Function and application research of transcription factor HSFA4a

Abstract

The invention provides functions and application research of a transcription factor HSFA4a, and discloses a novel gene HSFA4a closely related to regulation and control of phytoalexin, volatile monoterpene and sesquiterpene of plants. HSFA4a is not only subjected to constitutive regulation and control of PGF, but also induced by infection of plant diseases and insect pests. The invention provides a new action target for improving the insect resistance of plants.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and botany, and more specifically, the present invention relates to the function and application research of transcription factor HSFA4a. Background Art

[0002] During the long process of evolution, plants have formed complex and efficient regulatory networks to adapt to the environment and resist stress, among which transcription factor regulation plays a key role.

[0003] Upland cotton (Gossypium hirsutum) is an allotetraploid cotton species belonging to the Malvaceae family (Malvaceae) genus (Gossypium), and is one of the most important economic crops widely planted in the world. Its cotton fiber is an important raw material for the textile industry, and cotton seeds are rich in protein, oil and carbohydrates and are the raw material for cottonseed oil. Although genetically modified technology has been used to control lepidopteran pests, cotton still faces the threat of infection by a variety of pests and diseases and pathogens. A large amount of terpenoid phytoalexins accumulate in cotton glands, including gossypol, hemigosssypolene (HGQ), heliocides, and volatile monoterpenes and sesquiterpenes such as α-pinene, β-pinene, β-myrcene, ocimene, β-caryophyllene, and α-humulene (Yang et al., 2013). They can protect plants, reduce the infection of pathogens and the feeding of herbivorous insects, and play an important role in the insect and disease resistance of cotton (Tian et al., 2016). The biosynthesis of cottonpol starts from farnesyl pyrophosphate (FPP), a common precursor of sesquiterpene synthesis. FPP is synthesized by the mevalonate pathway (MVA), and then catalyzed and modified by downstream cadinene synthase and a series of oxidases, dehydrogenases, cyclases, etc. (Huang et al., 2020; Lin et al., 2023; Tian et al., 2018). The biosynthesis of volatile monoterpenes originates from the MEP pathway in the plastid, and the corresponding monoterpenes are generated by terpene synthase (Bouvier et al., 2005; Chen et al., 2003; Tholl et al., 2005). The development of cotton glands is usually accompanied by the accumulation of cottonpol and volatile terpenes in the glands (Xu et al., 2004).Cotton bHLH transcription factor GoPGF has been reported to be able to regulate the development of cotton glands. When GoPGF is inhibited, not only the gland development is affected, but the content of sesquiterpenoid compounds such as gossypol is also significantly reduced (Ma et al., 2016); NAC transcription factors CGF1 and CGF2 (Janga et al., 2019) and MYB transcription factor CGP1 (Gao et al., 2019) have been reported to play an important role in the gland formation of cotton plants and affect the accumulation of terpene compounds such as gossypol; GhERF105 encodes an ERF protein containing an AP2 domain, which regulates the gland development of upland cotton (Wu et al., 2021); At present, there are few reports on research on directly regulating the biosynthesis of cotton terpene phytoalexins without affecting gland development.

[0004] Currently, there are few reports on the direct regulation of the biosynthesis of cotton terpenoid phytoalexins without affecting gland development. Summary of the invention

[0005] The purpose of the present invention is to provide a function and application study of transcription factor HSFA4a.

[0006] In the first aspect of the present invention, there is provided an application of HSFA4a or its up-regulator, which is used for: regulating (improving / increasing / restoring in low-level plants) phytoalexin levels of cotton plants (Gossypium Linn) or regulating (improving / increasing / restoring in low-level plants) volatile monoterpenes and sesquiterpenes levels; preparing a preparation for regulating phytoalexin levels of cotton plants or regulating (promoting) volatile monoterpenes and sesquiterpenes levels; or, as a molecular marker for identifying phytoalexin levels, volatile monoterpenes and sesquiterpenes levels or insect resistance of cotton plants.

[0007] In one or more embodiments, the up-regulator includes (but is not limited to): a polynucleotide or expression construct encoding HSFA4a, a gene editing agent that promotes the expression of the HSFA4a gene, an up-regulator that promotes the driving ability of the HSFA4a gene promoter, an up-regulator that interacts with the HSFA4a protein to increase its expression or activity, a down-regulator of HSFA4a gene-specific microRNA, a chemical up-regulator of HSFA4a, or a combination thereof.

