​to form a greenhouse. The greenhouse is closed every morning, and the temperature inside the greenhouse will rise. After the temperature reaches above 45 degrees Celsius, the two sides of the greenhouse are opened and the temperature drops. The treatment continues during the grain filling stage until the rice matures.

[0131] 7. Investigation of agronomic characteristics of rice after alkaline stress and heat stress

[0132] After the rice matured after alkali treatment in the field and greenhouse, alkali stress in the field and heat stress, individual rice plants were collected for agronomic character examination, including single plant yield, plant height, number of tillers, panicle length, number of panicles, fruit setting rate, 1000-grain weight, grain length and grain width. An appropriate number of plants were selected in the field to form yield measurement plots, and the plot area and plot yield were examined to further calculate the rice yield per unit area.

[0133] 8. ATT1 Conservation Analysis

[0134] To determine the conservation of ATT1, the protein sequence of ATT1 was used for blast analysis (https: / / blast.ncbi.nlm.nih.gov / ) and the corresponding homologous gene sequence was obtained. Then, MEGA10.0 was used for sequence alignment analysis and a phylogenetic tree was constructed. The phylogenetic tree was constructed using the NJ neighbor-joining method with 1000 bootstrap values.

[0135] Under normal field conditions, NIL-ATT1 LTP and NIL-ATT1 BART , NIL-ATT1 NIP and NIL-ATT1 9311 Field yield and yield traits were investigated. BART, NIL-ATT1 LTP It showed a 40.91% increase in single plant yield and a 29.89% increase in plot yield. Figure 6 As shown, among them, (A) near isogenic line NIL-ATT1 LTP and NIL-ATT1 BART Plant type (scale bar, 1 m) and ear type (scale bar, 10 cm) at maturity. (BC) NIL-ATT1 under normal field conditions LTP and NIL-ATT1 BAR Statistical analysis of single plant yield (B) and plot yield (C), n = 20 plants or n = 3 plots (180 plants in each plot). (DK) NIL-ATT1 under normal field conditions LTP and NIL-ATT1 BART Statistical analysis of plant height (D), tiller number (E), ear length (F), number of grains per ear (G), fruit set rate (H), 1000-grain weight (I), grain length (J), and grain width (K). n = 20 plants. Values ​​in BK represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences by Student's t-tests; ns, no significant differences.

[0136] Further investigation of agronomic traits revealed that LTP In contrast, although NIL-ATT1 BART The main ear length increased, but the number of grains per ear did not change significantly. BART In comparison, NIL-ATT1 LTP The seed setting rate and grain length of the strains decreased slightly, the thousand-grain weight and grain width were not affected, but the tiller number increased significantly, resulting in a significant increase in yield ( Figure 6 ). In summary, NIL-ATT1 LTP The ATT1 allele in has the ability to increase rice yield.

[0137] To investigate the effect of ATT1 on rice yield under alkaline field conditions, a near-isogenic line of ATT1 (NIL-ATT1 LTP and NIL-ATT1 BART ) were planted in alkaline soils, and yield and other related agronomic traits were investigated at maturity. We transplanted near-isogenic lines at the seedling stage on soils where we have conducted rice alkali tolerance experiments for many years. In the early stage, we covered the soil with water to transfer the alkali content in the soil to the lower layer to ensure that the transplanted rice seedlings can survive and produce tillers. After the tillering period, the water content was reduced to restore the alkaline stress (pH8.5-9). During this period, normal fertilizer management was carried out. Surprisingly, under alkaline stress, NIL-ATT1BART NIL-ATT1 LTP The yield per plant increased by 19.3%, NIL-ATT1 BART NIL-ATT1 LTP The yield of the plot increased by 8.17% ( Figure 7 AC); Effect of alkaline stress on NIL-ATT1 BART The yield loss caused by NIL-ATT1 was 4.23%. LTP The yield loss caused by the plot was lower (32.29%) Figure 7 D) Further investigation of agronomic traits revealed that LTP In comparison, NIL-ATT1 BART The main ear length and 1000-grain weight increased significantly, the number of grains per ear and the fruiting rate increased slightly, the plant height and grain length also increased significantly, the tiller number decreased, and there was no significant difference in grain width ( Figure 7 These data suggest that under alkaline stress, NIL-ATT1 BART The ATT1 allele in the genotype can reduce the yield loss caused by alkaline stress to a certain extent ( Figure 7 ).

