CN116790640A

CN116790640A - Novel gene STH1 for synergistically regulating and controlling salt tolerance, yield and growth period of plants and application thereof

Info

Publication number: CN116790640A
Application number: CN202210271351.0A
Authority: CN
Inventors: 林鸿宣; 项友煌; 单军祥; 叶汪薇; 董乃乾
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2023-09-22

Abstract

The invention provides a novel gene STH1 (Salt Tolerance and Heading date 1) for synergistically regulating and controlling plant salt tolerance, yield and growth period and application thereof. The STH1 codes a polypeptide with important biological functions and can regulate and control the salt tolerance, yield traits or flowering phase traits of plants. Furthermore, the invention also discloses a novel STH1-D3-HAL3 pathway or STH1-Hd1-Hd3 pathway containing STH1 for the first time and the role of the pathway in the synergistic regulation of plant traits. The invention has important significance for genetic improvement of plant traits.

Description

Novel gene STH1 for synergistically regulating and controlling salt tolerance, yield and growth period of plants and application thereof

Technical Field

The invention belongs to the field of agriculture and molecular biology; more particularly, the invention relates to a novel gene STH1 for synergistically regulating and controlling salt tolerance, yield and growth period of plants and application thereof.

Background

In the global climate change background, the increasingly accelerated salinization of soil severely restricts the production and development of crops, wherein salt stress is one of main abiotic stress factors for limiting the yield of grain crops such as rice and the like. It is counted that 6% of the land (about 120 hundred million mu) on the world is being subjected to salt stress, while China is a large country of saline-alkali lands, which is the third world. In recent years, due to factors such as irrational agricultural irrigation and seawater backflow, the problem of secondary salinization in an agricultural area is increasingly severe, and the sustainable development of grain production and ecological safety are seriously threatened. Therefore, the genetic germplasm resource with salt and alkali tolerance is excavated, which is beneficial to enlarging the usable land use area for tillage, improving the crop yield and playing a positive guarantee role for national and even world grain safety. On the other hand, the molecular mechanism of plant response to salt stress and tolerance is deeply analyzed, and important theoretical basis and gene resources can be provided for cultivating new varieties of crops with salt tolerance.

Crops are often subjected to environmental stresses such as drought, high salt, high temperature and the like in the growth and development process, and the crop yield is greatly influenced. How to improve the crop yield and the resistance of the crop to environmental stress at the same time and realize the high and stable yield of the crop is a great subject faced by scientists and breeders.

Disclosure of Invention

The invention aims to provide a novel gene STH1 for synergistically regulating and controlling salt tolerance, yield and growth period of plants and application thereof.

In a first aspect of the invention there is provided a method of modulating salt tolerance, yield traits or flowering phase traits in a plant comprising: modulating expression or activity of STH1 in a plant, or modulating an STH1-D3-HAL3 pathway in a plant, or modulating an STH1-Hd1-Hd3 pathway in a plant; the plant is a monocot.

In one or more embodiments, the flowering phase trait comprises a heading trait of a cereal plant; preferably, the earlier flowering phase includes earlier heading phase, and the later flowering phase includes later heading phase.

In one or more embodiments, the modulating a salt tolerance, yield trait, or flowering phase trait in a plant comprises a plant selected from the group consisting of:

(a) Down-regulating the expression or activity of STH1, or down-regulating the D3-mediated interaction of STH1 with HAL3 in the STH1-D3-HAL3 pathway, thereby improving salt tolerance, increasing yield or delaying flowering phase of plants;

(b) Up-regulating the expression or activity of STH1 (preferably up-regulating in plants with low expression of STH 1), or up-regulating the interaction of STH1 with Hd1 in the STH1-Hd 3 pathway, thereby promoting early flowering in plants.

In one or more embodiments, downregulating the expression or activity of STH1 comprises: knocking out or silencing a gene encoding STH1 in a plant, or inhibiting the activity of STH 1; preferably, it includes: gene editing using CRISPR system to knock out STH1 encoding gene, and gene of STH1 having loss-of-function mutation (e.g. STH1 ^HP46 Alleles) into plants, subjecting STH1 to a loss-of-function mutation in plants containing STH1 to specifically interfere with expression of a coding gene for STH 1; preferably, the loss-of-function mutations include (but are not limited to): the 115 th nucleotide C of the 1 st exon of STH1 gene is mutated into T, so that the coded protein is terminated in advance.

In one or more embodiments, up-regulating expression or activity of STH1 comprises: transferring STH1 coding gene or expression construct or vector containing the coding gene into plant; performing functional acquired mutation on STH 1; promoting STH1 expression with an expression-enhanced promoter or a tissue-specific promoter; alternatively, STH1 expression is promoted with an enhancer.

In one or more embodiments, downregulating D3-mediated interaction of STH1 with HAL3 in the STH1-D3-HAL3 pathway comprises: down-regulating the expression or activity of STH1, thereby down-regulating D3-mediated degradation of HAL3 by STH1 as a positive regulator of salt tolerance.

In one or more embodiments, up-regulating the interaction of STH1 with Hd1 in the STH1-Hd1-Hd3 pathway comprises: up-regulating expression or activity of STH1, thereby up-regulating transcriptional activation of Hd3a by STH1 and promoting transcriptional levels of Hd3 a.

In one or more embodiments, the STH1 is a fatty acid hydrolase, and the downregulating STH1 improves salt tolerance by downregulating the fatty acid hydrolase function of STH1, allowing plants to accumulate fatty acids, protecting plasma membrane (including cell membrane) structures from damage by salt stress.

In one or more embodiments, the D3-mediated interaction of STH1 with HAL3 is D3-mediated ubiquitination degradation of HAL 3; the down-regulation of D3 mediated interaction of STH1 with HAL3 increases HAL3 expression or activity by reducing D3 mediated ubiquitination degradation of HAL3, increasing salt tolerance.

In a further aspect of the invention there is provided the use of STH1, an STH1-D3-HAL3 pathway comprising the same or an STH1-Hd1-Hd3 pathway or a modulator thereof for modulating salt tolerance, yield traits or flowering phase traits in plants; wherein the regulator comprises an up regulator or a down regulator; the plant is a monocot.

In one or more embodiments, the STH1 downregulator is used to increase salt tolerance, increase yield or delay flowering phase of a plant, comprising: agents that knock out or silence STH1, agents that inhibit STH1 activity; preferably, it includes: an interfering molecule that specifically interferes with expression of a coding gene of STH1, a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent for STH1, said reagent subjecting STH1 to a loss-of-function mutation; preferably, the downregulator includes (but is not limited to): the reagent for changing the 115 th nucleotide C of the 1 st exon of STH1 gene into T or the DNA formed by annealing the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 are used as the gene editing reagent of sgRNA.

In one or more embodiments, the STH1 upregulators are used to promote early flowering in plants, comprising: exogenous STH1 encoding gene or expression construct or vector containing the encoding gene; preferably, the expression construct comprises an enhanced promoter, a tissue specific promoter or an enhancer; or, an agent that performs a functional point mutation on STH 1; preferably, the agent reverts the mutant to STH1 wild type in plants having the STH1 mutation.

In one or more embodiments, the plant is a cereal crop, or the STH1, STH1-D3-HAL3 pathway, or STH1-Hd1-Hd3 pathway is from a cereal crop; preferably, the cereal crop comprises a grass; more preferably, it comprises: rice (Oryza sativa), corn (Zea mays), millet (Setaria itaica), barley (Hordeum vulgare), wheat (Triticum aestivum), millet (Panicum miliaceum), sorghum (Sorghum bicolor), rye (Secale cereale), oat (Avena sativa l.), brachypodium distachum (Brachypodium distachyum).

In one or more embodiments, the plant is a cereal crop and the increasing plant yield comprises: increasing ear length, increasing number of branches (including number of primary branches and number of primary branches), increasing seed setting rate, increasing number of seeds (ear number), increasing seed length, increasing seed width, and/or increasing thousand seed weight of seeds.

In one or more embodiments, the amino acid sequence of the STH1 polypeptide is selected from the group consisting of: (i) a polypeptide having the amino acid sequence shown in SEQ ID NO. 3; (ii) The polypeptide which is formed by substituting, deleting or adding one or a plurality of (such as 1-20, 1-10, 1-5 and 1-3) amino acid residues of the amino acid sequence shown as SEQ ID NO. 3 and has the regulatory character function and is derived from (i); (iii) The homology of the amino acid sequence with the amino acid sequence shown in SEQ ID NO. 3 is more than or equal to 80 percent (preferably more than or equal to 85 percent, more than or equal to 90 percent, more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has the function of regulating and controlling the characters; (iv) An active fragment of a polypeptide of the amino acid sequence shown in SEQ ID NO. 3; or, (v) a polypeptide comprising a tag sequence or an enzyme cleavage site sequence added to the N-terminus or the C-terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO. 3, or a signal peptide sequence added to the N-terminus thereof.

In one or more embodiments, the STH1 comprises a cDNA sequence, a genomic sequence (gDNA), or a sequence that is artificially optimized or engineered based thereon, or further comprises a promoter upstream thereof.

In one or more embodiments, the expression construct or vector includes an STH1 promoter, preferably the promoter has the nucleotide sequence shown in SEQ ID NO. 4; a polynucleotide which hybridizes with the polynucleotide sequence shown in SEQ ID No. 4 under stringent conditions and has a function of promoting the expression of a target gene; or, a polynucleotide having 80% or more (preferably 90% or more, more preferably 95% or more, most preferably 98% or more) identity with the polynucleotide sequence shown in SEQ ID NO. 4 and having a function of promoting expression of a target gene.

In one or more embodiments, the coding gene for STH1 includes a full-length gene sequence, a coding region gene sequence; preferably, a promoter sequence is also included.

In one or more embodiments, the STH1 loss-of-function mutation further comprises: so that the translation of STH1 protein is terminated in advance, thereby losing the function; preferably, by gene editing, insertion, deletion or mutation of a base is performed in the STH1 protein-encoding gene, so that translation of the STH1 protein is terminated prematurely.

In one or more embodiments, the up-regulation, promotion, increase or enhancement means up-regulation, promotion, increase or enhancement of significance, such as up-regulation, promotion, increase or enhancement by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or more.

In one or more embodiments, the reduction, decrease, attenuation, inhibition, or downregulation means a significant reduction, decrease, attenuation, inhibition, or downregulation, such as a reduction, decrease, attenuation, inhibition, or downregulation of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or less.

In one or more embodiments, the rice comprises a plant selected from the group consisting of: indica rice and japonica rice.

In one or more embodiments, the STH1 or STH1-D3-HAL3 pathway comprising the same; or a protein or gene in the STH1-Hd1-Hd3 pathway comprising the same, and homologues thereof.

In a further aspect of the invention there is provided the use of a plant STH1, an STH1-D3-HAL3 pathway comprising the same or an STH1-Hd1-Hd3 pathway as a molecular marker for identifying salt tolerance, yield traits or flowering traits in plants, or as a molecular marker for the directed screening of plants; the plant is a monocot.

In another aspect of the invention, there is provided a method of selecting or identifying a plant, the method comprising: identifying expression or sequence characteristics of STH1 in the test plant, or identifying interaction conditions of the STH1-D3-HAL3 pathway or the STH1-Hd1-Hd3 pathway in the plant; if the STH1 of the test plant is expressed low or not, or the test plant is a plant with high salt tolerance, high yield or delayed flowering phase; if STH1 is highly expressed, the test plant is a plant with low salt tolerance, low yield or early flowering phase.

In one or more embodiments, the high expression or activity refers to an increase in expression or activity that is statistically significant compared to the average value of the expression or activity of the same class or species of plant.

In one or more embodiments, the low expression or activity refers to a statistically significant reduction in expression or activity as compared to the average value of expression or activity of the same class or species of plant.

In another aspect of the present invention, there is provided a method of screening for a substance (potential substance) that modulates salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising: (1) adding a candidate substance to a system expressing STH 1; (2) Detecting the system, observing the expression or activity of STH1 therein, and if the expression or activity is reduced, indicating that the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants; if its expression or activity is increased, it is indicated that the candidate substance is a substance useful for promoting the early flowering phase of plants.

In another aspect of the present invention, there is provided a method of screening for a substance (potential substance) that modulates salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising: (1) Adding a candidate substance to a system expressing the STH1-D3-HAL3 pathway; (2) Observing the interaction of D3-mediated STH1 and HAL3 in the STH1-D3-HAL3 pathway, if the degradation of HAL3 by D3-mediated STH1 is reduced, the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants.

In another aspect of the present invention, there is provided a method of screening for a substance (potential substance) that modulates salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising: (1) Adding a candidate substance to a system expressing the STH1-Hd1-Hd3 pathway; (2) Observing the interaction of STH1 and Hd1 in the STH1-Hd1-Hd3 pathway, if the transcription stimulus of STH1 to Hd3a, preferably the transcription level of Hd3a, is enhanced, the candidate substance is a substance which can be used for promoting the early flowering phase of plants.

In one or more embodiments, the method further comprises providing a control group to specifically identify differences in protein expression, activity, transcription or ubiquitination in the STH1 or pathway in which it participates in the test group, from the control group.

In one or more embodiments, the candidate substance includes (but is not limited to): regulatory molecules designed for STH1, HAL3, D3, hd1, hd3 proteins or genes encoding them or upstream or downstream proteins or genes thereof (e.g., such as modulators, small molecule compound gene editing constructs, etc.).

In one or more embodiments, the system is selected from the group consisting of: a cell system (e.g., a cell or cell culture that expresses STH1 and/or a pathway protein in which it participates), a subcellular (culture) system, a solution system, a tissue system, an organ system, or an animal system.

In another aspect of the present invention, there is provided a downregulator of STH1 for a substance for improving salt tolerance, increasing yield or delaying flowering phase of a plant, which is: reagents for mutating nucleotide C at position 115 of exon 1 of STH1 gene into T; or CRISPR gene editing reagent for down-regulating STH1 gene, preferably, the gene editing reagent takes DNA annealed by the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 as sgRNA.

In another aspect of the invention there is provided a construct or plant cell, tissue or organ comprising exogenous said agent for increasing salt tolerance, increasing yield or delaying flowering phase of a plant under STH 1.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, primary localization linkage segment of STH1, and genetic background analysis; (a) genotype of CSSL (STH 1); (b) substitution HPC020 salt stress tolerance differences.

FIG. 2, positional cloning of STH 1; (a) Fine localization of STH1 and natural mutation sites of candidate genes; (b) NIL-STH1 ^HJX74 And NIL-STH1 ^HP46 Salt stress tolerance comparison; (c-e) transgenic knockout lines and controls (c), overexpressing lines and controls (d), and genetically complementary transgenic positive lines and NIL-STH1 ^HP46 (e) Salt tolerance phenotype and survival rate statistics before and after salt treatment; (f) Variation of STH1 expression levels at different times in salt (120 mM) treatment; (g) subcellular localization form of STH 1.