[0008] In one or more embodiments, the phytoalexin includes: gossypol, hemiscotton, noctuidin (noctuidin 1-4) (etc.).

[0009] In one or more embodiments, the volatile monoterpenes and sesquiterpenes include: α-pinene, β-pinene, β-myrcene, trans-β-ocimene, β-caryophyllene, α-humulene (etc.).

[0010] In one or more embodiments, the Gossypium plant includes Gossypium hirsutum.

[0011] In one or more embodiments, the cotton plant includes a plant that under-expresses HSFA4a; for example, the plant is a plant that under-expresses HSFA4a naturally or due to a disease state.

[0012] In one or more embodiments, the regulation of phytoalexin levels in cotton plants is a direct regulation without affecting gland development.

[0013] In one or more embodiments, the modulation of volatile monoterpenes and sesquiterpenes is a direct modulation without affecting glandular development.

[0014] In one or more embodiments, the insect is a herbivorous insect.

[0015] In one or more embodiments, the herbivorous insect is a Lepidoptera insect.

[0016] In one or more embodiments, the Lepidoptera insects include: Noctuidae insects such as cotton bollworm, Pyralidae insects such as Asian corn borer of the genus Sesamia, Glechomae insects such as pink bollworm, Pyralidae insects such as navel orange borer, Plutella xylostella insects such as Plutella xylostella, Papilionidae insects such as Swallowtail butterfly, etc.

[0017] In one or more embodiments, high levels of phytoalexins are equivalent to enhanced phytoalexin synthesis.

[0018] In one or more embodiments, the HSFA4a or its up-regulator is also used to regulate the upstream MVA pathway enzyme genes of cottonypol synthesis, cottonypol pathway synthase genes and terpene synthase GhTPS1 / 3 / 6 (maintain expression or increase expression).

[0019] In another aspect of the present invention, a method for regulating (improving / increasing / restoring in low-level plants) phytoalexin levels or volatile monoterpenes and sesquiterpenes in cotton plants is provided, comprising: upregulating the expression or activity of HSFA4a in cotton plants; preferably, the cotton plants are plants with insufficient expression or activity of HSFA4a.

[0020] In one or more embodiments, the upregulation of HSFA4a expression or activity includes: administering an upregulator of HSFA4a, thereby increasing its expression or activity; preferably, the upregulator includes (but is not limited to): a polynucleotide or expression construct encoding HSFA4a, an upregulator that promotes the driving ability of the HSFA4a gene promoter, an upregulator that interacts with the HSFA4a protein to increase its expression or activity, a downregulator of HSFA4a gene-specific microRNA, a chemical upregulator of HSFA4a, or a combination thereof.

[0021] In one or more embodiments, the HSFA4a is selected from the following group:

[0022] (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2;

[0023] (b) a HSFA4a derivative having the polypeptide function of (a) or (b), formed by substituting, deleting or adding one or more (e.g., 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 1 to 2) amino acid residues in the amino acid sequence shown in (a), or an active fragment thereof;

[0024] (c) a HSFA4a derivative having a sequence homology ≥80% (≥82%, ≥85%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98% or ≥99%) to the amino acid sequence of SEQ ID NO: 2, or an active fragment thereof; or

[0025] (d) A polypeptide formed by adding a tag sequence or an enzyme cleavage site sequence to the N- or C-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO: 2, or by adding a signal peptide sequence to its N-terminus.

[0026] In one or more embodiments, the expression of HSFA4a is regulated by upstream PGF; preferably, the PGF binds to the G-box in the promoter region of HSFA4a.

[0027] In one or more embodiments, the HSFA4a is induced by pest and disease infection and its expression is increased.

[0028] In one or more embodiments, the HSFA4a includes homologs thereof, which are proteins or encoding genes from different species (species other than upland cotton) that have sequence homology with SEQ ID NO: 2 or a polynucleotide encoding the same from upland cotton.

[0029] In one or more embodiments, the expression construct (expression vector) includes: a cotton plant expression vector.

[0030] In one or more embodiments, the expression construct (expression vector) includes: a non-viral vector and a viral vector.

[0031] In one or more embodiments, the up-regulated expression includes overexpression.

[0032] In one or more embodiments, the upregulation refers to a significant upregulation, such as an upregulation of 20%, 40%, 60%, 80%, 90% or more.

[0033] In one or more embodiments, the cotton plant is a cotton plant expressing HSFA4a or a homolog thereof.

