Application of HTT1 gene in regulation and control of high-temperature tolerance of rice

Abstract

The invention provides an HTT1 gene and application of protein coded by the HTT1 gene in regulation and control of high-temperature tolerance of rice. The amino acid sequence of the protein coded by the HTT1 gene is shown as SEQ ID No.2, and the nucleotide sequence of a coding region of the HTT1 gene is shown as SEQ ID No.1. The invention further provides an application of the protein coded by the HTT1 gene in regulation and control of high-temperature tolerance of rice. The HTT1 gene is a target gene of OsHsfA2d, can negatively regulate and control the high-temperature tolerance of the rice, and can be applied to breeding of the high-temperature-resistant rice, so that a new gene resource is provided for breeding of the high-temperature-resistant rice.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of HTT1 gene in regulating high temperature tolerance of rice. Background Art

[0002] my country is an important grain production base in the world, and the production level of rice is closely related to people's quality of life. The Yangtze River Basin is an important grain production area in my country. It is extremely vulnerable to extreme high temperature weather caused by subtropical high pressure, resulting in large-scale yield reduction or even crop failure. Rice is an important food crop in China and the staple food of more than half of the world's population. With the increase in population and the acceleration of industrialization, the warming of globalization is inevitable, which has posed a serious threat to rice production (Quint et al, 2016; Janni et al, 2020; Xu et al, 2021). Rice is very sensitive to high temperature throughout the growth period, including the vegetative growth period, reproductive growth period and filling period (Li et al, 2022). Specifically, high temperature reduces pollen viability, resulting in low fruiting rate, poor milling quality, low gel consistency, high chalkiness rate and other grain qualities (De Storme & Geelen, 2014; Nevame et al, 2018; Xu et al, 2020; Kan & Lin, 2021). Studies have shown that for every 1 degree Celsius increase in global average temperature, global rice production is estimated to decrease by 3.2% (Zhao et al, 2017). Therefore, in-depth analysis of the molecular mechanism of rice heat-resistant genes will help breed heat-resistant rice varieties, which is of great significance to food security (Chen et al, 2021). Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an application of the HTT1 gene in regulating the high temperature tolerance of rice. The function of the HTT1 gene in regulating the high temperature tolerance of rice has not been reported in the prior art. Therefore, research on the function of the HTT1 gene in regulating the high temperature tolerance of rice will enrich the regulatory network and regulatory mechanism of rice high temperature tolerance and provide genetic resources for breeding high temperature resistant rice.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] The present invention provides an application of an HTT1 gene in regulating high temperature tolerance of rice. The amino acid sequence of a protein encoded by the HTT1 gene is shown in SEQ ID No.2.

[0006] Furthermore, the nucleotide sequence of the HTT1 gene coding region is shown in SEQ ID No.1.

[0007] Furthermore, the application is that the HTT1 gene negatively regulates the high temperature tolerance of rice.

[0008] The present invention also provides the use of a protein encoded by the HTT1 gene in regulating the high temperature tolerance of rice. The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.2.

[0009] Furthermore, the application is that the protein encoded by the HTT1 gene negatively regulates the high temperature tolerance of rice.

[0010] The present invention also provides the use of the HTT1 gene in the breeding of high temperature resistant rice. The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.2.

[0011] Furthermore, the nucleotide sequence of the HTT1 gene coding region is shown in SEQ ID No.1.

[0012] The present invention also provides the use of a protein encoded by the HTT1 gene in breeding high temperature resistant rice. The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.2.

[0013] The present invention also provides a method for improving the high temperature tolerance of rice, which is to improve the high temperature tolerance of rice by lowering the expression level of the HTT1 gene or the protein encoded by the HTT1 gene, wherein the amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.2, and the nucleotide sequence of the HTT1 gene coding region is shown in SEQ ID No.1.

[0014] Furthermore, the method is to reduce the expression of HTT1 gene by reducing the expression of OsHsfA2d.