FIG. 3, STH1 function to control plant salt tolerance by fatty acid hydrolase; (a) Detecting and analyzing the obtained differential significant metabolites by the broad targeted metabolome; (b) Identification of the substrate LC-MS after the enzymatic reaction of MBP-STH1 and MBP; (c) Comparing the peak areas of the substrate after MBP-STH1 fusion protein and MBP are incubated in a no-load mode; (d-f) salt tolerance phenotype (d) and quantitative data of the plants after fatty acid treatment, aerial height (e) and fresh weight (f); (g-i) determination of NILs (STH 1) relative conductivity (g), malondialdehyde content (h) and chlorophyll content (i) before and after salt treatment; (j) The leaves of NILs (STH 1) plants were observed for cytological changes before and after salt treatment by transmission electron microscopy.

FIG. 4, STH1 has broad spectrum fatty acid hydrolase activity; (a-c) detecting and analyzing the widely targeted metabolome to obtain different significant metabolites, namely, stearic acid (a), elaidic acid (b) and palmitoleic acid (c); (d-f) substrate LC-MS identification after MBP-STH1 and MBP enzymatic reaction, stearic acid (d), elaidic acid (e), palmitoleic acid (f).

FIG. 5, STH1 enhances D3-mediated ubiquitination modification of OsHAL3 to affect salt tolerance of plants; (a-c) STH1, D3 and OsHAL3 interact two by two to form a protein complex, and BiFC (a), SFLC (b) and pull down (c) experiments verify the interaction relationship between proteins; (D) co-localization of STH1, D3 and OsHAL3 in the nucleus; (e) Quantitative comparison of endogenous OsHAL3 protein content of NILs (STH 1); (f) Comparison of the degree of ubiquitination modification of OsHAL3 in different NILs (STH 1) genetic backgrounds; (g) OsHAL3 in vitro protein degradation rate comparison; (h, i) genetic episomal analysis of STH1 and OsHAL 3.

FIG. 6, interaction of STH1 and Hd1 in nucleus to enhance transcription activity of Hd3a and regulate flowering time of rice; (a-c) NILs (STH 1) heading date phenotype (a) and flowering-time quantification data for (c) under different planting environments (b) and photoperiod conditions; (d) Analysis of expression level of related representative marker genes in NILs (STH 1) in the photoperiod pathway; (e) STH1, hd1 and Hd3a expression levels varied under different photoperiod environments; (f, g) BiFC (f) and SFLC (g) experiments verify the interaction relationship between proteins; (h) co-localization of STH1, hd1 and Hd3a in the nucleus; (i) a map of different plasmid construction modes for a dual fluorescence reporting system; (j-k) wild type (j) and different NILs (STH 1) background (k) transcriptional activity of Hd1 on Hd3a was examined.

FIG. 7, agronomic trait comparisons of NILs (STH 1); (a-c) quantitative statistics of NILs (STH 1) plant height (a), tillering number (b) and main stem diameter (c).

FIG. 8, comparison of NILs (STH 1) yield performance under normal conditions and under salt stress; (a-c) comparing the mature stage plant type (a), the main ear type (b) and the grain type (c) of the representative seed of NILs (STH 1); (d, e) statistics of NILs (STH 1) individual yield (d) and cell yield (e); (f, g) salt stress tolerance comparison of NILs (STH 1) seedling stage (f) and maturity stage (g); (h-j) comparison of ear (h), seed setting (i) and individual yield (j) of NILs (STH 1) representative plants in salt pond planting environments.

FIG. 9, hd3a, balances rice salt tolerance and flowering time; (a) The expression level of Hd3a varied at different time points under salt (120 mM) treatment conditions; (b-d) salt tolerance phenotype identification of Hd1 mutants under long-day (LD) (b) and short-day (SD) (c) conditions, post-recovery survival statistics (d); (e, f) heading stage phenotype under NILs (STH 1) salt treatment (e) and flowering time quantification of results (f).

FIG. 10, hd3a negatively regulated rice salt tolerance; (a-c) target knockout of transgenic line that knocks out Hd3a under salt (120 mM) treatment conditions (a), salt tolerance phenotype (b), and survival statistics (c); (d) salt stress inhibits transcriptional activation of Hd3a by Hd 1.

FIG. 11, STH1 and homologous genes thereof are functionally differentiated; (a) protein secondary structure analysis of STH1 and homologous genes; (b) Colinear analysis of STH1 and its homologous genes in different species; (c) STH1 and homologous gene expression pattern comparison; (d, e) transgenic lines after STH1 homologous knockout had a heading stage phenotype (d) and a target knockout form (e).

FIG. 12, STH1 is not involved in signal transduction, synthesis and metabolic pathways of SL; (a, b) a rootstock binding part branch number phenotype (a) and a tillering number quantification result (b) under NIL (STH 1) hydroponic conditions; (c) NIL (STH 1) seedling stage tillering primordium scanning electron microscope results; (d, e) rootstock binding portion branch phenotype (d) and tillering quantification result (e) after NIL (STH 1) SL analogue treatment; (f) comparison of endogenous SL levels in the aerial parts of NIL (STH 1) seedlings; (g) Identification of the substrate LC-MS after the enzymatic reaction of MBP-STH1 and MBP; (h) Comparison of the substrate peak area after MBP-STH1 fusion protein and MBP no-load incubation.

FIG. 13 sequence comparison analysis of STH1 orthologous genes in various monocots.

Detailed Description

The invention firstly researches and reveals a novel gene STH1 (Salt Tolerance and Heading date 1) which codes a polypeptide with important biological functions and can regulate and control the salt tolerance, yield traits or flowering phase traits of plants. Furthermore, the invention also discloses a novel STH1-D3-HAL3 pathway or STH1-Hd1-Hd3 pathway containing STH1 for the first time and the role of the pathway in synergistically regulating salt tolerance and heading stage balance. The invention has important significance for genetic improvement of plant traits.

Terminology

As used herein, the terms "pathway," "signal pathway," and "regulation pathway" are used interchangeably.

As used herein, the "plant" is a plant comprising in its genome the STH1 or STH1-D3-HAL3 pathway or STH1-Hd 3 pathway of the invention or a homolog of a gene/protein involved in the signal pathway (homologous gene/homologous polypeptide (protein)), said plant being a monocot; such as: gramineous plants, liliaceae, lycoris, etc. Preferably comprising a gramineous plant. In some preferred embodiments, the plant is a crop, preferably a cereal crop. Preferably, the gramineous plant is such as rice of the genus gramineous rice, the gramineous wheat plant is such as wheat, the gramineous maize plant is such as maize, etc. Examples include: rice, sorghum, maize, barley, wheat, oats, rye. It will be appreciated by the person skilled in the art that plants suitable for use in the present invention are not limited to the above list, and that suitable plants may be determined by identifying the presence of the STH1, STH1-D3-HAL3 pathway or STH1-Hd1-Hd3 pathway therein.

As used herein, the term "flowering trait" includes the "heading trait" of cereal plants.

As used herein, the term "kernel" refers to the fruit or seed of a plant, also known as a spike in crops such as rice, maize, wheat, barley, and the like.

As used herein and as will be appreciated by those skilled in the art, selection of an appropriate "control plant" is a routine part of an experimental design and may include a corresponding wild-type plant or a corresponding transgenic plant without the gene of interest. The control plants are generally of the same plant species or even varieties which are identical to or belong to the same class as the plants to be evaluated. The control plant may also be an individual who has lost the transgenic plant due to isolation. Control plants as used herein refer not only to whole plants, but also to plant parts, including seeds and seed parts.

As used herein, the terms "salt tolerance" and "salt stress tolerance" are used interchangeably with "salt tolerance".

As used herein, the terms "maintaining yield under high salt", "protecting plants" or "maintaining plants' high salt yield" refer to plants that remain viable, maintain normal growth, and have relatively normal, substantially normal, or higher yields in a high salt (soil or medium having a salt content significantly higher than their normal culture conditions) environment. In some ways, the term "maintaining yield under high salt" means that after a particular plant is cultivated under high salt environment, its yield is maintained at 30% or more, 40% or more, 50% or more, 60% or more, etc., of the latter as compared to the yield of "non-high salt environment (normal/suitable temperature environment)"; preferably maintained at 70% or more, 80% or more, 90% or more, or 100% of the latter.

As used herein, the term "high salt" or "salt (environment)" refers to an environment that is significantly higher than the suitable salt content for plant growth; for example, by "high salt" or "salt (environment)" is generally meant that the soil or medium contains a salt content that is greater than that in normal soil or medium, such as 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or greater than that in normal soil; for example, the soil or medium contains NaCl levels above 50mM, 75mM, 100mM, 120mM, 150mM, 200mM, 250mM or more.

As used herein, the term "high salt tolerance" refers to a statistically significant increase in salt tolerance, such as a 5%, 10%, 20%, 40%, 60%, 80%, 90% or more increase in survival or yield, of a plant (e.g., an engineered plant) as compared to the salt tolerance of a similar or identical plant.

As used herein, the term "up-regulation" includes: promotion, overexpression, enhancement, etc., which is statistically or significantly up-regulated, promoted, enhanced, or enhanced by 20%, 40%, 60%, 80%, 90% or more as up-regulated, promoted, enhanced, or enhanced.

As used herein, the term "down-regulating" includes: weakening, lowering, inhibiting; represents a down-regulation, attenuation, reduction, inhibition of significance, such as a down-regulation, attenuation, reduction, inhibition or down-regulation of 20%, 40%, 60%, 80%, 90% or less.

As used herein, "salt tolerance" refers to the ability of a plant to withstand a salt environment. In general, the salt tolerance of a test plant is determined by comparing the test plant to a control plant in the same salt environment. Test plants are generally considered to have better salt tolerance when they survive higher at high salinity, longer time to survive, and higher yield.

As used herein, the term "high expression or high activity" is intended to refer to a gene/protein of interest whose expression or activity in a particular plant (e.g., an engineered plant) is statistically increased, e.g., by 10%, 20%, 40%, 60%, 80%, 90% or more, as compared to the average value of the expression or activity of the same or similar plant.

As used herein, the term "low expression or low activity" is intended to refer to a gene/protein of interest whose expression or activity in a particular plant (e.g., an engineered plant) is statistically reduced, e.g., by 10%, 20%, 40%, 60%, 80%, 90% or less, as compared to the average value of the expression or activity of the same or similar plant.

As used herein, the "loss of function mutation" includes: such that the target protein is rendered nonfunctional, e.g., the critical region of its protein chain is mutated, deleted or inserted resulting in the loss of function. In some embodiments, insertion, deletion, or mutation of bases is performed in the gene encoding the target protein by gene editing, such that translation of the target protein is prematurely terminated.

As used herein, the "function-gain mutation" includes: so that the target protein which is originally expressed in a limited or non-expressed way is expressed normally; in some embodiments, reversion of bases (e.g., reversion to the same or degenerate sequence as wild-type) is performed in the gene encoding the protein of interest by gene editing, such that the protein of interest is reverted to expression.

STH1

The invention adopts a method of map cloning to locate and clone a new QTL which can synergistically regulate and control the balance of salt tolerance and heading stage of rice and is named as Salt Tolerance and Heading date 1 (STH 1) by earlier-stage identification and screening of a strain of the African rice chromosome substitution with a salt tolerance late flowering phenotype. STH1 encodes a hydrolase that contains an alpha/beta sheet domain and degrades fatty acids as metabolic substrates. Natural mutation sites in african rice cause premature termination of STH1 transcription and loss of enzyme activity, accumulation of endogenous fatty acid content, and elevation of protein content level of salt tolerance key regulatory factor OsHAL3 to further enhance salt tolerance of rice. Knock-out and overexpression of STH1 respectively increased and decreased resistance of plants to salt stress. In a rice photoperiod mediated flowering regulation network, STH1 plays a role of a zinc finger protein Hd1 transcriptional coactivator, regulates the expression of a florigen gene Hd3a, and further influences the heading period and yield of plants. Introduction of the STH1 allelic variant site form derived from African rice delays the heading time of the rice and further improves the yield performance. Under salt treatment conditions, STH1 will further inhibit Hd3a expression and plant floral transformation by down-regulating its own expression levels in response to salt stress. These results show that STH1 is a gene with multiple effects, which is used as a negative genetic factor for regulating the salt tolerance of rice and a positive regulatory factor for the flowering time of rice, and the expression quantity of Hd3a under normal and high salt conditions is balanced to synergistically regulate the yield and salt tolerance of rice.

As used herein, unless otherwise specified, STH1 refers to a polypeptide having the sequence of SEQ ID NO. 3 or a gene encoding the same, and includes a variant of the sequence having the same function as the STH1 polypeptide. The coding gene can be gDNA or cDNA, and can also comprise a promoter. For example, the gDNA has the nucleotide sequence shown in SEQ ID NO. 1, the cDNA has the nucleotide sequence shown in SEQ ID NO. 2, and the promoter has the nucleotide sequence shown in SEQ ID NO. 4. The sequences of the coding genes also include sequences that are degenerate to the sequences provided herein.

Variant forms of the STH1 polypeptide include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus. Any protein having high homology (e.g., 70% or more homology to the polypeptide sequence shown in SEQ ID NO: 3; preferably 80% or more homology; more preferably 90% or more homology, e.g., 95%,98% or 99%) to the STH1 polypeptide and having the same function as the STH1 polypeptide is also included in the present invention. Polypeptides derived from other species than rice that have higher homology to the polypeptide sequence of SEQ ID NO. 3 or that exert the same or similar effect in the same or similar regulatory pathways are also encompassed by the present invention.

In the present invention, the term "STH1" also includes homologues thereof. It should be understood that while STH1 obtained from rice of a particular species is preferably studied in the present invention, other polypeptides or genes obtained from other species that are highly homologous (e.g., have greater than 60%, such as 70%,80%,85%, 90%, 95%, or even 98% sequence identity) to the STH1 are also within the contemplation of the present invention.

The invention also comprises a mutant STH1 polypeptide truncated body, the gene sequence of which is coded by the mutant STH1 polypeptide truncated body is mutated into T at the 115 th nucleotide C of the 1 st exon corresponding to the STH1 gene (SEQ ID NO: 2), and the coded protein is terminated in advance, so that the polypeptide truncated body is obtained. The truncations do not have the function of wild STH1 polypeptide, and the seed number and the seed shape of the plants with the mutation are obviously changed.

The polynucleotides (genes) encoding the STH1 polypeptides may be natural genes from plants or degenerate sequences thereof.

Vectors comprising the coding sequences and host cells genetically engineered with the vectors or polypeptide coding sequences are also included in the invention. Methods well known to those skilled in the art can be used to construct vectors containing suitable expression.

The host cell is typically a plant cell. The transformed plants can be transformed by agrobacterium transformation or gene gun transformation, such as leaf disc method, young embryo transformation method, etc.; preferred is the Agrobacterium method. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain plants with altered traits relative to the wild type.