[0034] In another aspect of the present invention, a cotton plant cell, tissue or organ is provided, wherein the cotton plant is a cotton plant expressing HSFA4a, and contains an exogenous HSFA4a up-regulator, wherein the up-regulator includes: a polynucleotide or expression construct (including an expression vector) encoding HSFA4a, an up-regulator that promotes the driving ability of the HSFA4a gene promoter, an up-regulator that interacts with the HSFA4a protein to increase its expression or activity, a down-regulator of HSFA4a gene-specific microRNA, a chemical up-regulator of HSFA4a, or a combination thereof.

[0035] In one or more embodiments, the cotton plant cell, tissue or organ does not directly generate a living cotton plant, or is not used as a cotton plant propagation material.

[0036] In one or more embodiments, the high expression or high activity refers to a statistically significant increase in expression or activity compared to the average value of expression or activity of the same type or species of cotton, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or more.

[0037] In another aspect of the present invention, a method for identifying phytoalexins, volatile monoterpenes or sesquiterpenes, or insect resistance of cotton plants is provided, the method comprising: identifying the expression or activity of HSFA4a in the tested cotton plants; if the HSFA4a expression or activity of the tested cotton plants is high (e.g., higher than the average value of the cotton plants of this species) or normal, then the phytoalexin, volatile monoterpenes or sesquiterpenes level is normal, or the plants have insect resistance; if the HSFA4a expression or activity of the tested cotton plants is low (e.g., lower than the average value of the cotton plants of this species), then the phytoalexin, volatile monoterpenes or sesquiterpenes level is low, or the plants have poor insect resistance; preferably, the plants are identified as cotton plants that need to be regulated with HSFA4a or its up-regulator to restore the expression or activity of HSFA4a to normal.

[0038] In another aspect of the present invention, a method for screening potential substances with high levels of phytoalexins, volatile monoterpenes or sesquiterpenes, or insect resistance is provided, the method comprising: (1) treating an expression system expressing HSFA4a with a candidate substance; and (2) detecting the expression or activity of HSFA4a in the system; if the candidate substance statistically increases (e.g., increases by 5%, 10%, 15%, 20%, 30% or more, preferably increases by 50% or more; more preferably increases by more than 80%) the expression or activity of HSFA4a, it indicates that the candidate substance is a potential substance for regulating the level of phytoalexins, volatile monoterpenes or sesquiterpenes in cotton plants to maintain them at normal levels or promoting their increase.

[0039] In one or more embodiments, the screening method further comprises setting a control group to clearly distinguish the expression or activity of HSFA4a.

[0040] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules targeting HSFA4a protein or its encoding gene, or its upstream or downstream proteins or genes (such as upregulators, interfering molecules (for example, they can interfere with the upstream gene that "inhibits HSFA4a expression"), nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, etc.

[0041] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 , VIGS verified the function of GoHSFA4a. (A) Expression pattern of GoHSFA4a in different types of cells in true leaves of upland cotton; (B) GUS staining of pGoHSFA4a::GUS transgenic cotton leaves; (C) There was no change in the glands of cotton plant leaves and stems after GoHSFA4a silencing, TRV:GoHSFA4a represents the leaves of plants after the GoHSFA4a gene was suppressed, TRV:00 is the control group injected with TRV2 empty vector, the same below; (D) The relative content of terpenoid compounds in the gossypol pathway, such as gossypol, hemi-gossypolone and noctuidin 1-4, in the leaves of plants with suppressed GoHSFA4a, with the content of compounds in the leaves of TRV:00 plants as 1; (E) The relative content of volatile terpenoid compounds in the leaves of plants with suppressed GoHSFA4a, with the content of compounds in the leaves of TRV:00 plants as 1; (F) The relative expression of VIGS targeted genes, with the gene expression in the leaves of TRV:00 plants as 1.

[0043] Figure 2, Gene editing verifies the function of GoHSFA4a. (A) CRISPR editing type of GoHSFA4a; (B) Relative contents of gossypol, hemi-gossypolone and noctuidin in sepals of GoHSFA4a gene-edited plants, with the content of compounds in sepals of wild-type cotton plants at the same period as 1; (C) Relative contents of volatile monoterpenes and sesquiterpenes in leaves of GoHSFA4a-inhibited plants, with the content of compounds in leaves of wild-type cotton plants as 1.