[0015] The beneficial effects of the present invention are as follows: the present invention provides the use of the HTT1 gene and the protein encoded by the HTT1 gene in regulating the high temperature tolerance of rice, the amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No. 2, and the nucleotide sequence of the coding region of the HTT1 gene is shown in SEQ ID No. 1. The HTT1 gene is a target gene of OsHsfA2d, can negatively regulate the high temperature tolerance of rice, and can be applied to the breeding of high temperature resistant rice, which provides a new gene resource for breeding high temperature resistant rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1are the trait analysis results of the wild type and htt1 mutant after high temperature treatment, where a is the phenotype of the wild type and htt1 mutant plants after high temperature treatment, b is the statistical result of the survival rate of the wild type and htt1 mutant after 45℃ treatment, c is the leaves of the wild type and htt1 mutant after 28℃ and 45℃ treatment, and d is the hydrogen peroxide content of the leaves of the wild type and htt1 mutant after 28℃ and 45℃ treatment.

[0017] Figure 2 Statistical results of rice panicle phenotype and seed setting rate of wild type and htt1 mutant after high temperature treatment, where a is the rice panicle of wild type and htt1 mutant under normal temperature and high temperature treatment, and b is the statistical results of seed setting rate of wild type and htt1 mutant under normal temperature and high temperature treatment.

[0018] Figure 3 Figure 2 is the result of positional cloning and verification of the HTT1 gene, wherein a is the result of positional cloning of the HTT1 gene, b is the phenotype of the wild type, htt1 mutant, and functionally complemented plant COM, c is the genetic status of the wild type, htt1 mutant, and functionally complemented plant COM, d is the survival rate statistics of the wild type, htt1 mutant, and functionally complemented plant COM after high temperature treatment, e is the high temperature treatment phenotype of the wild type, htt1 mutant, and functionally complemented plant COM, and f is the leaf phenotype and leaf hydrogen peroxide content results of the wild type, htt1 mutant, and functionally complemented plant COM.

[0019] Figure 4 This is the Cas variation of the HTT1 gene in the HTT1 knockout strain.

[0020] Figure 5 These are the verification results of HTT1 overexpression and HTT1 knockout strains, where a is the high temperature resistance of the overexpression strain, b is the high temperature resistance of the HTT1 knockout strain, c is the statistical result of the survival rate of the overexpression strain under high temperature treatment, and d is the statistical result of the survival rate of the HTT1 knockout strain under high temperature treatment.

[0021] Figure 6 This is the analysis of HTT1 encoded protein, where a is the evolutionary tree, b is the result of protein multiple sequence alignment, and cf is the result of functional complementation experiment in Saccharomyces cerevisiae.

[0022] Figure 7 The differences in lipidome between the wild type and the htt1 mutant, where a and b are the results of lipidomics analysis and c is the result of fatty acid content determination.

[0023] Figure 8 HTT1 affects the integrity of cell membrane under high temperature stress, where a is the ion permeability result and b is the change in cell membrane morphology.

[0024] Fig. 9 The subcellular localization and high temperature expression analysis of HTT1, where a is the subcellular localization result of HTT1, b is the expression of HTT1 under high temperature treatment, and ce is the change of GUS staining of pHTT1-GUS transgenic plants with high temperature treatment time.

[0025] Fig.10 are the results of QPCR and GUS staining of HTT1, wherein a is the result of QPCR detection of the expression of HTT1 in roots, stems, leaves, leaf sheaths, stems, stalks, panicles, and branches, and b is the result of GUS staining.

[0026] Fig.11 Verification that HTT1 is the target gene of OsHsfA2d, where a is the functional element analysis of the HTT1 promoter region, b is the Y1H result, cd are the dual luciferase analysis of rice protoplasts, e is the expression of OsHsfA2d and HTT1 in the oshsfa2d-Cas9 mutant, and fg are the high temperature tolerance results of the oshsfa2d-Cas9 mutant, htt1-Cas9 mutant and htt1-Cas9 double mutant.