STH 1-involved pathways

As used herein, the term "signaling pathway" refers to a pathway system formed by a series of proteins or genes that interact or interact with each other, which generally results in the occurrence of some cellular event. The STH1-D3-HAL3 pathway includes (but is not limited to): the STH1 gene (and/or protein encoded thereby), the D3 gene (and/or protein encoded thereby), the HAL3 gene (and/or protein encoded thereby). The STH1-Hd1-Hd3 pathway includes (but is not limited to): STH1 gene (and/or protein encoded thereby), hd3 gene (and/or protein encoded thereby).

As used herein, the term "STH1-D3-HAL3 pathway" is used interchangeably with "STH1/D3/HAL3 pathway".

As used herein, the term "STH1-Hd1-Hd3 pathway" is used interchangeably with "STH1/Hd1/Hd3 pathway".

The nucleotide sequence of the D3 gene is shown as LOC_Os06g 06050; the protein amino acid sequence is shown in LOC_Os06g 06050.

The nucleotide sequence of the HAL3 gene is shown as LOC_Os06g 09910; the amino acid sequence of the protein is shown as LOC_Os06g 09910.

The nucleotide sequence of the Hd1 gene is shown as LOC_Os06g 16370; the protein amino acid sequence is shown in LOC_Os06g 16370.

The nucleotide sequence of the Hd3a gene is shown as LOC_Os06g 06320; the amino acid sequence of the protein is shown as LOC_Os06g 06320.

When used as a target for artificial regulation or when a screening system is artificially established, the above protein or coding gene may be naturally occurring, for example, it may be purified and isolated from a mammal; it may also be recombinantly produced, e.g., recombinant proteins may be produced according to conventional genetic recombination techniques. In addition, any variant that does not affect the biological activity of these proteins, such as derivatives or variants whose function is not altered, may be used.

Variants of the above-described pathway proteins are also encompassed by the present invention, and include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus. Any protein having high homology to the polypeptide (e.g., 70% or more homology to the polypeptide sequence shown in SEQ ID NO: 4; preferably 80% or more homology, more preferably 90% or more homology, e.g., 95%,98% or 99%) and having the same function as the polypeptide is also included in the present invention. Polypeptides derived from other species than rice that have higher homology to the polypeptide sequence or that exert the same or similar effect in the same or similar regulatory pathways are also encompassed by the present invention.

Homologues of the pathway genes/proteins described above are also encompassed by the present invention. It should be understood that while the corresponding pathway gene/protein obtained from rice of a particular species is preferably studied in the present invention, other polypeptides or genes obtained from other species that are homologous (e.g., have more than 60%, such as 70%,80%,85%, 90%, 95%, or even 98% sequence identity) to the pathway gene/protein are also within the contemplation of the present invention.

The inventors found that downregulating the expression or activity of STH1 in the pathway can downregulate D3-mediated degradation of HAL3 by STH1 (as a positive regulator of salt tolerance), thereby improving salt tolerance of plants, increasing yield of plants or delaying flowering phase of plants. Conversely, up-regulating STH1 expression or activity promotes D3-mediated STH1 degradation of HAL3 (as a salt-tolerant positive regulator).

The inventor finds that the up-regulation of STH1 expression or activity in the channel can up-regulate the interaction between STH1 and Hd1 in the STH1-Hd1-Hd3 channel, up-regulate the transcriptional activation of STH1 on Hd3a and promote the transcriptional level of Hd3a, thereby promoting the early flowering phase of plants.

Method for improving plants

Based on the new findings of the present inventors, the present invention provides a method of improving a plant, the method comprising: regulating the expression or activity of STH1 in plants, regulating STH1-D3-HAL3 passage or regulating STH1-Hd1-Hd3 passage, and regulating salt tolerance, yield or flowering phase of plants.

In one aspect, the invention provides a method of conferring a trait on a plant that is salt tolerant, high in yield or late in flowering (heading stage), comprising: down-regulating the expression or activity of STH1, or down-regulating D3-mediated interaction of STH1 with HAL3 in the STH1-D3-HAL3 pathway. Wherein a preferred way of down-regulating the STH1-D3-HAL3 pathway is: down-regulating the expression or activity of STH1, thereby down-regulating D3-mediated interaction of STH1 with HAL3 in the STH1-D3-HAL3 pathway (D3-mediated ubiquitination degradation of HAL 3). Wherein HAL3 is used as a positive regulator of salt tolerance and yield.

In another aspect, the present invention provides a method of causing plants, particularly plants that express low (including non-expressed) STH1, to exhibit advanced flowering (heading stage), comprising: up-regulating the expression or activity of STH1, and/or up-regulating the STH1-Hd1-Hd3 pathway. Wherein, a preferred mode of up-regulating STH1-Hd1-Hd3 pathway is: up-regulating expression or activity of STH1, thereby up-regulating transcriptional activation of Hd3a by STH1 and promoting transcriptional levels of Hd3 a.

It will be appreciated that after the function of the STH1 and the STH1-D3-HAL3 pathway or STH1-Hd1-Hd3 pathway (preferably including genes upstream and downstream of the pathway) comprising STH1 is known, various methods known to those skilled in the art may be employed to modulate the expression or activity of the STH1 or to modulate the STH1-D3-HAL3 pathway or STH1-Hd1-Hd3 pathway. For example, STH1 expression may be reduced, deleted or STH1 overexpressed by a variety of methods well known to those skilled in the art. Or by methods well known to those skilled in the art to promote or attenuate ubiquitination of downstream proteins and to enhance or attenuate transcriptional activation of downstream genes.

In the present invention, the down-regulator of STH1 protein includes inhibitor, antagonist, blocker, etc. The downregulator of STH1 protein or gene encoding the same refers to any substance which can reduce STH1 protein activity, reduce STH1 stability, downregulate STH1 expression, reduce STH1 effective action time, inhibit STH1 transcription and translation, reduce STH1 protein-induced degradation of HAL3 (as a salt-tolerant positive regulatory factor), and reduce interaction of STH1 protein with D3, and can be used in the present invention as a substance useful for downregulating STH1. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecules may be nucleic acid-level (including DNA, RNA) or protein-level. For example, the downregulator is: a gene editing reagent that specifically edits STH1, or an interfering RNA molecule or antisense nucleotide that specifically interferes with STH 1; etc.

As a preferred mode, the present invention provides a method for down-regulating STH1 in a plant, comprising targeted mutation, gene editing or gene recombination of STH1, thereby effecting down-regulation. As a more specific embodiment, STH1 is transformed into its truncations, whose function is reduced or lost, by any of the methods described above, thereby altering the traits of the plant. As a more specific example, gene editing is performed using the CRISPR/Cas9 system to knock out or down target genes. Suitable sgRNA target sites will lead to higher gene editing efficiency, so suitable target sites can be designed and found before proceeding with gene editing. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.

As another embodiment of the present invention, there is provided a method for down-regulating expression of STH1 in a plant, comprising: (1) Transferring an interfering molecule interfering with STH1 gene expression into a plant cell, tissue, organ or seed to obtain the plant cell, tissue, organ or seed into which the interfering molecule is transferred; (2) Regenerating a plant from the plant cell, tissue, organ or seed obtained in step (1) into which the interfering molecule has been introduced. Preferably, the method further comprises: (3) Selecting a plant cell, tissue or organ into which the vector has been transferred; and (4) regenerating the plant cells, tissues or organs of step (3) into a plant.

In the present invention, the up-regulator of STH1 protein includes promoter, agonist, activator, etc. The "up-regulation", "promotion" includes "up-regulation", "promotion" of protein activity or "up-regulation", "promotion" of protein expression, and they are "up-regulation", "promotion" of protein activity in a statistical sense. Any agent that increases the activity of STH1 or other signaling pathway protein, increases the stability of STH1 or other signaling pathway protein, up-regulates the expression of STH1 or other signaling pathway gene, increases the effective duration of action of STH1 or other signaling pathway protein, increases the phosphorylation/activation level of the respective protein may be used in the present invention as an agent useful for up-regulating STH1 or signaling pathway. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecules may be nucleic acid-level (including DNA, RNA) or protein-level.

As one embodiment, the present invention provides a method of up-regulating STH1 expression in a plant, said method comprising: the coding gene of STH1 or an expression construct or vector containing the coding gene is transferred into plants.

The technical scheme of the invention can be applied to molecular design breeding for various ways. For example, the STH1 gene or its promoter region or intron region is edited/interfered/modified by CRISPR/Cas9 or the like, or the members in the STH1-D3-HAL3 pathway or the STH1-Hd1-Hd3 pathway are edited/interfered/modified, or the proper alleles are introduced into the current main cultivated variety by means of crossing to create excellent gene loci, so as to cultivate the ideal high-yield plant variety.

In the specific examples of the present invention, a detailed study was conducted on rice, and found that: (1) STH1 is a negative regulator of salt stress tolerance, and knockout of STH1 gene significantly enhances salt tolerance of plants, while overexpression of STH1 ^HJX74 Or complementary proSTH1 gSTH1 results in plant saltIncreased sensitivity; (2) The rice STH1 gene codes a hydrolase containing alpha/beta folding structural domain, belongs to a global positioning form in cells, is specifically and highly expressed in leaves, but has low expression level in other tissues and is subjected to salt stress induced expression down regulation; (3) STH1 has the function of fatty acid hydrolase, participates in the fatty acid metabolic pathway of rice, influences the integrity and fluidity of biomembrane components including cell membranes under adverse conditions, and further regulates and controls the resistance of rice to salt stress; (4) STH1 can interact with F-box protein D3 and flavin mononucleotide binding protein OsHAL3 in pairs to form a protein complex in the nucleus, so that D3-mediated ubiquitination modification of OsHAL3 is enhanced, and the stability of salt-tolerant protein OsHAL3 is influenced to regulate and control the salt tolerance of plants; (5) D3 and OsHAL3 are negative and positive regulatory factors, respectively, of salt stress tolerance, wherein OsHAL3 is located genetically downstream of STH1 in altering salt stress tolerance in rice; overexpression of the D3 gene and OsHAL3 gene in other rice varieties reduces or enhances the salt tolerance of plants, respectively, while in NIL-STH1 ^HP46 Knockout of OsHAL3 in the background or in NIL-STH1 ^HJX74 Over-expression of OsHAL3 in the background promotes the plant to return to NIL-STH1 ^HJX74 Or NIL-STH1 ^HP46 A similar salt-sensitive or salt-tolerant phenotype; (6) STH1 is a positive control factor of flowering time (heading stage) of rice, and enhances the transcriptional activation of the STH1 on a florigen gene Hd3a through interaction with a transcription factor Hd1, so that the transcriptional level of Hd3a is promoted, and the heading stage of the rice is advanced; (7) Hd3a is a negative regulatory factor of salt stress tolerance, responds to salt stress and is subjected to down regulation of inhibition expression of the salt stress, and knockout of Hd3a gene can remarkably enhance the salt tolerance of plants under different photoperiod conditions; (8) Salt stress can inhibit the transcriptional activity of Hd1 on Hd3a, influence the expression level of Hd3a so as to delay the conversion of plant from vegetative growth to reproductive growth, and STH1 can further inhibit the expression of Hd3a by reducing the expression quantity of the STH1, so that the heading date is delayed to cope with adverse environment (high-salt environment); (9) Mutation site STH1 from African rice ^HP46 Has good application prospect and NIL-STH1 ^HP46 The yield is increased by about 30% under the normal field planting environment, the yield is increased by about 70% in the artificial simulated saline-alkali soil environment, and the gene locus is used for the cultivation of the plantIs an important gene resource for high-yield and high-salt-tolerance rice breeding. In addition, since the STH1 gene has conservation in monocots, it is possible to modify the promoter region or coding region of the STH1 gene or to modify STH1 gene using CRISPR/Cas9 gene editing technology ^HP46 The alleles are introduced into the current main cultivated varieties in a hybridization mode, so that the salt tolerance and the yield of plants are improved, and then new varieties of crops with high yield and stress resistance (including but not limited to rice, wheat, corn, sorghum, millet, soybean and the like) are cultivated. These specific findings fully support the feasibility of the technical solution of the invention.

Molecular marker and application thereof

After the STH1 and the regulation and control channels involved in the STH1 are known, the STH1 can be used as a molecular marker to perform directional screening of plants. Substances or potential substances that modulate plant grain traits or hormonal traits in a targeted manner by modulating this mechanism can also be screened based on this new discovery.

In one aspect, the invention provides a method of directionally selecting or identifying plants, the method comprising: identifying expression or sequence characteristics of STH1 in the test plant, or identifying interaction conditions of the STH1-D3-HAL3 pathway or the STH1-Hd1-Hd3 pathway in the plant; if the STH1 of the test plant is expressed low or not, or the test plant is a plant with high salt tolerance, high yield or delayed flowering phase; if STH1 is highly expressed, the test plant is a plant with low salt tolerance, low yield or early flowering phase.

In another aspect, the invention provides a method of screening for a substance (potential substance) that modulates a salt tolerance, yield trait or flowering phase trait in a plant, comprising: (1) adding a candidate substance to a system expressing STH 1; (2) Detecting the system, observing the expression or activity of STH1 therein, and if the expression or activity is reduced, indicating that the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants; if its expression or activity is increased, it is indicated that the candidate substance is a substance useful for promoting the early flowering phase of plants.

The present invention also provides a method of screening a substance (potential substance) that modulates a salt tolerance, yield trait or flowering phase trait of a plant, comprising: (1) Adding a candidate substance to a system expressing the STH1-D3-HAL3 pathway; (2) Observing the interaction of D3-mediated STH1 and HAL3 in the STH1-D3-HAL3 pathway, if the degradation of HAL3 by D3-mediated STH1 is reduced, the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants.

The present invention also provides a method of screening a substance (potential substance) that modulates a salt tolerance, yield trait or flowering phase trait of a plant, comprising: (1) Adding a candidate substance to a system expressing the STH1-Hd1-Hd3 pathway; (2) Observing the interaction of STH1 and Hd1 in the STH1-Hd1-Hd3 pathway, if the transcription excitation of STH1 to Hd3a is enhanced, preferably the transcription level of Hd3a is enhanced, the candidate substance is a substance which can be used for promoting the early flowering phase of plants.

Methods for screening for substances that act on a target site, either on a protein or on a gene or on a specific region thereof, are well known to those skilled in the art and can be used in the present invention. The candidate substance may be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the kind of substance to be screened, it is clear to the person skilled in the art how to select a suitable screening method.

Through large-scale screening, a kind of potential substances which specifically act on STH1 or a path participated in the STH1 and have regulation and control effects on plant salt tolerance, yield traits or flowering phase traits can be obtained.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which are not specifically noted in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.