[0044] Figure 3 , GoHSFA4a regulates the expression of MVA pathway genes and cottonyphenol pathway biosynthetic enzyme genes. (A) Relative expression of MVA pathway genes in leaves of plants inhibited by GoHSFA4a, with the gene expression level in leaves of TRV:00 plants as unit 1, the same below; (B) Expression of cottonyphenol biosynthetic pathway genes in leaves of plants inhibited by GoHSFA4a; (C) Relative expression of sesquiterpene synthase GhTPS1, monoterpene synthase GhTPS3 and GhTPS6 in leaves of plants inhibited by GoHSFA4a; (D) Dual-LUC analysis showed that GoHSFA4a activated some MVA pathway genes. The ratio of LUC to REN was used for normalization, and the LUC value of the empty vector was defined as unit 1, the same below; (E) Dual-LUC analysis showed that GoHSFA4a activated some cottonyphenol biosynthetic pathway genes.

[0045] Figure 4 , GoHSFA4a expression is regulated by GoPGF and induced by pests and diseases. (A) The expression of GoHSFA4a in leaves of plants inhibited by GoPGF was downregulated, with the gene expression level in leaves of TRV:00 plants as 1; (B) Dual-LUC analysis showed that GoPGF could directly activate GoHSFA4a expression; (C) Schematic diagram of the 2,000bp promoter region upstream of GoHSFA4a, with the black vertical line representing the E-box and the blue vertical line representing the G-box; (D-E) MST and EMSA respectively proved that GoHSFA4a could bind to the G-box in the promoter region of GoHSFA4a; (F) Yeast single hybrid showed that GoPGF interacted with the G-box in the promoter region of GoHSFA4a. SD / -LTH, minimal medium lacking leucine, tryptophan, and histidine; SD / -LT, minimal medium lacking leucine and tryptophan; 3AT, 3-aminotriazole; (G) The expression level of GoHSFA4a in the true leaves of cotton fed by cotton bollworms increased, with the expression level of the gene in the unfed cotton leaves as 1; (H) The expression level of GoHSFA4a in the true leaves of cotton infected by Verticillium dahliae increased, with the expression level of the gene in the uninfected cotton leaves as 1. DETAILED DESCRIPTION

[0046] Based on extensive research and screening, the inventors have discovered a new gene HSFA4a that is closely related to regulating plant phytoalexins, volatile monoterpenes and sesquiterpenes (and thus can regulate insect resistance). The present invention provides a new target for improving the insect resistance of plants.

[0047] the term

[0048] As used herein, the insects are herbivorous insects; preferably include Noctuidae insects such as cotton bollworm, Pyralidae insects such as Asian corn borer of the genus Sesamia, Glechomae insects such as pink bollworm, Pyralidae insects such as navel orange borer, Leptospiridae insects such as Plutella xylostella, Papilionidae insects such as Swallowtail butterfly, etc.

[0049] As used herein, the "plant" includes plants expressing HSFA4a or containing HSFA4a and the signaling pathways involved. According to the knowledge in the art, the plant expressing HSFA4a has the mechanism of action claimed by the present invention, and can achieve the technical effects claimed by the present invention. In some preferred embodiments, the plant is an economic crop, preferably a cotton crop. More specifically, for example, upland cotton.

[0050] About "control plant", it is a routine part of experimental design to select suitable control plant, and can comprise corresponding wild-type plant or corresponding transgenic plant without target gene.Control plant is generally the same plant species or even the variety that is the same as plant to be evaluated or belongs to the same class.Control plant can also be the individual that lost transgenic plant because of separation.Control plant as used herein refers not only to complete plant, but also to plant part, comprises blade, gland, seed or their part.

[0051] As used herein, the terms "increase", "promote", "improve" or "enhance" are interchangeable and shall mean in the applied sense at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15%, 20%, 25%, 30% or 40%, more preferably 50%, 60%, 80%, 90% or more regulation compared to control plants as defined herein.

[0052] HSFA4a gene and its encoded protein

[0053] In the present invention, unless otherwise specified, the HSFA4a protein includes its homologues (homologous proteins). The HSFA4a is a polypeptide (protein) having an amino acid sequence as shown in SEQ ID NO: 2. The present invention also includes sequence variants having the same function as the HSFA4a protein.

[0054] The variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. Any protein with high homology to the HSFA4a protein (such as 70% or higher homology to the polypeptide sequence shown in SEQ ID NO: 2; preferably 80% or higher homology; more preferably 90% or higher homology, such as 95%, 98% or 99% homology) and having the same functions as the HSFA4a protein is also included in the present invention.