[0027] Fig.12 Haplotype and nucleotide diversity analysis of HTT1. DETAILED DESCRIPTION

[0028] The present invention is described clearly and completely below through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the protection scope of the present invention.

[0029] Example 1

[0030] 1. Obtaining mutants

[0031] A heat-resistant mutant htt1 was screened from the EMS mutant library of the indica rice restorer line "Jinhui No. 10". After high temperature treatment at 45℃, the wild type showed more severe wilting than the htt1 mutant ( Figure 1 a), whose survival rate was significantly lower than that of the htt1 mutant ( Figure 1 b). The leaves of the htt1 mutant are greener than those of the wild type after high temperature treatment ( Figure 1 c). Since high temperature stress can cause cell damage and thus increase antioxidant activity, the H2O2 content in wild-type leaves increased significantly after high temperature treatment, while the H2O2 content in htt1 leaves did not increase significantly ( Figure 1d); Compared with the wild type, the detached leaves of the htt1 mutant were not stained strongly with trypan blue after high temperature treatment, indicating that there were fewer dead cells in the htt1 mutant ( Figure 1 d) The high temperature tolerance of htt1 mutant may also be due to its oxidative stress response.

[0032] 2. htt1 increases the fruit set rate of rice under high temperature

[0033] When sown in the field at different times, htt1 showed semi-dwarfism compared to the wild type at all developmental stages from the seedling stage to the maturity stage. The length of the first to fourth internodes and the ear of the htt1 mutant and the wild type were measured at maturity. The results showed that htt1 was significantly shorter than the corresponding parts of the wild type, among which the third internode and ear length were extremely significantly shortened. There was no significant difference in the grain size between htt1 and the wild type. After high temperature treatment, the fruit setting rate of the wild type decreased extremely significantly, while the fruit setting rate of the htt1 mutant was basically unaffected ( Figure 2 a, b). Therefore, htt1 increases the rice seed setting rate under high temperature conditions.

[0034] 3. Map-based cloning of the HTT1 gene

[0035] Using the positional cloning strategy, HTT1 was located between the molecular markers Ind08-6 and RM25572 on the short arm of chromosome 8, with a physical distance of 115 kb (http: / / rice.plantbiology.msu.edu / ) ( Figure 3 a). The corresponding ORFs were amplified and sequenced from the wild type and htt1 mutant. The results showed that LOC_Os08g10010 (SEQ ID No.1) in htt1 had a GA base substitution in the exon region, resulting in premature termination of coding ( Figure 3 a), therefore, LOC_Os08g10010 was preliminarily identified as a candidate gene for HTT1. In order to verify whether the mutation of LOC_Os08g10010 caused the phenotype of htt1, a 5104bp genomic fragment of the wild-type LOC_Os08g10010 (including the entire coding region and 2662bp upstream and 1155bp downstream sequences) was constructed into a plant expression vector and transformed into the htt1 mutant. The gene function complementation positive strain (htt1-com) restored the high temperature sensitive phenotype under high temperature stress ( Figure 3 df). At the same time, the phenotype of the functionally complemented plants also returned to normal ( Figure 3 b, c). These results indicate that the mutant phenotype of the htt1 mutant is caused by the LOC_Os08g10010 mutation.

[0036] 4. HTT1 negatively regulates high temperature tolerance

[0037] In order to explore the role of HTT1 in high temperature stress, an HTT1 overexpression strain (HTT1-OE) in the wild-type background and an HTT1 knockout strain (htt1-Cas9) in the Zhonghua 11 (ZH11) background were constructed. Compared with Zhonghua 11, the htt1-cas9 mutant has a 113 bp deletion in the HTT1 coding region, resulting in HTT1 htt1-Cas9 The protein undergoes frameshift mutations and conformational changes ( Figure 4 ac). Compared with the wild type, HTT1-OE transgenic plants showed a heat-sensitive phenotype ( Figure 5 a, c), the survival rate of the wild type was about 70%, while the survival rate of the HTT1-OE transgenic plants was only about 20% ( Figure 5 c). In contrast, the htt1-Cas9 mutant exhibited a heat-resistant phenotype and had a higher survival rate than ZH11 ( Figure 5 b, d). These results indicate that HTT1 negatively regulates high temperature stress in rice.