Materials and methods

1. Positional cloning of genes

The inventor constructs a set of chromosome fragment substitution systems (CSSLs) by taking the African cultivated rice HP46 as a donor parent and the Asian cultivated rice Hua Jingxian (HJX 74) as an acceptor parent, and the chromosome fragment substitution systems are used for positioning, cloning and controlling the rice salt stress tolerance and the QTLs related to the heading period. The QTL identified by initial positioning to the substitution line HPC020 comprising the balance of salt tolerance and heading date of the synergistic regulation of rice was designated STH1 and was backcrossed with the recurrent parent HJX 74. Selection of STH 1-nearby segments as heterozygotes and construction of BC for plants with other background regions of HJX74 ₄ F ₂ Populations were selected at 16664 BC with the aid of molecular markers ₄ F ₂ The STH1 was finely mapped in the plants and the STH1 candidate segment was reduced to a 3.034kb region of chromosome 5, which contained only 1 predicted gene, sequenced and aligned for further analysis. Simultaneous use of BC ₅ F ₂ The generation plant constructs near isogenic line NIL-STH1 with target segment containing very small (60 kb) HP46 chromosome segment and most of other genetic background of HJX74 ^HP46 And its control NIL-STH1 ^HJX74 。

CRISPR/Cas9 Gene editing, overexpression and genetic complementation

(1) For STH1

In order to further verify candidate gene LOC_Os05g51240, the inventor uses CRISPR/Cas9 technology for gene editing of target gene STH1, designs a knockout target point aiming at the functional domain of LOC_Os05g51240, namely the vicinity of a key nucleotide variation site C115T, anneals with primers of SEQ ID NO. 7 and SEQ ID NO. 8 to form DNA, and constructs the DNA into a CRISPR/Cas9 vector for knockout of target gene. A constitutive strong promoter UBI is adopted to drive strong expression LOC_Os05g51240 ORF from HJX74 background, and an over-expression strain is obtained. LOC_Os05g51240 full-length genomic sequence derived from HJX74 and comprising 1.8kb promoter, all exons and introns and 729bp 3' UTR (untranslated region) was constructed on the transgenic complementation vector pCAMBIA1300 in NIL-STH1 ^HP46 Genetic transformation was performed in the background. Genetic transformation is carried out by a rice mature embryo transformation method mediated by agrobacterium tumefaciens EHA105, a transgenic positive strain is screened, and the transgenic positive strain is planted in a field and is subjected to transgenic T ₂ Examination for substitutionAnd (5) observing the phenotype. Knock-out and over-expression plasmid construction was introduced as shown in Table 1.

TABLE 1

(2) Against OsHAL3

TABLE 2

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(3) For Hd1

TABLE 3 Table 3

(4) For Hd3a or D14

Mutant materials were ordered from the hundred biotechnology company.

GUS staining to detect STH1 expression Pattern

And (3) taking GUS transgenic plants in the booting stage, performing tissue dissection separation, immersing the in-vitro tissues to be detected in a pre-configured GUS dye solution, vacuumizing to enable the samples to be completely immersed below the liquid level, and standing at 37 ℃ for overnight. The dye solution was discarded, decolorized with 95% ethanol in a 80 ℃ water bath, and after the green color of the sample had completely faded, observed under a dissecting scope and photographed.

4. Subcellular localization

The tobacco leaves were transiently expressed by Agrobacterium-mediated transformation using Benshi tobacco Nicotiana benthamiana in the vigorous growth period (about 4 weeks without flowering), and the Agrobacterium strain GV3101 transformed with the expression plasmid (2 x 35S::: STH 1-GFP) was picked up, and after 48 hours of cultivation, the inner epidermis of the tobacco leaf marker region was torn off and fluorescence imaging was performed using a laser confocal microscope. Coding regions of STH1, D3, osHAL3, hd1 and Hd3a genes are respectively constructed on corresponding pA7-CFP/YFP/RFP vectors, HJX74 yellowing seedlings are used for separating protoplasts by dark culture for 2 weeks, the purified and concentrated expression plasmids are transformed into receptor cell protoplasts by a PEG mediated method to carry out instantaneous expression of exogenous genes, and finally, the expression plasmids are observed and photographed by a laser confocal microscope after dark culture for 14-16 hours.

The sequences for plasmid construction are shown in Table 4

TABLE 4 Table 4

5. Extensive targeted metabolome detection

The present inventors explored the in vivo metabolic processes involved in STH1 and the role played in salt stress resistance through a broad targeted metabonomics assay. NILs (STH 1) grown in water for 20 days are taken as experimental materials, frozen at a low temperature by liquid nitrogen and dried, and then ground into powder by a grinder (30 Hz,1.5 minutes), wherein the overground parts of 24 seedlings are repeatedly taken as tissue samples from each biology, and then the tissue samples are sent to the Wuhanmai Metabolic Biotechnology company for wide targeted metabolome technical service. Based on UPLC-MS/MS detection platform and self-built database, 528 metabolites were finally detected in total.

STH1 in vitro expression and enzyme Activity detection

To verify whether fatty acids are hydrolysis substrates for STH1, in vitro expression of STH1 and enzyme activity detection experiments were performed. The coding region sequence of STH1 was constructed into pMal-c5x vector and transferred into E.coli BL21 (DE 3), cultured until 0D600 was added with 0.1mM IPTG at about 0.6-0.8, and shake-cultured at 16℃for 20 hours to induce protein expression. After the cells are broken by ultrasound, the supernatant is separated by ultracentrifugation and purified by an affinity chromatography medium (MBPSep Dextrin Agarose Resin) to obtain MBP-STH1 fusion protein. 200 μl of the enzymatic reaction system was as follows: 100. Mu.M fatty acid or epi-5DS as potential targeted hydrolysis substrate and 50. Mu.g/ml recombinant protein. The reaction system is incubated for 3 hours at 25 ℃ and 22 ℃ respectively, then an equal volume of precooled methanol or acetone is added for terminating the reaction, and finally, the supernatant is sucked by high-speed centrifugation for liquid phase tandem mass spectrometry (LC-MS) analysis, and the obtained total ion flow diagram, mass spectrogram and metabolite peak area are quantitatively analyzed by using Xcalibur software.

The 5' end oligonucleotide primer sequence constructed by the pMal-c5x fusion protein expression vector is as follows:

5’-gcgatatcgtcgacggatccATGAAGAAGATGTGGCGCGC-3’(SEQ ID NO:37)；

the 3' end primer sequence is as follows:

5’-gaattccctgcaggtaattaCTATATGGCAACATCGATGC-3’(SEQ ID NO:38)。

7. exogenous fatty acid treatment and quantitative analysis of plant endogenous fatty acid

The method comprises the steps of firstly taking wild HJX74 seeds after dehulling through exogenous fatty acid treatment, soaking the wild HJX74 seeds in 75% ethanol for 90 seconds, sterilizing the wild HJX74 seeds in a 2% NaClO solution (1-2 drops of Tween-20 are added) for more than 120 minutes, flushing the wild HJX74 seeds with sterile water for 4-5 times, draining the wild HJX74 seeds through filter paper, inoculating the wild HJX74 seeds on a 1/2MS culture medium through a camera, and pre-germinating the wild HJX74 seeds in an illumination incubator for 3 days. Seeds with substantially uniform germination conditions were then and randomly sown on 1/2MS medium with excess salt (200 mM or 250mM NaCl) and fatty acid added for further growth for 30 days to evaluate the change in salt stress tolerance. NIL-STH1 ^HP46 With NIL-STH1 ^HJX74 In contrast, the unsaturated fatty acid content is obviously enriched, so that the endogenous fatty acid content of wild plants before and after salt treatment is detected to study the relationship between plant salt stress response and fatty acid metabolism. Taking wild plants HJX74 as a material, and taking overground parts of rice seedlings as detection samples respectively before treatment and after salt treatment (120 mM NaCl) for 7 days after water culture in an illumination incubator for 20 days, and rapidly freezing with liquid nitrogen, wherein 24 seedlings are taken from each biological repetition. Samples were then sent to the wu-kanmai metabolic biotechnology company for quantitative analysis of fatty acids based on GC-MS/MS platform, containing 48 fatty acids in total.

8. Adverse-condition related physiological index measurement and cytological observation

Rice leaves before and after 4 days of salt treatment with NILs (STH 1) seedlings grown in water for 20 days were used as control and experimental groups, respectively, and each biological repeat contained 2 leaves of seedlings. The relative conductivity was measured by rinsing the leaves of the treatment and experimental groups with ultrapure water for more than 2 times, then placing the leaves in a test tube containing deionized water, sufficiently evacuating, and shaking on a shaker for more than 3 hours to allow the leaves to settle below the liquid surface. After measuring the initial conductivity S1 with a conductivity meter, the plant tissue was killed by boiling water bath for more than 10 minutes, and after cooling to room temperature, the final conductivity S2 was measured. Malondialdehyde (MDA) content is determined by dissolving plant tissue powder ground in advance with liquid nitrogen in 10% TCA solution, centrifuging, collecting supernatant as sample extractive solution, adding 0.67% TBA solution of equal volume, mixing well, boiling for 30 min, cooling to room temperature, and centrifuging again. Chlorophyll extraction experiment the chlorophyll of plant tissue is extracted by acetone-ethanol mixing method, rice leaf is soaked in dark place at room temperature until the material becomes white completely (overnight), and colorimetric determination is carried out by high-speed centrifugation the next day. The preparation method comprises the steps of glutaraldehyde fixation, ethanol gradual dehydration, resin embedding, solidification, slicing and staining, and finally observing the cell membrane and chloroplast change under a transmission electron microscope.

9. Transcriptome sequencing

To screen NIL-STH1 ^HJX74 With NIL-STH1 ^HP46 Further revealing the related molecular mechanism of STH1 for regulating and controlling the salt tolerance of rice, taking 10cM young ears of NILs (STH 1) in the booting stage as a tissue sample to be detected, and carrying out transcriptome sequencing by utilizing a BGISEQ-500 platform of Huada genes, wherein 38,226 genes are detected in total.

10. Protein interaction technology

To verify the protein-protein interaction relationship, one-to-one interaction verification was performed for the target protein by means of a bimolecular fluorescence complementation assay (BiFC), a luciferase enzyme activity recovery method (SFLC), a pull-down assay, co-immunoprecipitation (Co-IP), and a yeast two-hybrid (Y2H) assay.

11. Protein in vitro degradation experiment

NILs (STH 1) were pretreated with a plant nutrient solution supplemented with a protease inhibitor (MG 132) for 3h to prevent protein degradation by 26S proteasome ubiquitination, and then total protein was extracted with degradation buffer. Dissolving plant tissue powder at 4deg.C for 30 min, centrifuging at high speed, collecting supernatant, packaging into multiple tubes, and incubating at 28deg.C in water bath for corresponding time. Protein loading buffer was added at designated time points to terminate the reaction, boiled for 10 min and centrifuged again at high speed for immunoblotting experiments, i.e., western Blot.

12. Ubiquitination experiment

Endogenous total proteins of stably transformed genetic material (OE-HAL 3/NILs) are extracted using a vegetable protein extraction buffer, the target proteins are IP-isolated by co-immunoprecipitation method, and then the ubiquitination level is detected by SDS electrophoresis and total ubiquitination antibodies.

13. Dual fluorescence reporting system

Constructing expression vectors connected with different target genes, and adding Hd3a promoter with the size of 2k fragments in front of a Luciferase reporter gene as a reporter gene. The different combinations of plasmids were transfected into rice protoplasts or the Agrobacterium strain GV3101 (pSoup) carrying the vector was transformed into tobacco using the Agrobacterium-mediated transient expression method, and finally the two luciferase activities were examined using dual-luciferase reporter assay system (Promega).

14. Sequence information

STH1 ^HJX74 Gene sequence (SEQ ID NO:1; underlined bases represent start codon and stop codon):

>actgcaccagcttaagcttattagctcctgagagtgaagcccctatagctgatcgaagctatgaagaagatgtggcgcgccaacgcgagagtggtggaaaggagaggtggtcgcgaggccgaggagagagggggagtaattagcgtggtgcttgcccatggctatggcgcgagccaggcggtgtgggacaagctcgtgccctccctctccaagagccacaacctcctcctcttcgactgggacttcaccggcgcaggcgccgggaaggacgacgacgagtacacttacggcaggttcgccgacgagctcatcgcggtgatggaggagcggggcgtgggcgcgtcgggcgcggtggtggtggcgcactccatgtccgccatggccgcctgcatcgccgcccaacggagacccgacctcttcgcccacatcttcctcgtctgcgcctccccgaggttaattaattatcgatcactagatttcattcatttttgctgttccttaattaattaagcccctcaagatcaaggatgacatatatagcctaattaccgttattattattacagctatttaatttaattagttatttttaagcacaattatattagttagtcgttgtgtaacacgtgtttggttcatccagatatcactcccagtagatagcagctatgctagcttttgagttttatcattgtcaactttgtgtgttaattgtcagcgtgtgtacgtccacgtcagcacactaatcgcgaccatgcagcagcagtcacgttcactgttgctcacgactcacccaacatctagaaacatcatcaattgcatagcttcattcacaggaagaaaacaaacgcctccttttcgattatttttccctagatagcgcttgtcaaatctgaccaatgaacagcaattaagatttggccggatcgaaccgggacgtcacaacaaacacttgctgctagctactctagattagatcaaagctagctctagtccggatgagatgaagcaggatcgatcaatcgtggcctcatgttagttagtccataacctgtacgtgtggctaaaaaataggaacacaagaaattatccatgtatcaatcagtcaagatgtttcagatccttgcaactaactaattaatattctattgccaaaacatgcatgatcaagcatctaattgattacttagcatttctactcgattgcatgcaagtacctgttgaaaattcaattgggcatgcagctgccagataggagtaagctactctgacagtacaagaaactgatgaacaaaattaaaactgaactagagaacgtatagctgatgaaaacgggtggtggatcgatcgatcaggtacataaacttggaggaggaagggtatgtaggaggctttgaggaggcggcgatccacggcatgctggcagccatggagtcggacttcgacggctgggtcaggagcttcctccccaacgccgccggcgacgcgtccgccgtggagcacctcctcaagagcttcctcgccatggacccgaccgtcgcgctcaagctggccaagatgatcttcctcggcgaccagcgggaggtcctcgacggcgtgaagacgccgtgcaccatcgtccaggtgaaggccgacttcgcggcgccgcccagcgtggcggagtacatgcacctcaggatgaagggcgccgccacggccgtcgagatcatcggctccgtcgggcacttcccccagctcgtcgcgccgcagcagctgctggacatactcgccggcgtcctgcgcctccgggaggcggcggcggaggcggagcacgacgacgccggcaccgtggagattgccggtggcatcgatgttgccatataggttgtgcatcatgcatgcatgcttgtaaaaagtctgcacatatatgcgtcagagctagctgcatgtgtgctcccttgtatgtatgtttgtttgttttgttttcagagcatgcattagttgtactattatgttttcctgtgtaaacatatcatagaggttatgttacttgtacacctgcatgcatggtttgggtatgctgatggatgtaagtaccattttcaatgggacatataccttatt