[0055] In the present invention, the "HSFA4a protein" also includes their homologs. It should be understood that, although the HSFA4a protein obtained from a specific species is preferably studied in the present invention, other polypeptides or genes obtained from other species, especially Malvaceae plants, which are highly homologous to the HSFA4a protein (e.g., having a sequence identity of more than 70%, more particularly 80%, 85%, 90%, 95%, or even more than 98%) are also within the scope of the present invention.

[0056] Polypeptides derived from species other than upland cotton that have a high homology to the polypeptide sequence shown in SEQ ID NO: 2, or that play the same or similar functions in the same or similar signal pathways are also included in the present invention.

[0057] The present invention also provides an isolated protein, which is a fragment of the HSFA4a protein or is formed by adding other proteins or tags at both ends.

[0058] The present invention also relates to a polynucleotide sequence encoding the HSFA4a protein of the present invention or a sequence variant thereof. The polynucleotide may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO:2 or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a polypeptide having a sequence of SEQ ID NO:2 but differs from the gene sequence shown in SEQ ID NO:1. The present invention also relates to variants (variants) of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.

[0059] The present invention also relates to a vector comprising the polynucleotide, and a host cell produced by genetic engineering using the vector or the polypeptide encoding nucleic acid.

[0060] In the present invention, the polynucleotide sequence encoding the polypeptide of the present invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element. Preferably, the expression vector can also selectively add resistance elements, screening (selection) elements or reporter gene elements, such as Bar, GUS.

[0061] When the polynucleotide is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription.

[0062] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. Plant transformation can be carried out using methods such as Agrobacterium transformation or gene gun transformation.

[0063] Changes in phytoalexins, volatile monoterpenes and sesquiterpenes

[0064] Based on extensive research screening and systematic analysis, the present inventors identified the HSFA4a gene, which is capable of regulating plant phytoalexins, volatile monoterpenes and sesquiterpenes.

[0065] In a specific embodiment, by analyzing the single-cell transcriptome data of glandular cotton leaves, the inventors screened out that the HSF transcription factor GoHSFA4a of upland cotton is specifically expressed in cotton glands. The pGoHSFA4a::GUS transgenic cotton leaves were histochemically stained using X-Gluc dye, and GUS spots only appeared in cotton glands, indicating that it may regulate the biosynthesis of phytoalexins such as gossypol. Using virus-induced transgenic silencing technology (VIGS) to silence GoHSFA4a, there was no difference between the cotton glands of GoHSFA4a-inhibited plants and wild-type cotton, but the contents of gossypol, hemi-gossypolone and noctuidin, as well as volatile monoterpenes and sesquiterpenes in its true leaves were significantly reduced, indicating that GoHSFA4a regulates the biosynthesis of terpenoid phytoalexins such as gossypol in cotton but does not affect gland development. Correspondingly, the expression levels of MVA pathway enzyme genes, cottonpol pathway synthase genes and terpene synthase GhTPS1 / 3 / 6 upstream of cottonpol synthesis were reduced to varying degrees, and GoHSFA4a was able to activate the expression of some MVA pathway enzyme genes and cottonpol pathway synthase genes. Using CRISPR-Cas9 gene editing technology to specifically knock out GoHSFA4a, the content of cottonpol and other phytoalexins in gene-edited cotton sepals also decreased significantly, further proving that GoHSFA4a can regulate the biosynthesis of cottonpol and its related sesquiterpenoid aldehydes, volatile monoterpenes and sesquiterpenes in glands.

[0066] GoPGF is the main transcription factor reported to control cotton gland development and belongs to the bHLH protein family. The expression of GoHSFA4a in leaves of GoPGF-silenced plants decreased significantly. There is a bHLH transcription factor-binding G-box (CACGTG) and multiple E-boxes (usually CANNTG) in the promoter region of GoHSFA4a. GoPGF can directly bind to the G-box on the GoHSFA4a promoter and activate its expression, indicating that GoHSFA4a is a downstream gene of GoPGF and is regulated by GoPGF. In addition, the expression of GoHSFA4a is also induced by infection with Verticillium dahliae and feeding by cotton bollworm, which is directly related to cotton resistance to diseases and pests.

[0067] It is well known to those skilled in the art that the phytoalexin content of crops is positively correlated with their disease and insect resistance. Sesquiterpenoid compounds such as gossypol are important phytoalexins for cotton, which can help plants resist infection by pathogens and feeding by herbivorous insects, and play an important role in cotton's insect and disease resistance.