[0138] NIL-ATT1 LTP and NIL-ATT1 BART High temperature treatment was carried out during the field filling period, and yield and related agronomic traits were investigated during the maturity period ( Figure 8 ). The results showed that under high temperature stress, NIL-ATT1 LTP NIL-ATT1 BART The yield per plant increased by 49.72%, NIL-ATT1 LTP NIL-ATT1 BART The yield of the plot increased by 84.76% ( Figure 8 AC); Effect of high temperature stress on NIL-ATT1 LTP The yield loss caused by NIL-ATT1 was 18.49%. BART The yield loss caused by the plot (42.70%) was lower ( Figure 8 D) Further investigation of agronomic traits revealed that BART In comparison, NIL-ATT1 LTP The tillering number, seed setting rate and 1000-grain weight increased significantly, the plant height and grain length decreased significantly, and there was no significant difference in grain width and number of grains per ear ( Figure 8 These data suggest that under heat stress, NIL-ATT1 LTPThe ATT1 allele in the genotype can reduce the yield loss caused by heat stress to a certain extent ( Figure 8 ).

[0139] In order to determine whether ATT1 can be applied to other crops to regulate yield and alkali-heat tolerance, the conservation of ATT1 in other species was investigated. The protein sequences of ATT1 in common monocotyledonous and dicotyledonous plants were compared, and a phylogenetic tree was constructed ( Fig. 9 ). The sequence alignment results show that ATT1 is significantly conserved in a variety of crops, including but not limited to wheat (homology 90%), corn (homology 91%), sorghum (homology 89%) ( Fig. 9 This indicates that ATT1 is highly conserved in a variety of important crops. Through gene introgression or gene editing technology, ATT1 can be widely used in the alkali and heat resistance improvement breeding of other crops, and has a high breeding application value ( Fig. 9 ).

[0140] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. ATT1 LTP A gene characterized by The sequence is shown as SEQ ID NO.

1.

2. ATT1 LTP A promoter, characterized in that Its sequence is shown in SEQ ID NO.

2.

3. ATT1 LTP The coding region is characterized in that Its sequence is shown in SEQ ID NO.

3.

4. ATT1 as claimed in claim 1 LTP Gene encoding ATT1 LTP A protein, characterized in that Its sequence is shown in SEQ ID NO.

4.

5. ATT1 BART A gene characterized by The sequence is shown as SEQ ID NO.

5.

6. ATT1 BART A promoter, characterized in that Its sequence is shown in SEQ ID NO.

6.

7. ATT1 BART The coding region is characterized in that Its sequence is shown in SEQ ID NO.

7.

8. ATT1 as claimed in claim 5 BART Gene encoding ATT1 BART A protein, characterized in that Its sequence is shown in SEQ ID NO.

8.

9. Application of gene ATT1 in regulating rice yield and alkali-heat tolerance, characterized in that: Gene ATT1 is selected from ATT1 LTP ATT1 BART Gene, ATT1 LTP The gene sequence is shown in SEQ ID NO.1, ATT1 BART The gene sequence is shown as SEQ ID NO.

5.

10. The use according to claim 9, characterized in that: Under normal field conditions or high temperature conditions, NIL-ATT1 LTP Used to increase rice yield per plant and plot; Under alkaline field conditions, NIL-ATT1 BART Used to increase the yield of rice per plant and the yield of each plot.

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