STH1 ^HJX74 coding region sequence (SEQ ID NO: 2):

>atgaagaagatgtggcgcgccaacgcgagagtggtggaaaggagaggtggtcgcgaggccgaggagagagggggagtaattagcgtggtgcttgcccatggctatggcgcgagccaggcggtgtgggacaagctcgtgccctccctctccaagagccacaacctcctcctcttcgactgggacttcaccggcgcaggcgccgggaaggacgacgacgagtacacttacggcaggttcgccgacgagctcatcgcggtgatggaggagcggggcgtgggcgcgtcgggcgcggtggtggtggcgcactccatgtccgccatggccgcctgcatcgccgcccaacggagacccgacctcttcgcccacatcttcctcgtctgcgcctccccgaggtacataaacttggaggaggaagggtatgtaggaggctttgaggaggcggcgatccacggcatgctggcagccatggagtcggacttcgacggctgggtcaggagcttcctccccaacgccgccggcgacgcgtccgccgtggagcacctcctcaagagcttcctcgccatggacccgaccgtcgcgctcaagctggccaagatgatcttcctcggcgaccagcgggaggtcctcgacggcgtgaagacgccgtgcaccatcgtccaggtgaaggccgacttcgcggcgccgcccagcgtggcggagtacatgcacctcaggatgaagggcgccgccacggccgtcgagatcatcggctccgtcgggcacttcccccagctcgtcgcgccgcagcagctgctggacatactcgccggcgtcctgcgcctccgggaggcggcggcggaggcggagcacgacgacgccggcaccgtggagattgccggtggcatcgatgttgccatatag

STH1 ^HJX74 protein sequence (SEQ ID NO: 3):

>MKKMWRANARVVERRGGREAEERGGVISVVLAHGYGASQAVWDKLVPSLSKSHNLLLFDWDFTGAGAGKDDDEYTYGRFADELIAVMEERGVGASGAVVVAHSMSAMAACIAAQRRPDLFAHIFLVCASPRYINLEEEGYVGGFEEAAIHGMLAAMESDFDGWVRSFLPNAAGDASAVEHLLKSFLAMDPTVALKLAKMIFLGDQREVLDGVKTPCTIVQVKADFAAPPSVAEYMHLRMKGAATAVEIIGSVGHFPQLVAPQQLLDILAGVLRLREAAAEAEHDDAGTVEIAGGIDVAI

STH1 ^HJX74 promoter sequence (SEQ ID NO: 4):

>ccttgagtggtacacgtccttattatatatccaagttttcagctagctagctctactcataatgatctagtataatgcatgaggtcatgacatattccttccgtaaaaaaaaaagttctagtattcacaaaaatcaaaaaatataattctttttagataatttttaaaattgtaattttttaagaacaaagggaatgctagttaatactatcatgcttaattaatctgcttgcttatggatgtagaggatcccagcatgtaatgaactaatgatatgatgctgatatataactccaaatctatcattttaattgggtagcagagtactgtatttgcctaaatgttatactaatatgcggacagtgatgacgtgttcagcccagcaatttaacattttagcccttttacaaacattattctccaaataaatccccatacaattataactaaaaatagagctcaacattagcgtcaggtccatggcgacgaagtctaacatctcagcaccaaggggcaatacaatagtaagcaccagtatattttgtacttttctatataggacagctaatataattagaacgctttctacagaattcgatcagctaatatcaccagaacacttcaattagaaaagagaatgaaaaacagaggcatgggaagtcgatcatgccagggagagattgcataagaaagccactaagctactccctccattttagtaggttataagaagatttgattttggtcaaagtcaaactactctaagtttgactaattttatagaaaaaaaatagtatatttttaaccaagacaaatatattataaaaatttactcaattataaatttaataaaactatgctagtgttgtaaatattactacatttttctacagaattagtcaaacttagaataccaaagtcaaaacgtcttataacctaaaacggagtatagtaaaaaaatagcacatttatatttatcaagtactataaagctgagcttcacatagctaagtggcaagactgatgacgctgagatgttgggcttccgcgtcataggtgtgactgctgaggtttagctttagtatttatgaggatctatttatagattaaattttatgaagggactaaaatgtaaaaattgttgatgttcagtcagagatgatatgttctacggagtatctacgacctggcaaccaccattggtgatgctagctaggcgacaaagcagcactttcccgggagagactgggaatctaggctatctgtactgtagctcccggccacttgctacctgccttcttgcaccttttctagcccttctgtccttttttcttgcttgtcagtattcagaaagaaagatgcactataatttatgcatatccaagtcaatcatttattgtaaaagtgcgcgtggttttatttgtaactaatcgttgcaatttcttatagaatctttagctctttcaaagaaaataaaatataagtctgtttagggaagccttaaactctaaaaatcaactagtgagaattcgaatatcctgagaaaaccaatttaagtttggcttgtagtttattttttaaattctgtaaccgagcatttttatattttaataaggcagctatcaattggttacttctcaaaacagagggctatattaattgctagcccttaattttgttgacacaaagtcgggctggcatctgctgagttcctataaatacaggcgtacgtggttgcgcagtttactgcaccagcttaagcttattagctcctgagagtgaagcccctatagctgatcgaagct

STH1 ^HP46 coding region sequence (SEQ ID NO:5; bold bases indicate that natural variation sites are bold and underlined bases represent bases that terminate prematurely):

>atgaagaagatgtggcgcgccaacgcgagagtggtggaaaggagaggtggtcgcgaggccgaggagagagggggagtaattagcgtggtgcttgcccatggctatggcgcgagctaggcggtgtgggacaagctcgtgccctccctctccaagagccacaacctcctcctcttcgactgggacttcaccggcgccggcgccgggaaggacgacgacgagtacactttcggcaggttcgccgacgagctcatcgcggtgatggaggagcggggcgtgggcgcgtcgggcgcggtggtggtggcgcactccatgtccgccatggccgcctgcatcgccgcccaacggagacccgacctcttcgcccacatcttcctcgtctgcgcctccccgaggtacataaacttggaggaggaagggtatgtaggaggctttgaggaggcggcgatccacggcatgctggcagccatggagtcggacttcgacggctgggtcaggagcttcctccccaacgccgccggcgacgcgtccgccgtggagcacctcctcaagagcttcctcgccatggacccgaccgtcgcgctcaagctggccaagatgatcttcctcggcgaccagcgggaggtcctcgacggcgtgaagacgccgtgcaccatcgtccaggtgaaggccgacttcgcggcgccgcccagcgtggcggagtacatgcacctcaggatgaagggcgccgccacggccgtcgagatcatcggctccgtcgggcacttcccccagctcgtcgcgccgcagcagctgctggacatactcgccggcgtcctgcgcctccgggaggcggcggcggaggcggagcacgacgacgccggcaccgtggagattgccggtggcatcgatgttgccatatag

STH1 ^HP46 protein sequence (SEQ ID NO: 6):

>MKKMWRANARVVERRGGREAEERGGVISVVLAHGYGAS

example 1 localization and cloning of target genes

The invention takes the QTL related to the salt tolerance of positioning cloned rice as a starting point, utilizes a set of chromosome substitution lines constructed by taking Asian cultivated indica rice (Oryza Sativa L.ssp. Indica) variety Huajing-shaped indica 74 (Huajingxin 74; HJX 74) as recurrent parent and African rice HP46 (Oryza glaberma) as donor parent to carry out salt tolerance phenotype identification of substitution lines, and successfully screens a strain HPC020 (CSSL-STH 1) containing an African rice substitution segment at the tail end of a long arm of chromosome 5 ^HP46 ) The strain exhibits a higher degree of resistance than the recurrent parent (CSSL-STH 1) ^HJX74 ) Significantly stronger salt stress tolerance. Based on this the inventors selected the HPC020 strain as subject of investigation, F was constructed by re-crossing with recurrent parent ₂ The population was isolated, further refined positioned and QTL located in this segment that controls rice salt stress tolerance was designated STH1 (fig. 1 a-b).

To clone the STH1 gene, the STH1 candidate segment was reduced to a 3.034kb region (between the S543 and ID556 molecular markers) by high density molecular marker-assisted selection, which contained only one predicted candidate gene LOC_Os05g51240 encoding an alpha/beta sheet domain-containing hydrolase. The LOC_Os05g51240 was identified by alignment as 9 variations between the two parents, including SNP (Single Nucleotide Polymorphism) and InDel (Insertion/delivery) sequence differences on the introns. Wherein only 3 mutation sites occur on exons (see positions 115, 195, 227 in SEQ ID NO: 4), and nucleotide C at position 115 on exon 1 in HJX74 is mutated to T in the allele of HP46, directly resulting in premature termination of the protein encoded by the HP46 allele. While this variant is found only in a small fraction of african rice material, indicating STH1 ^HP46 Is a rare mutation site. In addition, the mutations "A" to "C" (position 195) and "A" to "T" (position 227) located at two positions on exon 1, respectively (i.e., from A in HJX74 to C and T in HP 46) were only one (A227T) resulting in a difference in amino acid level between the two (FIGS. 2 a-b).

To further confirm the accuracy of the localization results and confirm the candidate gene for STH1, the present inventors constructed related transgenic vectors for genetic verification. At HUnder the genetic background of JX74, gene knockout is carried out on LOC_Os05g51240 by adopting a CRISPR/Cas9 gene editing system; at the same time to LOC_Os05g51240 ^HJX74 Is overexpressed. The results showed that the knockout of loc_os05g51240 did not result in a significant change in plant growth but the plant resistance to salt stress was significantly improved (fig. 2 c), whereas the salt resistance of the overexpressed line was significantly reduced (fig. 2 d). The result of genetic complementation shows that in NIL-STH1 ^HP46 Loc_os05g51240 is complemented in the background ^HJX74 Transgenic positive plants with full-length genomic sequences reverted to a salt-sensitive phenotype consistent with recurrent parent HJX74 (fig. 2 e).

Therefore, by screening and identifying the substitution line of the chromosome fragment, the gene is positioned at the tail end of a long arm of a No. 5 chromosome of rice, the QTL-STH1 related to the salt tolerance of the rice is regulated, and the STH1 gene for controlling the phenotype of the rice is cloned. The transgenic result fully shows that the inventor successfully clones and regulates the rice salt tolerance related QTL-STH1, and the STH1 gene is a negative regulation factor of rice salt stress response.

Example 2 STH1 is specifically highly expressed in leaf tissue and globally localized in cells in response to salt stress

To investigate the tissue expression pattern of STH1, the present inventors cloned a sequence of about 1.8kb before the STH1 translation initiation site as the self promoter of STH1 and driven the expression of GUS gene in the medium flower 11 background, and examined the expression site of GUS gene in transgenic plants. The results of GUS activity analysis showed that STH1 was expressed in the leaf at the highest level, and also expressed to some extent in the ear and glume, but barely detected in other sites. The results of qRT-PCR also confirm that STH1 gene is specifically highly expressed in leaf blades, while the expression level in other tissues is very low. In addition, under salt treatment conditions, STH1 responds rapidly to salt stress, while high salt environments significantly inhibit transcription of STH1 gene mRNA, resulting in a significant down-regulation of its expression level, and eventually remain at a relatively stable lower level after about 10 hours of salt treatment. Subcellular localization of STH1 protein was studied using rice protoplast system and tobacco system, respectively, and laser confocal observations showed that the fluorescent signal of fusion protein was similar to that of no-load control, but was also ubiquitously expressed throughout the cell, with the exception of being able to significantly overlap with the red fluorescence of nuclear localization signal (FIGS. 2 f-g).

The above results indicate that STH1 is down-regulated by salt treatment induced expression, its time-space expression characteristics are specific, and its subcellular localization belongs to a globally targeted expression form.

Example 3, STH1 functioning as a fatty acid hydrolase involved in fatty acid metabolism in plants, thereby affecting the integrity and fluidity of plasma membrane components under salt stress

To further explore the potential targeted metabolic substrates of STH1, the present inventors found that NIL-STH1 was detected by a broad targeted metabolome assay ^HP46 Large amounts of unsaturated fatty acid metabolites, and NIL-STH1 in the aerial parts of seedlings ^HJX74 A significant increase in the material content compared to that occurs (fig. 3 a). The study data of the metabolome indicate that STH1 may be involved in the regulation of fatty acid metabolic pathways in plants. The MBP-STH1 fusion protein and the control MBP protein are respectively expressed and purified in vitro by a prokaryotic expression system of escherichia coli, and the maximum differential metabolite Eicosenoic acid (Eicosenoic acid) is used as a substrate for in vitro enzymatic reaction, and finally, the reaction product is subjected to high-resolution liquid chromatography-mass spectrometry (LC-MS) detection. The final enzyme activity experiment showed that the eicosenoic acid peak area after MBP-STH1 catalytic reaction was significantly smaller than that of control MBP and the detected reaction substrate was also completely consistent with the standard in retention time (FIGS. 3 b-c). Next, the same experimental analysis of enzyme activity was carried out using Elaidic acid (Elaidic acid), stearic acid (Stearic acid) and Palmitoleic acid (Palmitoleic acid) as reaction substrates, respectively, in agreement with the above results, the peak areas of fatty acids after MBP-STH1 catalytic reaction were smaller to different extents than those of the control group (fig. 4 a-f). The above results indicate that STH1 may possess a broad spectrum of hydrolase activity on fatty acids.

The inventor performs unified growth in an illumination environment by sowing wild seeds with consistent pre-germination states on a plant culture medium containing salt with exogenous fatty acid, and finally counts the height or fresh weight of the overground parts of the wild seeds as a quantitative index of plant salt tolerance. Statistical results showed that rice seedlings grown in plant medium with applied fatty acids (eicosenoic acid, elaidic acid, stearic acid or palmitoleic acid) were significantly higher in either aerial height or aerial fresh weight than the control (fig. 3 d-f), indicating that fatty acids as a primary metabolite were able to enhance plant resistance to salt stress. Quantitative determination of fatty acid content of rice seedlings before and after salt treatment, most species of fatty acids among all 48 fatty acids detected, whose substance content was up-expressed to different degrees in plant samples after salt treatment, indicated that high salt environment induced rice to accumulate fatty acids to resist stress (fig. 3).