[0068] The experimental results of the present invention show that GoHSFA4a regulates the expression of MVA pathway genes and cottonypol pathway synthase genes, and the relative contents of cottonypol pathway terpenoid phytoalexin compounds such as cottonypol, hemi-gossypolone and noctuidin 1-4 and volatile monoterpenes and sesquiterpenes in the leaves of plants in which GoHSFA4a is inhibited are significantly reduced. Therefore, it can be understood that GoHSFA4a is an essential regulatory factor for the biosynthesis of cotton sesquiterpenoid phytoalexins such as cotton gossypol, and promoting the biosynthesis of the latter can improve the disease and insect resistance of cotton.

[0069] In summary, the inventors' research has found the transcription factor GoHSFA4a that regulates cotton terpenoid phytoalexins. Silencing GoHSFA4a does not affect cotton gland development, but the accumulation of gossypol, hemi-gossypolone, noctuidin, volatile monoterpenes and sesquiterpenes in cotton glands are reduced. GoHSFA4a is both constitutively regulated by GoPGF and induced by pests and diseases. It provides a new target for breeding insect-resistant and disease-resistant cotton varieties.

[0070] Based on the above new discovery of the inventors, a use of HSFA4a protein or its regulatory molecule is provided, which is used for: regulating (improving / increasing / restoring in low-level plants) phytoalexin levels of cotton plants or regulating (improving / increasing / restoring in low-level plants) volatile monoterpenes and sesquiterpenes levels; preparing preparations for regulating phytoalexin levels of cotton plants or regulating (promoting) volatile monoterpenes and sesquiterpenes levels; or as molecular markers for identifying phytoalexin levels, volatile monoterpenes and sesquiterpenes levels or insect resistance of cotton plants.

[0071] After knowing the function of HSFA4a, various methods known to those skilled in the art can be used to overexpress HSFA4a, thereby increasing the phytoalexins, volatile monoterpenes and sesquiterpenes of plants. For example, an expression unit (such as an expression vector or virus, etc.) carrying the HSFA4a gene can be delivered to the target site through methods known to those skilled in the art, and active HSFA4a can be expressed.

[0072] As a preferred method, a method for preparing a transgenic plant is provided, comprising: (1) transferring an exogenous HSFA4a encoding nucleic acid into a plant tissue, organ or tissue to obtain a plant tissue, organ or seed transformed with an HSFA4a encoding polynucleotide; and (2) regenerating the plant tissue, organ or seed into which the exogenous encoding nucleic acid is transferred obtained in step (1) into a plant plant. In addition, the present invention can also obtain plant cells that do not reproduce into plants.

[0073] Other methods for increasing the expression of HSFA4a gene or its homologous gene are well known in the art. For example, the expression of HSFA4a gene or its homologous gene can be enhanced by driving with a strong promoter. Alternatively, the expression of HSFA4a can be enhanced by an enhancer (such as the first intron of rice waxy gene, the first intron of Actin gene, etc.). Suitable strong promoters include but are not limited to: 35s promoter, Ubi promoter of rice and corn, etc.

[0074] In the present invention, the up-regulator of the HSFA4a polypeptide or its encoding gene includes promoters, agonists, and activators. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of polypeptide activity or "up-regulation" and "promotion" of polypeptide expression. Any substance that can increase the activity of HSFA4a, increase the stability of HSFA4a, up-regulate the expression of HSFA4a, and increase the effective action time of HSFA4a can be used in the present invention as a substance useful for up-regulating HSFA4a. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. The HSFA4a or its up-regulator is particularly suitable for application to a type of plant whose HSFA4a expression is lower than the average value of the plant or whose HSFA4a is not expressed; thus, the use of the HSFA4a protein or its up-regulator can restore the plant to a wild-type phenotype or a better phenotype.

[0075] The present invention also includes plants obtained by any of the aforementioned methods, wherein the plants include: transgenic plants into which the nucleic acid encoding the polypeptide is transferred.

[0076] Plant breeding screening or targeted screening of regulatory molecules

[0077] Based on the inventors' new discovery, the present invention provides a molecular marker suitable for identifying phytoalexin levels, volatile monoterpenes and sesquiterpenes levels or insect resistance, namely the HSFA4a gene. The present invention also relates to specific molecular markers designed for the HSFA4a gene, and identification strategies. Early determination of the phytoalexin, volatile monoterpenes and sesquiterpenes energy of a plant, or its insect resistance.