[0038] 5. HTT1 encodes an acyl desaturase SAD

[0039] NCBI analysis showed that HTT1 encodes an acyl desaturase (SAD) (SEQ ID No. 2), which catalyzes the formation of double bonds. Phylogenetic analysis showed that HTT1 has high sequence similarity with SAD family members in Arabidopsis and rice ( Figure 6 a) Predicted WT HTT1 The protein structure is very similar to the RcSAD protein in Ricinus communis (Lindqvist et al., 1996), but in the httt1 mutant, the mutation of HTT1 causes a conformational change in its protein structure. Protein multiple sequence alignment results show that HTT1 has the same conserved domain as SAD proteins in multiple different species, such as RcSAD, AtSSI2, OsSSI2 and TaSSI2 ( Figure 6 b) These results suggest that HTT1 belongs to the SAD family and may be involved in the biosynthesis of unsaturated fatty acids.

[0040] To verify the role of HTT1, functional complementation experiments were performed in Saccharomyces cerevisiae. ScOLE1 is the first gene isolated from Saccharomyces cerevisiae that encodes a Δ9 fatty acid desaturase, which converts saturated C18:0 to unsaturated C18:1. Saccharomyces cerevisiae strains with ScOLE1 mutations are defective in unsaturated fatty acid biosynthesis and cannot grow in the absence of exogenous C18:1, making it an ideal system for identifying the function of SAD genes in other species ( Kajiwara, 2002 ; Sakai&Kajiwara,2003 ; Stukey et al., 1990). Using the homologous recombination knockout strategy, the scole1 knockout mutant of the haploid Saccharomyces cerevisiae BY4741 strain (BY4741-scole1) was obtained ( Figure 6 c, d). The yeast expression vectors pYES2-ScOLE1, pYES2-HTT1 WT , pYES2-HTT1 htt1 and pYES2 control plasmids were transformed into BY4741-scole1 strains. The results showed that only strains containing pYES2-HTT1 WT Alternatively, BY4741-scole1 strain of pYES2-ScOLE1 can grow on CM-Ura medium lacking C18:1 ( Figure 6 e) These results indicate that HTT1 has the function of fatty acid desaturase. WT The BY4741-scole1 strain contained lower 16:1 fatty acid content and higher 18:1 fatty acid content than the strain containing pYES2-ScOLE1 ( Figure 6 f), indicating that HTT1 prefers C18:0 as a substrate rather than C16:0 as Sc-OLE1 does.

[0041] 6. Differences in lipid profiles between wild-type WT and mutant htt1

[0042] Since HTT1 is involved in the biosynthesis of unsaturated fatty acids, plasmomic analysis of wild-type and htt1 mutant seedlings was performed, and a total of 47 lipids and 2942 lipid species were identified. As key components of cell membranes, the contents of major glycerophospholipid molecules such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylethanolamine (PE) in htt1 mutants were significantly reduced compared with those in wild-type, as was the content of sphingomyelin (SM) ( Figure 7 a). In contrast, the contents of diglyceride (DG) and digalactosylmonoglycerol (DGMG) in glycerolipids were significantly increased in the htt1 mutant ( Figure 7 b). Further fatty acid content determination results showed that the contents of C18:0, C18:1 and C18:2 in the htt1 mutant were significantly reduced compared with the wild type ( Figure 7 c). After high temperature treatment, the C18:3 content in the htt1 mutant was significantly reduced compared with the wild type ( Figure 7 c). Comparison of the ratio of fatty acids at 45°C and 28°C showed that these fatty acids, especially C18:0 and C18:1, changed relatively slowly in the htt1 mutant after high temperature treatment. At the same time, the fatty acid content of the COM strain before and after high temperature treatment was consistent with that of the wild type ( Figure 7 c).