Furthermore, the salt tolerance and the membrane damage degree of the near isogenic line are evaluated by using the stress related physiological indexes. Wherein the relative conductivity (REC) represents the extent of ion leakage, the Malondialdehyde (MDA) content reflects the extent of membrane lipid peroxidation, and the chlorophyll content characterizes the extent of inhibition of photosynthesis. As a result of measurement, it was found that the above physiological indexes were not different between NILs before the salt treatment, but NIL-STH1 after the salt treatment ^HJX74 But showed higher relative conductivity and MDA content and lower chlorophyll content, indicating NIL-STH1 ^HJX74 The extent of cell membrane disruption was more severe under high salt conditions (FIG. 3 g-i). And (5) observing the cross-section ultrastructure of the NILs (near field plasma) blade before and after salt treatment by using a transmission electron microscope. Results were in agreement with expectations, comparison of NIL-STH1 ^HP46 Relatively intact membrane structure, NIL-STH1 after salt treatment ^HJX74 The smoothness of the cell membrane profile was reduced and the number of thylakoids in chloroplasts was significantly reduced, resulting in a decrease in the number and volume of basal ganglia, with numerous vesicular structures in the center of the cytoplasm, presumably due to the breakdown and digestion of numerous damaged organelles induced by salt stress by intracellular vacuoles (FIG. 3 j). The transcriptome sequencing result further shows that the expression level of the membrane component related genes and the genes involved in the plant stress response pathway are obviously changed between NILs, so the inventor considers that the content of endogenous fatty acid in the plant can be influenced by the integrity and fluidity of a biological membrane system including a cell membrane, therebyMediating the strength of the plant to the tolerance of salt stress.

In conclusion, NIL-STH1 ^HP46 It is because STH1 function loss causes fatty acid content accumulation in plants, thus causing plasma membrane structures in cell membranes to be not easily damaged by salt stress, and the improvement of salt treatment resistance of plants is shown.

Example 4 STH1 enhances D3-mediated degradation of OsHAL3 ubiquitination and thus regulates salt tolerance in plants

Through the bimolecular fluorescence complementation assay (BiFC), luciferase enzyme recovery (SFLC) and Pull-Down experiments, it was verified that STH1 can interact with a known salt-tolerant protein OsHAL3 (LOC_Os 06g 09910) and a protein D3 containing an F-box domain (LOC_Os 06g 06050) more strongly in pairs, respectively, in vivo and in vitro. The same results were obtained by one-to-one interaction verification of Co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) experiments against the target protein, which further increases the confidence of the three interactions at the protein level. In addition, co-localization analysis was performed with different fluorescence for each of STH1, D3 and OsHAL3, and as a result, a good overlap of fluorescence signals was observed in the nuclei of the three (FIGS. 5 a-D), which indicated that the three could form a protein complex by interaction and exert its function in the nuclei.

Extracting total protein of near-isogenic line rice seedlings, and performing semi-quantitative analysis on the protein abundance by utilizing an OsHAL3 autoantibody. The results show that the protein content of OsHAL3 is NIL-STH1 under the condition that the content of the reference beta-actin in the protein is basically consistent whether salt treatment is carried out or not ^HP46 Higher in (3). MG132 (26S proteasome inhibitor) treatment of seedlings of rice in different NIL contexts, NIL-STH1 ^HJX74 In which OsHAL3 protein level is markedly increased, while NIL-STH1 ^HP46 This suggests that the OsHAL3 protein is not very stable in plants and its degradation may be related to 26S proteasome, while STH1 may be involved in regulating the ubiquitination process of OsHAL 3. Furthermore, the transgenic material of stable conversion of OsHAL3-GFP is utilized to carry out immune co-precipitation on the OsHAL3-GFP fusion protein through agarose beads of covalent coupling GFP antibodyThe extent of ubiquitination of OsHAL3 proteins in different near isogenic line genetic backgrounds was measured with anti-ubiquitin antibodies (FIGS. 5 e-f). As a result of comparison, NIL-STH1 was found ^HP46 The color of the bands of the OsHAL3 polyubiquitination in the medium was lighter, indicating NIL-STH1 ^HP46 Has relatively lower level of OsHAL3 ubiquitination.

Comparing the in vitro degradation rates of OsHAL3 protein in different near isogenic line backgrounds, extracting and aliquoting total protein of the rice seedlings after MG132 pretreatment, terminating the reaction at different incubation time nodes in a degradation system, and finally detecting the protein level of the substrate protein OsHAL3 after internal reference correction. West Blotting (WB) results showed that compared to NIL-STH1 ^HP46 The OsHAL3 protein in the medium has no obvious change at all time points, and NIL-STH1 ^HJX74 The protein levels of OsHAL3 protein in the culture medium were significantly reduced at 2h, 3h and 4h, which indicates NIL-STH1 ^HP46 The OsHAL3 protein in the (1) is higher in stability and is not easily degraded by a proteasome (figure 5 g). Based on the above findings, the present inventors believe that the presence of STH1 exacerbates the D3-mediated ubiquitination of OsHAL3, which in turn leads to down-regulation of OsHAL3 protein levels, while NIL-STH1 ^HP46 As a result of the loss of STH1 function, osHAL3 protein content is promoted to be accumulated and stability is improved.

Salt tolerance phenotypic analysis of STH1, D3 and OsHAL3 transgenic material showed that the transgenic lines overexpressing D3 in the medium flower 11 background had a greatly increased salt sensitivity compared to the wild type, whereas the transgenic lines overexpressing OsHAL3 in the HJX74 background showed an enhanced resistance to salt treatment, indicating that D3 and OsHAL3 exert the effects of negative and positive regulatory factors, respectively, mediating the tolerance of rice to salt stress. At the same time, at NIL-STH1 ^HP46 None of the 3 independent OsHAL3 knockout homozygous lines obtained in the genetic background maintained their original salt-resistant phenotype, but restored NIL-STH1 ^HJX74 Salt-sensitive phenotype. In contrast, in NIL-STH1 ^HJX74 Up-regulating OsHAL3 expression level in background makes plant show NIL-STH1 expression level ^HP46 A similar salt-tolerant phenotype (fig. 5 h-i).

These results suggest that OsHAL3 is located genetically downstream of STH1 in terms of altering rice salt stress tolerance, and also in agreement with the above-described findings that STH1 promotes degradation of OsHAL 3.

Example 5 STH1 as transcriptional coactivator of Hd1 to regulate expression level of Hd3a to affect flowering time in Rice

The length of time from sowing in the field to the heading stage of the near isogenic line was counted, and as a result, significant heading period differences were found between NILs. NIL-STH1 in either Shanghai or three-in-one field environment ^HP46 Always with late flowering phenotype, average flowering time was delayed by 10 and 5 days, respectively (FIGS. 6 a-d). The complementation successful transgenic lines are also subjected to quantitative investigation of heading stage phenotype, and the result also shows that the flowering time of the transgenic positive lines returns to the early flowering phenotype of the recurrent parent HJX 74. Based on the analysis results, the STH1 not only affects the salt tolerance of rice, but also is a positive genetic factor for controlling the heading time of rice, and is regulated by a photoperiod route, thus being an important gene in a plant flowering molecule regulation network.

Considering that STH1 may play a role in the photoperiod flowering pathway, the present inventors selected 5 representative marker genes (OsGI, hd1, ehd1, hd3a and RFT 1) in the photoperiod pathway for the detection of the expression level. The results showed that NIL-STH1 ^HP46 mRNA levels of OsGI and Hd1 were not significantly altered in the plants, but the expression levels of Ehd1, hd3a and RFT1 were significantly down-regulated (FIG. 6 d). These results suggest that STH1 positively regulates the flowering process in rice by regulating the expression level of the florigen gene.

The analysis of the transcription levels of STH1, hd1 and Hd3a in rice seedling leaves under different photoperiod growth conditions showed that the reduction of sunshine time induces the accumulation of STH1 and Hd3a transcripts, and the transcription levels of both reach the highest value under the growth environment of short sunshine (10 h illumination, 14h darkness), while Hd1 is expressed in a double induction form, namely, the short sunshine and the long sunshine promote the increase of the expression level (FIG. 6 e).

From the above results, it can be seen that STH1 and Hd3a have similar photoperiod induced expression patterns, which suggests that STH1 acts as an upstream regulator of Hd3a to regulate the change of its expression level, and finally plays a central role in the plant flowering transformation process.

Further screening of STH1 potential interacting proteins by using IP-MS technology means has unexpectedly found that the candidate interacting proteins comprise a key protein Hd1 in the photoperiod control network. The verification of the protein interaction relationship by the method of BiFC and SFLC shows that STH1 and Hd1 have obvious interaction in plant body as expected, and the result of fluorescence co-localization also shows that the two have obvious overlap of fluorescence signals in cell nucleus (FIGS. 6 f-h). The results of these experiments suggest that there is a possibility: the process by which STH1 promotes flowering in rice is mediated by the interaction with Hd1 in the nucleus.

Furthermore, the inventor constructs a double fluorescence report system plasmid, namely STH1 and Hd1 which are expressed by a tobacco mosaic virus 35S promoter in a driving way are used as effector factors, and a DNA fragment with the size of 2kb before the Hd3a translation initiation site is connected in front of a Luciferase report gene as a report gene. The ratio of LUC to REN was finally calculated by transferring the plasmids of different combinations into rice protoplasts, dark culturing for 14-16h and measuring the activity of two luciferases using Promega detection kit. The statistical analysis results show that Hd1 has transcription activity per se, and that the transcription activity is higher in the case where STH1 and Hd1 exist simultaneously. More convincingly, hd1 is transformed in protoplast prepared by separation under different near isogenic system backgrounds, the LUC/REN ratio obtained by calculation has obvious difference, and NIL-STH1 ^HP46 The transcriptional activity of Hd1 in protoplasts was lower, which is a full indication that STH1 has the function of enhancing the transcriptional activity of Hd1 (FIG. 6 i-k).

In conclusion, STH1 serves as an upstream component of the Hd1-Hd3a regulation module, and further promotes the transcriptional expression of Hd3a by playing the role of the Hd1 transcriptional coactivator, so that the rice is induced to finish the transformation of reproductive growth, and the heading period is advanced.

Example 6, STH1 ^HP46 The allele is helpful to maintain high and stable yield of rice, and has application potential in production

Examination of near isogenic line related field agronomic traits showed that there was no significant difference in plant height, tillering number and stalk thickness between NILs at maturity (FIGS. 7 a-c).

However, near isogenic lines are significantly different in ear types, and mainly show differences in ear length and primary branch number. NIL-STH1 ^HP46 The number of primary branches is larger, and the number of primary branches is obviously increased, wherein the number of primary branches of the main spike is increased by 1-2 branches on average, and the number of the primary branches per spike is increased by about 20 seeds. In the aspect of grain size, grain width or thousand grain weight, NIL-STH1 ^HP46 Also the grain size and grain weight of the seeds are increased to different extents. Due to the improvement of the yield components such as the grain number, the grain weight and the like, the NIL-STH1 ^HP46 The final yield increases also reach 41.55% and 21.58% under Shanghai and Santa planting conditions, respectively. Further, cell measurement data also demonstrate STH1 ^HP46 Alleles had a higher stimulation potential, and cell production was also increased by nearly 30% (fig. 8 a-e). Analysis of changes in yield-constituting factors, changes in spike number and grain weight factors may be indicative of NIL-STH1 ^HP46 Important reasons for yield improvement.

Transplanting work in a salt pond (cultivation soil pre-mixed with salt) is carried out by randomly selecting rice seedlings with basically consistent growth states after seedling raising so as to evaluate the salt stress tolerance of NILs in the seedling stage. The survival rate of seedlings was counted after 2 weeks of growth in a salt pond environment, and as a result, NIL-STH1 was found ^HP46 The survival rate of the plants is obviously higher than that of NIL-STH1 ^HJX74 At the same time, the yield of the single plant is improved by approximately 52.44 percent. In addition, high concentration saline (75 mM NaCl) was irrigated against NILs at maturity and plant viability was counted after one month of continued treatment. Similar to the result of salt treatment in seedling stage, NIL-STH1 in mature stage ^HP46 The final survival rate of the plants after being subjected to salt stress is still obviously higher than that of NIL-STH1 ^HJX74 (FIG. 8 g). The statistics fully prove that the NIL-STH1 is in the vegetative growth or reproductive growth stage of rice ^HP46 Has stronger resistance to salt stress all the time.

Based on the above judgment, the present inventors further quantitatively analyzed NILs production after salt stress during the reproductive growth phase. Due to the difference in heading time between NILs, NIL-STH1 was obtained by late sowing ^HJX74 Flowering time of the two is regulated in the mode of (1), 24 NILs plants in early spike development are selected and transplanted into a salt pond, and after the 50 th day of transplantation, the plants are harvested and the measurement of the fruiting rate and the yield of the single plant is carried out. Statistical results show that the seed setting rate of the main rice ears among NILs is obviously different from that of NIL-STH1 ^HP46 With a relatively higher setting rate, which in turn resulted in a further expansion of the yield differences between NILs to 67.14% compared to normal conditions (FIG. 8 h-j), indicating STH1 ^HP46 Is favorable for maintaining high and stable yield of rice under salt stress, is a very excellent natural variation site, and has important application potential.

Example 7, STH1 influences the expression of Hd3a to integrate the balance between salt tolerance and heading stage of rice.

The qPCR assay showed that Hd3a was induced to rapidly down-regulate by salt treatment, and its expression level decreased by more than 100-fold after 10 hours of treatment, and eventually decreased to an almost undetectable level, whereas the transcription level of the transcription factor Hd1 upstream of Hd3a expression was not responsive to salt stress (fig. 9 a).

Gene editing is carried out on Hd1 by using CRISPR/Cas9 technology, and salt-tolerant phenotype identification is carried out on the obtained successful knockout transgenic strain. Because Hd1 has dual functions in the flowering regulation of rice, under the condition of short sunshine, if the Hd1 function is lost, the expression quantity of Hd3a is reduced, so that the flowering is delayed; in contrast, under long-day conditions, hd3a is no longer inhibited by Hd1, so that the expression level of Hd3a is promoted to be up-regulated and flowering is advanced. Consistent with the above reasoning, the Hd1 knockout transgenic lines did show an increase or decrease in Hd3a expression level under long and short sun conditions, respectively, with a concomitant decrease and increase in salt stress tolerance (FIGS. 9 b-d).

Meanwhile, the transgenic lines obtained by directly carrying out targeted knockout on Hd3a show extremely strong resistance to salt stress under any photoperiod growth condition, wherein the difference between mutant and wild salt-resistant phenotypes is particularly obvious under a short-day environment (FIGS. 10 a-b). According to the result of genetic phenotype identification, hd3a can be judged to be a positive regulatory factor for regulating the flowering time of rice and also can be used as a negative regulatory factor for affecting the salt tolerance of the rice.

Since the expression level of Hd3a is inversely related to rice salt tolerance, a tobacco system was used to examine whether the transcriptional activity of Hd1 was affected by salt treatment. Firstly, injecting premixed bacterial solutions in different combination forms into the tobacco epicutaneous cells, after the plasmids are fully expressed for about 40 hours, carrying out exogenous injection of 200mM NaCl to continue expression for 3-5 hours, and finally, detecting the activity of luciferase by using a double-fluorescence reporting system kit. The results of the Luc/Ren data values show that the negative control, i.e. the empty values, did not change either before or after salt treatment, but expressed the Hd1 effector alone, or the co-transformed version of STH1 and Hd1, the Luc/Ren ratio was significantly reduced after salt treatment (fig. 10 c-d), which suggests that salt stress inhibited the transcriptional activation of Hd3a by Hd1, and also matched the dramatic down-regulation of Hd3a transcript levels after salt treatment.