[0078] Therefore, the present invention provides a method for specifically identifying phytoalexins, volatile monoterpenes and sesquiterpenes of plants, comprising: identifying the expression of HSFA4a of the test plant, and if HSFA4a of the test plant is highly expressed, it is a plant having phytoalexins, volatile monoterpenes and sesquiterpenes.

[0079] The present invention also provides a method for directed selection or identification of plants with regulated agronomic traits, comprising: identifying the expression or activity of HSFA4a protein in a test plant; if the expression or activity of HSFA4a protein in the test plant is higher than the average value of the expression or activity of HSFA4a protein in the plant of the same type (control plant), the plant is a plant with normal or relatively high phytoalexins, volatile monoterpenes and sesquiterpenes, which also means that the plant has good insect resistance; or, if the expression or activity of HSFA4a protein in the test plant is lower than the average value of the expression or activity of HSFA4a protein in the plant of the same type (control plant), the plant is a plant with abnormal or relatively low phytoalexins, volatile monoterpenes and sesquiterpenes, which also means that the plant has low insect resistance.

[0080] According to the new discovery of the present invention, those skilled in the art can use any of the various techniques known in the art or being developed to analyze nucleic acid sequences, and these techniques can all be included in the present invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe method, hybridization, restriction enzyme analysis, allele polymorphism analysis (such as melting curve method) to identify nucleic acid sequences, etc.

[0081] The identification method of the present invention only requires PCR reaction and / or agarose gel electrophoresis, and by determining the length of the corresponding PCR product, the phenotype of the sample to be tested can be accurately and quickly determined, the cost is low, it is suitable for large-scale identification, and the amount of sample required is very small. If necessary, those skilled in the art can design primers for identifying the molecular markers.

[0082] The method of obtaining DNA of the sample to be tested is a technique well known to those skilled in the art, for example, the conventional phenol / chloroform / isoamyl alcohol method can be adopted, or some commercially available DNA extraction kits can be adopted, and such kits are well known to those skilled in the art. The polymerase chain reaction (PCR) technique is a technique well known to those skilled in the art, and its basic principle is a method of enzymatically synthesizing specific DNA fragments in vitro. The method of the present invention can be carried out using conventional PCR techniques.

[0083] The invention has good application prospects in molecular design breeding and crop variety improvement using genetic engineering technology.

[0084] After knowing the function of HSFA4a gene, it can be used as a molecular marker for targeted screening of plants. Based on this new discovery, it is also possible to screen for substances or potential substances that can regulate phytoalexins, volatile monoterpenes and sesquiterpenes by regulating this mechanism.

[0085] The present invention provides a method for screening potential substances with high levels of phytoalexins, volatile monoterpenes or sesquiterpenes, or insect resistance, the method comprising: (1) treating an expression system with a candidate substance, the system expressing HSFA4a; (2) detecting the expression or activity of HSFA4a in the system; if the candidate substance statistically increases (e.g., increases by 5%, 10%, 15%, 20%, 30% or more, preferably increases by 50% or more; more preferably increases by more than 80%) the expression or activity of HSFA4a, it indicates that the candidate substance is a potential substance for regulating the level of phytoalexins, volatile monoterpenes or sesquiterpenes in cotton plants to maintain them at normal levels or promoting their increase.

[0086] The present invention provides a method for screening substances (potential substances) that promote plant trait improvement, wherein the trait improvement includes: enhanced insect resistance; the method comprises: (1) adding a candidate substance to a system expressing HSFA4a protein; (2) detecting the system, observing the expression or activity of the HSFA4a protein therein, and if the expression or activity is reduced, it indicates that the candidate substance is a substance that reduces the insect resistance of the plant.

[0087] Methods for screening substances acting on a protein or gene or a specific region thereof as a target are well known to those skilled in the art, and all of these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0088] The interaction between proteins and the strength of the interaction can be detected by various techniques well known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation experiment, yeast two-hybrid system or immunoprecipitation technology.

[0089] After large-scale screening, a class of substances can be obtained that specifically act on HSFA4a protein or its encoding gene, and have a regulatory effect on the levels of phytoalexins, volatile monoterpenes and sesquiterpenes (which is beneficial for regulating insect resistance).

[0090] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0091] Materials and methods

[0092] GoHSFA4a Gene ID

[0093] In upland cotton (Gossypium hirsutum), the GoHSFA4a gene IDs are: Gh_A05G3255 in the A subgenome and Gh_D04G0354 in the D subgenome.