[0043] 7. HTT1 affects the integrity of cell membranes under high temperature stress

[0044] Considering the differences in the main components of the cell membrane between the wild type and the htt1 mutant, the role of HTT1 in cell membrane integrity was further investigated. The ion permeability of the wild type, mutant htt1, and complementary COM strains was first tested. The results showed that under normal growth conditions, the ion permeability of the htt1 mutant was the same as that of the wild type and COM strains. However, after treatment at 45°C, the ion permeability of the htt1 mutant was significantly lower than that of the wild type and COM strains ( Figure 8 a), which indicates that after high temperature stress, the membrane fluidity of the htt1 mutant is more stable than that of the wild type and COM strains. Transmission electron microscopy was further used to observe the morphological changes of the cell membrane before and after high temperature treatment. The results showed that after 48 hours of treatment at 45°C, the degree of cell membrane damage of the wild type and COM strains was more serious than that of the htt1 mutant ( Figure 8 b). This indicates that the htt1 mutant has less chloroplast degradation induced by high temperature.

[0045] 8. HTT1 subcellular localization and expression analysis

[0046] To understand the localization of HTT1 in cells, the coding sequence of HTT1 was fused to the PAN580 vector containing green fluorescent protein (GFP), and the vector was transferred into rice protoplasts for transient expression. Fluorescence confocal electron microscopy showed that the fluorescence signal of the HTT1-GFP fusion protein overlapped with the chloroplast autofluorescence ( Fig. 9 a). At the same time, the coding sequence of HTT1 was fused to the s65T-2 plant expression vector containing GFP, and the tobacco was infected by Agrobacterium transformation. The results showed that the localization of the HTT1-GFP fusion protein was consistent with that of the HTT1-GFP fusion protein in rice protoplasts ( Fig. 9 a). These results indicate that HTT1 is localized in chloroplasts. The expression of HTT1 gene in various tissues of rice was studied by quantitative PCR. The results showed that it was expressed in roots, stems, leaves, leaf sheaths, stems, panicles, and branches ( Fig.10 a). At the same time, the temporal and spatial distribution of HTT1 expression was studied using the pHTT1-GUS transgenic line with the β-glucuronidase (GUS) reporter gene. The results showed that GUS signals were detected in the roots, stems, leaves and ears of the transgenic lines, which was basically consistent with the qPCR results ( Fig.10 b). Detect the expression of HTT1 under different abiotic stresses. qPCR results showed that the expression of HTT1 was significantly reduced after high temperature treatment ( Fig. 9b). The GUS staining of pHTT1-GUS transgenic plants also weakened with the extension of high temperature time ( Fig. 9 ce), indicating that high temperature inhibited the expression of HTT1.

[0047] 9. HTT1 is a target gene of OsHsfA2d

[0048] In order to explore the regulatory relationship of HTT1 in the high temperature tolerance network of rice, the functional elements in the promoter region of HTT1 were analyzed. The results showed that there were many heat stress response (HSE) elements ( Fig.11 a), indicating that it may be regulated by some HSF. Y1H results showed that OsHsfA2d could bind to the promoter of HTT1 gene ( Fig.11 b). In addition, dual-luciferase assays in rice protoplasts confirmed that OsHsfA2d inhibited the expression of HTT1 by binding to its promoter ( Fig.11 c, d), OsHsfA2d / HTG3 acts as an activating transcription factor and positively regulates the heat tolerance of rice ( Wu et al., 2022 ). The expression level of OsHsfA2d in the oshsfa2d-Cas9 mutant was significantly reduced, while the expression level of HTT1 was significantly increased ( Fig.11 e). To confirm the genetic relationship between OsHsfA2d and HTT1, double mutants of oshsfa2d-Cas9 and htt1-Cas9 were obtained by hybridization. Under high temperature treatment of 45°C, the htt1-Cas9 mutant showed a heat-resistant phenotype with a survival rate of about 70%, while the oshsfa2d-Cas9 mutant showed strong heat sensitivity with a survival rate of about 10%. It is worth noting that the loss of OsHsfA2d function did not completely suppress the heat-resistant phenotype of htt1-Cas9 in the oshsfa2d-Cas9 htt1-Cas9 double mutant, which had a survival rate of about 40% ( Fig.11 f,g). These genetic results suggest that HTT1 functions downstream of OsHsfA2d in the HSR.