In addition, the present inventors also irrigated NILs at the pre-spike-differentiation growth stage with relatively low concentration of saline (50 mM NaCl) for 2 months of soil culture growth, and as a result found that the flowering time of NILs was significantly delayed, wherein NIL-STH1 ^HP46 Further delayed heading stage, latest heading, and NIL-STH1 ^HJX74 The variation in heading time is more pronounced than this (FIGS. 9 e-f). This suggests, on the one hand, that plants inhibit the transformation from vegetative to reproductive growth by downregulating the expression of Hd3a to combat and alleviate the damage caused by salt stress, and on the other hand, that STH1 and Hd3a have a certain genetic correlation in the balance of salt tolerance and flowering in rice, and that plants further reduce the expression level of Hd3a in plants under salt stress by downregulating the expression level of STH1 itself.

Taken together, the above results support the following conclusions: hd3a is a key downstream switch for balancing the salt tolerance and heading stage of rice, while STH1 is used as a molecular hinge for integrating the two properties, and the balancing relationship between the heading stage and the salt tolerance is regulated and controlled synergistically by influencing the expression of Hd3a under salt stress.

Example 8 STH1 does not participate in SL biosynthesis or Signal transduction processes, while there is functional differentiation between Gene family members

STH1 and strigolactone receptor protein D14 are homologous proteins and belong to the superfamily of alpha/beta sheet hydrolases. Through database search and sequence alignment, a total of 5 α/β sheet domain-containing hydrolases were identified in rice, and all had the typical structure of esterases, i.e., contained one complete form of catalytic triplet required for function, suggesting that all of the 5 members were able to hydrolyze their ligands (fig. 11 a). The results of the inter-species colinear analysis showed that the homologous sequence of STH1 was distributed only in monocots compared to the presence of D14 in seed plants, indicating that STH1 was most likely produced independently during monocot evolution (FIG. 11 b). In combination with gene expression data in the transcriptome public database, the inventors found that the expression patterns of STH1 homologous genes were not identical. In contrast, D14 and D14L had higher expression levels in different tissues, whereas the overall expression levels of the other 3 genes were generally lower, where STH1 and D14L were relatively similar, and were specifically highly expressed in leaves (FIG. 11 c). Notably, analysis of the genetic phenotype of the STH1 homologous gene revealed that the LOC_Os01g41240 and LOC_Os05g08740 knocked-out lines were not significantly different from the wild type in plant type and growth period, except that the knocked-out D14 had the phenotype of multi-tiller dwarf and late flowers (FIG. 11D-e). In conclusion, it is suggested that STH1 is evolved from its homologous gene by gene replication, tandem repeat, and the like, and that although the protein conformation and some key features are similar, some important biochemical properties are retained, there is a possibility of new functionalization between different members of the gene family.

The branch number of NILs under the hydroponic growth condition is counted, and the result of tillering dynamic change shows that NIL-STH1 ^HP46 The number of branches of (C)There are few, but this difference is a periodic fluctuation over time (fig. 12 a-b). The observation by scanning electron microscopy further showed that morphological features of tillering primordia did not differ significantly between NILs, indicating that the tillering number differences of NILs at specific time periods were due to NIL-STH1 ^HP46 The tillering extension time was slow rather than that caused by abnormal formation of tillering primordia (FIG. 12 c). Quantitative statistics of tillering number variation of plants after application of strigolactone analog (rac-GR 24), the results showed that either NIL-STH1 was used ^HJX74 Whether NIL-STH1 ^HP46 The tillering numbers of the treated groups were all significantly reduced, indicating that the absence or absence of STH1 function did not affect the perception of SL signal by the plants (FIGS. 12 d-e). Quantitative mass spectrometry results for endogenous SL levels showed that neither 5-deoxystrigol (5-DS) nor strigol (stragol) were much different in content between NILs (fig. 12 f). The in vitro enzyme activity experiment result also proves that STH1 cannot carry out hydrolysis reaction by taking strigolactone epi-5DS as a substrate, and compared with a negative control, the peak area of the substrate generated after the reaction of the MBP-STH1 fusion protein and the substrate is incubated is not obviously reduced (figure 12 g).

The above research evidence fully demonstrates that STH1 does not function as a receptor protein and participates in the signal transduction pathway of SL, nor in the biosynthesis and metabolic processes of SL, and has a certain degree of functional differentiation with D14.

FIG. 13 is a sequence comparison analysis of STH1 orthologous genes in various monocots. The homology of STH1 of rice to two orthologous genes in maize, brevibacterium, millet, barley and sorghum was 56.86%, 53.18%, 55.85%, 56.21%, 41.18% and 58.33%, respectively. Thus, it can be seen that homologs of STH1 exist in other monocots and are conserved.

The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims. All documents referred to in this disclosure are incorporated by reference herein as if each was individually incorporated by reference.

Sequence listing

<110> molecular plant science Excellent innovation center of China academy of sciences

<120> novel gene STH1 for synergistically regulating and controlling salt tolerance, yield and growth period of plants and application thereof

<130> 220294

<160> 38

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2103

<212> DNA

<213> Oryza sativa L.

<400> 1

actgcaccag cttaagctta ttagctcctg agagtgaagc ccctatagct gatcgaagct 60

atgaagaaga tgtggcgcgc caacgcgaga gtggtggaaa ggagaggtgg tcgcgaggcc 120

gaggagagag ggggagtaat tagcgtggtg cttgcccatg gctatggcgc gagccaggcg 180

gtgtgggaca agctcgtgcc ctccctctcc aagagccaca acctcctcct cttcgactgg 240

gacttcaccg gcgcaggcgc cgggaaggac gacgacgagt acacttacgg caggttcgcc 300

gacgagctca tcgcggtgat ggaggagcgg ggcgtgggcg cgtcgggcgc ggtggtggtg 360

gcgcactcca tgtccgccat ggccgcctgc atcgccgccc aacggagacc cgacctcttc 420

gcccacatct tcctcgtctg cgcctccccg aggttaatta attatcgatc actagatttc 480

attcattttt gctgttcctt aattaattaa gcccctcaag atcaaggatg acatatatag 540

cctaattacc gttattatta ttacagctat ttaatttaat tagttatttt taagcacaat 600

tatattagtt agtcgttgtg taacacgtgt ttggttcatc cagatatcac tcccagtaga 660

tagcagctat gctagctttt gagttttatc attgtcaact ttgtgtgtta attgtcagcg 720

tgtgtacgtc cacgtcagca cactaatcgc gaccatgcag cagcagtcac gttcactgtt 780

gctcacgact cacccaacat ctagaaacat catcaattgc atagcttcat tcacaggaag 840

aaaacaaacg cctccttttc gattattttt ccctagatag cgcttgtcaa atctgaccaa 900

tgaacagcaa ttaagatttg gccggatcga accgggacgt cacaacaaac acttgctgct 960

agctactcta gattagatca aagctagctc tagtccggat gagatgaagc aggatcgatc 1020

aatcgtggcc tcatgttagt tagtccataa cctgtacgtg tggctaaaaa ataggaacac 1080

aagaaattat ccatgtatca atcagtcaag atgtttcaga tccttgcaac taactaatta 1140

atattctatt gccaaaacat gcatgatcaa gcatctaatt gattacttag catttctact 1200

cgattgcatg caagtacctg ttgaaaattc aattgggcat gcagctgcca gataggagta 1260

agctactctg acagtacaag aaactgatga acaaaattaa aactgaacta gagaacgtat 1320

agctgatgaa aacgggtggt ggatcgatcg atcaggtaca taaacttgga ggaggaaggg 1380

tatgtaggag gctttgagga ggcggcgatc cacggcatgc tggcagccat ggagtcggac 1440

ttcgacggct gggtcaggag cttcctcccc aacgccgccg gcgacgcgtc cgccgtggag 1500

cacctcctca agagcttcct cgccatggac ccgaccgtcg cgctcaagct ggccaagatg 1560

atcttcctcg gcgaccagcg ggaggtcctc gacggcgtga agacgccgtg caccatcgtc 1620

caggtgaagg ccgacttcgc ggcgccgccc agcgtggcgg agtacatgca cctcaggatg 1680

aagggcgccg ccacggccgt cgagatcatc ggctccgtcg ggcacttccc ccagctcgtc 1740

gcgccgcagc agctgctgga catactcgcc ggcgtcctgc gcctccggga ggcggcggcg 1800

gaggcggagc acgacgacgc cggcaccgtg gagattgccg gtggcatcga tgttgccata 1860

taggttgtgc atcatgcatg catgcttgta aaaagtctgc acatatatgc gtcagagcta 1920

gctgcatgtg tgctcccttg tatgtatgtt tgtttgtttt gttttcagag catgcattag 1980

ttgtactatt atgttttcct gtgtaaacat atcatagagg ttatgttact tgtacacctg 2040

catgcatggt ttgggtatgc tgatggatgt aagtaccatt ttcaatggga catatacctt 2100

att 2103

<210> 2

<211> 900

<212> DNA

<213> Oryza sativa L.

<400> 2

atgaagaaga tgtggcgcgc caacgcgaga gtggtggaaa ggagaggtgg tcgcgaggcc 60

gaggagagag ggggagtaat tagcgtggtg cttgcccatg gctatggcgc gagccaggcg 120

gtgtgggaca agctcgtgcc ctccctctcc aagagccaca acctcctcct cttcgactgg 180

gacttcaccg gcgcaggcgc cgggaaggac gacgacgagt acacttacgg caggttcgcc 240

gacgagctca tcgcggtgat ggaggagcgg ggcgtgggcg cgtcgggcgc ggtggtggtg 300

gcgcactcca tgtccgccat ggccgcctgc atcgccgccc aacggagacc cgacctcttc 360

gcccacatct tcctcgtctg cgcctccccg aggtacataa acttggagga ggaagggtat 420

gtaggaggct ttgaggaggc ggcgatccac ggcatgctgg cagccatgga gtcggacttc 480

gacggctggg tcaggagctt cctccccaac gccgccggcg acgcgtccgc cgtggagcac 540

ctcctcaaga gcttcctcgc catggacccg accgtcgcgc tcaagctggc caagatgatc 600

ttcctcggcg accagcggga ggtcctcgac ggcgtgaaga cgccgtgcac catcgtccag 660

gtgaaggccg acttcgcggc gccgcccagc gtggcggagt acatgcacct caggatgaag 720

ggcgccgcca cggccgtcga gatcatcggc tccgtcgggc acttccccca gctcgtcgcg 780

ccgcagcagc tgctggacat actcgccggc gtcctgcgcc tccgggaggc ggcggcggag 840

gcggagcacg acgacgccgg caccgtggag attgccggtg gcatcgatgt tgccatatag 900

<210> 3

<211> 299

<212> PRT

<213> Oryza sativa L.

<400> 3

Met Lys Lys Met Trp Arg Ala Asn Ala Arg Val Val Glu Arg Arg Gly

1 5 10 15

Gly Arg Glu Ala Glu Glu Arg Gly Gly Val Ile Ser Val Val Leu Ala

20 25 30

His Gly Tyr Gly Ala Ser Gln Ala Val Trp Asp Lys Leu Val Pro Ser

35 40 45

Leu Ser Lys Ser His Asn Leu Leu Leu Phe Asp Trp Asp Phe Thr Gly

50 55 60

Ala Gly Ala Gly Lys Asp Asp Asp Glu Tyr Thr Tyr Gly Arg Phe Ala

65 70 75 80

Asp Glu Leu Ile Ala Val Met Glu Glu Arg Gly Val Gly Ala Ser Gly

85 90 95

Ala Val Val Val Ala His Ser Met Ser Ala Met Ala Ala Cys Ile Ala

100 105 110

Ala Gln Arg Arg Pro Asp Leu Phe Ala His Ile Phe Leu Val Cys Ala

115 120 125

Ser Pro Arg Tyr Ile Asn Leu Glu Glu Glu Gly Tyr Val Gly Gly Phe

130 135 140

Glu Glu Ala Ala Ile His Gly Met Leu Ala Ala Met Glu Ser Asp Phe

145 150 155 160

Asp Gly Trp Val Arg Ser Phe Leu Pro Asn Ala Ala Gly Asp Ala Ser

165 170 175

Ala Val Glu His Leu Leu Lys Ser Phe Leu Ala Met Asp Pro Thr Val

180 185 190

Ala Leu Lys Leu Ala Lys Met Ile Phe Leu Gly Asp Gln Arg Glu Val

195 200 205

Leu Asp Gly Val Lys Thr Pro Cys Thr Ile Val Gln Val Lys Ala Asp

210 215 220

Phe Ala Ala Pro Pro Ser Val Ala Glu Tyr Met His Leu Arg Met Lys

225 230 235 240

Gly Ala Ala Thr Ala Val Glu Ile Ile Gly Ser Val Gly His Phe Pro

245 250 255

Gln Leu Val Ala Pro Gln Gln Leu Leu Asp Ile Leu Ala Gly Val Leu

260 265 270

Arg Leu Arg Glu Ala Ala Ala Glu Ala Glu His Asp Asp Ala Gly Thr

275 280 285

Val Glu Ile Ala Gly Gly Ile Asp Val Ala Ile

290 295

<210> 4

<211> 1805

<212> DNA

<213> Oryza sativa L.