[0049] 10. Haplotype and nucleotide diversity analysis of HTT1

[0050] In order to explore the role of HTT1 in the domestication and diversification of rice, the natural variation of HTT1 was analyzed using 4150 rice materials. The results showed that there were 6 SNPs in the promoter region and coding region of HTT1. Among the 6 SNPs in the coding region, 2 nucleotide substitutions of C186T and A967C caused amino acid conversion, and the remaining 4 were synonymous substitutions. According to these detected SNPs, the 4150 culture materials were divided into 8 haplotypes ( Fig.12a). Temperate japonica rice and tropical japonica rice mainly carry the Hap1 type, which is 97.22% and 94.22% respectively. Indica rice has three main haplotypes, namely Hap4 (41.98%), Hap2 (28.91%) and Hap3 (14.73%). Other haplotypes, including Hap5, Hap6, Hap7 and Hap8, are also mainly distributed in indica rice materials, but the frequency is relatively low and almost non-existent in japonica rice populations ( Fig.12 b) These results suggest that HTT1 may be differentiated between indica and japonica rice subspecies.

[0051] To further explore the evolutionary characteristics of HTT1 in indica and japonica rice subgroups, the fixation index (Fst) near HTT1 in the planting resources was determined. The results showed that there was a high degree of differentiation between indica rice and temperate and tropical japonica rice ( Fig.12 c). A decrease in nucleotide diversity (π) generally indicates positive selection (X uetal.,2020 ). In the region around HTT1, the π values ​​of the two japonica rice subspecies were lower than those of the indica rice subspecies, and the π value of tropical japonica rice was much lower than that of temperate japonica rice, indicating that there were characteristics of selection at the HTT1 locus ( Fig.12 d). These results suggest that HTT1 has the potential to be used as a breeding target for high temperature tolerance in rice.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of HTT1 gene in regulating high temperature tolerance of rice, characterized in that: The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the HTT1 gene coding region is shown in SEQ ID No.

1.

3. The use according to any one of claims 1-2, characterized in that: The application is that the HTT1 gene negatively regulates the high temperature tolerance of rice.

4. Application of the protein encoded by the HTT1 gene in regulating high temperature tolerance of rice, characterized in that: The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.

2.

5. The use according to claim 4, characterized in that: The application is that the protein encoded by the HTT1 gene negatively regulates the high temperature tolerance of rice.

6. Application of HTT1 gene in breeding of high temperature resistant rice, characterized in that: The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.

2.

7. The use according to claim 6, characterized in that: The nucleotide sequence of the HTT1 gene coding region is shown in SEQ ID No.

1.

8. Application of the protein encoded by the HTT1 gene in breeding of high temperature resistant rice, characterized in that: The amino acid sequence of the protein encoded by the HTT1 gene is shown in SEQ ID No.

2.

9. A method for improving high temperature tolerance of rice, characterized in that: The method is to improve the high temperature tolerance of rice by reducing the expression level of the HTT1 gene or the protein encoded by the HTT1 gene, wherein the amino acid sequence of the protein encoded by the HTT1 gene is shown as SEQ ID No.2, and the nucleotide sequence of the HTT1 gene coding region is shown as SEQ ID No.

1.

10. The method according to claim 9, characterized in that The method is to reduce the expression of HTT1 gene by reducing the expression of OsHsfA2d.

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