<400> 4

ccttgagtgg tacacgtcct tattatatat ccaagttttc agctagctag ctctactcat 60

aatgatctag tataatgcat gaggtcatga catattcctt ccgtaaaaaa aaaagttcta 120

gtattcacaa aaatcaaaaa atataattct ttttagataa tttttaaaat tgtaattttt 180

taagaacaaa gggaatgcta gttaatacta tcatgcttaa ttaatctgct tgcttatgga 240

tgtagaggat cccagcatgt aatgaactaa tgatatgatg ctgatatata actccaaatc 300

tatcatttta attgggtagc agagtactgt atttgcctaa atgttatact aatatgcgga 360

cagtgatgac gtgttcagcc cagcaattta acattttagc ccttttacaa acattattct 420

ccaaataaat ccccatacaa ttataactaa aaatagagct caacattagc gtcaggtcca 480

tggcgacgaa gtctaacatc tcagcaccaa ggggcaatac aatagtaagc accagtatat 540

tttgtacttt tctatatagg acagctaata taattagaac gctttctaca gaattcgatc 600

agctaatatc accagaacac ttcaattaga aaagagaatg aaaaacagag gcatgggaag 660

tcgatcatgc cagggagaga ttgcataaga aagccactaa gctactccct ccattttagt 720

aggttataag aagatttgat tttggtcaaa gtcaaactac tctaagtttg actaatttta 780

tagaaaaaaa atagtatatt tttaaccaag acaaatatat tataaaaatt tactcaatta 840

taaatttaat aaaactatgc tagtgttgta aatattacta catttttcta cagaattagt 900

caaacttaga ataccaaagt caaaacgtct tataacctaa aacggagtat agtaaaaaaa 960

tagcacattt atatttatca agtactataa agctgagctt cacatagcta agtggcaaga 1020

ctgatgacgc tgagatgttg ggcttccgcg tcataggtgt gactgctgag gtttagcttt 1080

agtatttatg aggatctatt tatagattaa attttatgaa gggactaaaa tgtaaaaatt 1140

gttgatgttc agtcagagat gatatgttct acggagtatc tacgacctgg caaccaccat 1200

tggtgatgct agctaggcga caaagcagca ctttcccggg agagactggg aatctaggct 1260

atctgtactg tagctcccgg ccacttgcta cctgccttct tgcacctttt ctagcccttc 1320

tgtccttttt tcttgcttgt cagtattcag aaagaaagat gcactataat ttatgcatat 1380

ccaagtcaat catttattgt aaaagtgcgc gtggttttat ttgtaactaa tcgttgcaat 1440

ttcttataga atctttagct ctttcaaaga aaataaaata taagtctgtt tagggaagcc 1500

ttaaactcta aaaatcaact agtgagaatt cgaatatcct gagaaaacca atttaagttt 1560

ggcttgtagt ttatttttta aattctgtaa ccgagcattt ttatatttta ataaggcagc 1620

tatcaattgg ttacttctca aaacagaggg ctatattaat tgctagccct taattttgtt 1680

gacacaaagt cgggctggca tctgctgagt tcctataaat acaggcgtac gtggttgcgc 1740

agtttactgc accagcttaa gcttattagc tcctgagagt gaagccccta tagctgatcg 1800

aagct 1805

<210> 5

<211> 900

<212> DNA

<213> Oryza sativa L.

<400> 5

atgaagaaga tgtggcgcgc caacgcgaga gtggtggaaa ggagaggtgg tcgcgaggcc 60

gaggagagag ggggagtaat tagcgtggtg cttgcccatg gctatggcgc gagctaggcg 120

gtgtgggaca agctcgtgcc ctccctctcc aagagccaca acctcctcct cttcgactgg 180

gacttcaccg gcgccggcgc cgggaaggac gacgacgagt acactttcgg caggttcgcc 240

gacgagctca tcgcggtgat ggaggagcgg ggcgtgggcg cgtcgggcgc ggtggtggtg 300

gcgcactcca tgtccgccat ggccgcctgc atcgccgccc aacggagacc cgacctcttc 360

gcccacatct tcctcgtctg cgcctccccg aggtacataa acttggagga ggaagggtat 420

gtaggaggct ttgaggaggc ggcgatccac ggcatgctgg cagccatgga gtcggacttc 480

gacggctggg tcaggagctt cctccccaac gccgccggcg acgcgtccgc cgtggagcac 540

ctcctcaaga gcttcctcgc catggacccg accgtcgcgc tcaagctggc caagatgatc 600

ttcctcggcg accagcggga ggtcctcgac ggcgtgaaga cgccgtgcac catcgtccag 660

gtgaaggccg acttcgcggc gccgcccagc gtggcggagt acatgcacct caggatgaag 720

ggcgccgcca cggccgtcga gatcatcggc tccgtcgggc acttccccca gctcgtcgcg 780

ccgcagcagc tgctggacat actcgccggc gtcctgcgcc tccgggaggc ggcggcggag 840

gcggagcacg acgacgccgg caccgtggag attgccggtg gcatcgatgt tgccatatag 900

<210> 6

<211> 38

<212> PRT

<213> Oryza sativa L.

<400> 6

Met Lys Lys Met Trp Arg Ala Asn Ala Arg Val Val Glu Arg Arg Gly

1 5 10 15

Gly Arg Glu Ala Glu Glu Arg Gly Gly Val Ile Ser Val Val Leu Ala

20 25 30

His Gly Tyr Gly Ala Ser

35

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

ggcatggcta tggcgcgagc cagg 24

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 8

aaaccctggc tcgcgccata gcca 24

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 9

tgcagcccgg gatccatgaa gaagatgtgg cgcgc 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 10

tccacccatc aattgtatgg caacatcgat gccac 35

<210> 11

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 11

aagcttgcat gcctgcaggt cgactatata tccaagtttt cagc 44

<210> 12

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 12

attcgagctc ggtacccggg tccattctgc agtaccacgc 40

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 13

ggcatccctc atcctagcaa acct 24

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 14

aaacaggttt gctaggatga ggga 24

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 15

gccgtctacc tggaagaaga tag 23

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 16

aaacctatct tcttccaggt aga 23

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 17

tgcagcccgg gatccatgac tacatcagag tcagt 35

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 18

tccacccatc aattggctag atgggaggtt tctgc 35

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 19

ggcaacgtgt tcgaccagga ggt 23

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 20

aaacacctcc tggtcgaaca cgt 23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 21

gccggagcgg ctgcccatgg gcg 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 22

aaaccgccca tgggcagccg ctc 23

<210> 23

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 23

accagtctct ctctcaagct atgaagaaga tgtggcgcgc 40

<210> 24

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 24

tcctgcagct cgaggatcca tatggcaaca tcgatgccac 40

<210> 25

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 25

ttacgaacga tactcgaggt cgacatgaag aagatgtggc gcgc 44

<210> 26

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 26

caccatacta gtggatcccc cgggtatggc aacatcgatg ccac 44

<210> 27

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 27

ttacgaacga tactcgaggt cgacatggcg gaagaggagg aggt 44

<210> 28

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 28

caccatacta gtggatcccc cgggatcatc aatttgccgg ctgt 44

<210> 29

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 29

ttacgaacga tactcgaggt cgacatgaat tataattttg gtgg 44

<210> 30

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 30

caccatacta gtggatcccc cgggaaccat ggaacagtac cat 43

<210> 31

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 31

accacctgtt cctgggtacc atgaagaaga tgtggcgcgc 40

<210> 32

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 32

tgatttttgc ggactctaga tatggcaaca tcgatgccac 40

<210> 33

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 33

tctattgcag caatttaaat atgactacat cagagtcagt 40

<210> 34

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 34

cccttgctca ccatctcgag gctagatggg aggtttctgc 40

<210> 35

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 35

tctattgcag caatttaaat atggccggaa gtggcaggga 40

<210> 36

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 36

cccttgctca ccatctcgag ggggtagacc ctcctgccgc 40

<210> 37

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 37

gcgatatcgt cgacggatcc atgaagaaga tgtggcgcgc 40

<210> 38

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 38

gaattccctg caggtaatta ctatatggca acatcgatgc 40

Claims

1. A method of modulating a salt tolerance, yield traits or flowering phase traits in plants comprising: modulating expression or activity of STH1 in a plant, or modulating an STH1-D3-HAL3 pathway in a plant, or modulating an STH1-Hd1-Hd3 pathway in a plant; the plant is a monocot.

2. The method of claim 1, wherein said modulating a plant salt tolerance, yield traits or flowering phase traits comprises a plant selected from the group consisting of:

(b) Up-regulating the expression or activity of STH1, or up-regulating the interaction of STH1 with Hd1 in STH1-Hd1-Hd3 pathway, so as to promote early flowering phase of plant.

3. The method of claim 1, wherein downregulating the expression or activity of STH1 comprises: knocking out or silencing a gene encoding STH1 in a plant, or inhibiting the activity of STH 1; preferably, it includes: gene editing with a CRISPR system to knock out the coding gene of STH1, introducing the STH1 gene with a loss-of-function mutation into a plant, and subjecting STH1 to a loss-of-function mutation in a plant containing STH1 to specifically interfere with expression of the coding gene of STH 1; preferably, the loss-of-function mutation comprises: the 115 th nucleotide C of the 1 st exon of the STH1 gene is mutated into T, so that the coded protein is terminated in advance;

Or up-regulating expression or activity of STH1 includes: transferring STH1 coding gene or expression construct or vector containing the coding gene into plant; performing functional acquired mutation on STH 1; promoting STH1 expression with an expression-enhanced promoter or a tissue-specific promoter; alternatively, STH1 expression is promoted with an enhancer.

4. The method of claim 2, wherein downregulating D3-mediated interaction of STH1 with HAL3 in the STH1-D3-HAL3 pathway comprises: down-regulating the expression or activity of STH1, thereby down-regulating D3-mediated degradation of HAL3 by STH 1; or (b)

Upregulating the interaction of STH1 with Hd1 in the STH1-Hd1-Hd3 pathway includes: up-regulating expression or activity of STH1, thereby up-regulating transcriptional activation of Hd3a by STH1 and promoting transcriptional levels of Hd3 a.

5. The method of claim 2, wherein the STH1 is a fatty acid hydrolase, and wherein the downregulating STH1 improves salt tolerance by downregulating the function of the STH1 fatty acid hydrolase to allow plants to accumulate fatty acids and protect plasma membrane structures from damage caused by salt stress; or (b)

The D3-mediated interaction of STH1 with HAL3 is D3-mediated ubiquitination degradation of HAL 3; the down-regulation of D3 mediated interaction of STH1 with HAL3 increases HAL3 expression or activity by reducing D3 mediated ubiquitination degradation of HAL3, increasing salt tolerance.

Use of STH1, STH1-D3-HAL3 pathway comprising the same or STH1-Hd 3 pathway or a modulator thereof for modulating salt tolerance, yield traits or flowering phase traits in plants; wherein the regulator comprises an up regulator or a down regulator; the plant is a monocot.

7. The use according to claim 6, wherein the STH1 down-regulator is used to increase salt tolerance, increase yield or delay flowering phase of plants, comprising: agents that knock out or silence STH1, agents that inhibit STH1 activity; preferably, it includes: an interfering molecule that specifically interferes with expression of a coding gene of STH1, a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent for STH1, said reagent subjecting STH1 to a loss-of-function mutation; preferably, the down-regulator includes: a reagent for changing the 115 th nucleotide C of the 1 st exon of the STH1 gene into T, or a DNA formed by annealing primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 is used as a gene editing reagent of sgRNA; or (b)

The STH1 up-regulator is used for promoting the early flowering phase of plants and comprises the following components: exogenous STH1 encoding gene or expression construct or vector containing the encoding gene; preferably, the expression construct comprises an enhanced promoter, a tissue specific promoter or an enhancer; or, an agent that performs a functional point mutation on STH 1; preferably, the agent reverts the mutant to STH1 wild type in plants having the STH1 mutation.

8. The method or use according to any one of claims 1 to 7, wherein the plant is a cereal crop, or the STH1, STH1-D3-HAL3 pathway or STH1-Hd 3 pathway is from a cereal crop; preferably, the cereal crop comprises a grass; more preferably, it comprises: rice (Oryza sativa), corn (Zea mays), millet (Setaria itaica), barley (Hordeum vulgare), wheat (Triticum aestivum), millet (Panicum miliaceum), sorghum (Sorghum bicolor), rye (Secale cereale), oat (Avena sativa l.), brachypodium distachyon (Brachypodium distachyum);

preferably, the plant is a cereal crop and the increasing plant yield comprises: increasing ear length, increasing number of branches, increasing seed setting rate, increasing number of kernels, increasing kernel length, increasing kernel width, and/or increasing thousand kernel weight of kernels.

9. The method or use according to any one of claims 1 to 7, wherein the amino acid sequence of the STH1 polypeptide is selected from the group consisting of: (i) a polypeptide having the amino acid sequence shown in SEQ ID NO. 3; (ii) The polypeptide which is formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown as SEQ ID NO. 3, has the regulatory character function and is derived from (i); (iii) The homology of the amino acid sequence with the amino acid sequence shown in SEQ ID NO. 3 is more than or equal to 80 percent, and the polypeptide has the function of regulating and controlling the characters; (iv) An active fragment of a polypeptide of the amino acid sequence shown in SEQ ID NO. 3; or, (v) a polypeptide comprising a tag sequence or an enzyme cleavage site sequence added to the N-terminus or the C-terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO. 3, or a signal peptide sequence added to the N-terminus thereof.

10. Use of a plant STH1, an STH1-D3-HAL3 pathway comprising the same or an STH1-Hd 3 pathway as a molecular marker for identifying salt tolerance, yield traits or flowering phase traits in plants, or as a molecular marker for directed screening of plants; the plant is a monocot.

11. A method of selecting or identifying a plant, the method comprising: identifying expression or sequence characteristics of STH1 in the test plant, or identifying interaction conditions of the STH1-D3-HAL3 pathway or the STH1-Hd1-Hd3 pathway in the plant; if the STH1 of the test plant is expressed low or not, or the test plant is a plant with high salt tolerance, high yield or delayed flowering phase; if STH1 is highly expressed, the test plant is a plant with low salt tolerance, low yield or early flowering phase.

12. A method of screening for a substance that modulates a salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising:

(1) Adding a candidate substance to a system expressing STH 1;

(2) Detecting the system, observing the expression or activity of STH1 therein, and if the expression or activity is reduced, indicating that the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants; if its expression or activity is increased, it is indicated that the candidate substance is a substance useful for promoting the early flowering phase of plants.

13. A method of screening for a substance that modulates a salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising:

(1) Adding a candidate substance to a system expressing the STH1-D3-HAL3 pathway;

(2) Observing the interaction of D3-mediated STH1 and HAL3 in the STH1-D3-HAL3 pathway, if the degradation of HAL3 by D3-mediated STH1 is reduced, the candidate substance is a substance which can be used for improving the salt tolerance of plants, improving the yield of plants or delaying the flowering phase of plants.

14. A method of screening for a substance that modulates a salt tolerance, yield traits or flowering traits in plants, which are monocotyledonous plants, comprising:

(1) Adding a candidate substance to a system expressing the STH1-Hd1-Hd3 pathway;

(2) Observing the interaction of STH1 and Hd1 in the STH1-Hd1-Hd3 pathway, if the transcription stimulus of STH1 to Hd3a, preferably the transcription level of Hd3a, is enhanced, the candidate substance is a substance which can be used for promoting the early flowering phase of plants.

15. A down-regulator of STH1 for a substance that increases salt tolerance, increases yield or delays flowering phase of a plant, which is:

reagents for mutating nucleotide C at position 115 of exon 1 of STH1 gene into T; or (b)

The CRISPR gene editing reagent for down-regulating STH1 gene is preferably the gene editing reagent with the primer shown in SEQ ID No. 7 and SEQ ID No. 8 annealed to form DNA as sgRNA.

16. A construct or plant cell, tissue or organ comprising the exogenous substance of claim 15 for increasing salt tolerance, increasing yield or delaying flowering phase of a plant.