[0094] GoHSFA4a (Gh_D04G0354) nucleotide sequence cDNA (SEQ ID NO: 1):

[0095] ATGGAAGGATCGCAGGGAAGTTGTAATGGGCCGCCGCCTTTTCTTACAAAGACATACGAGATGGTGGATGATCCAGTGACTGACTCGTTGGTGTCTTGGAGTGAAACTGGTTACAGTTTCGTTGTATGGAACCCCCCAGATTTCTCAAGAGATTTGCTCCCTAGATATTTCAAGCACAATAACTTCTCAAGCTTTGTCCGTCAGCTCAACACCTATGGGTTCAGAAAGATAGATCCTGATCAATGGGAGTTTGCAAACGAGGAGTTCATAAGAGGGCAGAAACATCTTTTAAAGAACATTTATCGACGCAAGCCAATCCATAGCCATTCCTTGAACCGGCAAGGAAGCTCTAGTGTGCCATTGACTGAGAAAGAGAAGAAGGAATTTGAGCAAGTGATCAAGATGTTGAGTGACGATAAGAACCGGCTTCAGTTACAGTTGCAGGGTCATCAAAAAGAGAACGAGGAATATCGGTGTCAAGTAAGGCTGTTATCGGAGCGTTTTCGGAACATGGAAGATCGACAACGACGAGTGATGGTCTCATTGGCTAGGATTATAGACAAACCCCATGTTCTGTCCCAATTTCATGGCAAAAAGAGAAAGTTATTGAACTGTAACGACTTCAATGATGAATGCAACATACAAGATCTTCACAGTTTGGCTTCTCTGAATGCAAACATGGGATTGGACTTGAAACAGATTGAAGAATTGGAATCATCTATAAGATGTTGGGAAACATTGTTTCTCGAAATCGGAGAAACTATAGGTGAAGAAGTGCGCGATTTCGGGACATCCTTACGGCCTTCTCCGGTTGTTGTTACCGAAATACAGACATCTTCAGGGGACTATGACATGGATGGTGAACTTTACTCACCTTCATCACACCATTGCTCCCCTTATTCAACCGATATTAACTCATCACCTGAGCTGGTTGCCCCTGCTTATCATCCTATCCATACACCATCATTTCACCATGTCGTAAATCTCAACCCAAAGCCTCCTGGAATTGGCCTAAACTCCCAACATGCTGGTCCACTGGAGACCGATCAGGCCTCAAAGAACCAGGCAGAAGCAGTGGCGACCGGTTATACCGTGCCAGGTAGTGTAAACGATGGCTTCTGGGAAAGGTTTCTAACCGAAGTTCCGGATACATCATCGTATGCACAAGAGATCCAGTCAGTTTAG

[0096] GoHSFA4a (Gh_D04G0354) encoded protein sequence (SEQ ID NO: 2):

[0097] MEGSQGSCNGPPPFLTKTYEMVDDPVTDSLVSWSETGYSFVVWNPPDFSRDLLPRYFKHNNFSSFVRQLNTYGFRKIDPDQWEFANEEFIRGQKHLLK NIYRRKPIHSHSLNRQGSSSVPLTEKEKKEFEQVIKMLSDDKNRLQLQLQGHQKENEEYRCQVRLLSERFRNMEDRQRRVMVSLARIIDKPHVLSQFHG KKRKLLNCNDFNDECNIQDLHSLASLNANMGLDLKQIEELESSIRCWETLFLEIGETIGEEVRDFGTSLRPSPVVVTEIQTSSGDYDMDGELYSPSSHHCSPYSTDINSSPELVAPAYHPIHTPSFHHVVNLNPKPPGIGLNSQHAGPLETDQASKNQAEAVATGYTVPGSVNDGFWERFLTEVPDTSSYAQEIQSV

[0098] The primers used in the present invention are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102] Example 1: GoHSFA4a regulates the biosynthesis of gossypol terpenoid phytoalexins

[0103] a: Single-cell data analysis suggests that GoHSFA4a regulates the biosynthesis of sesquiterpenoids such as cottonpol

[0104] In order to analyze the biosynthesis and transcriptional regulation of cottonpol, the inventors conducted single-cell transcriptome sequencing on glandular cotton leaves. Using 10x Genomics single-cell transcriptome sequencing technology, a total of more than 15,000 single-cell transcriptomes were obtained. The sequencing results were visualized and cell clustered, and six major cell types in cotton leaves were identified, including mesophyll cells, vascular cells, epidermal cells, proliferating cells, guard cells, and glandular secretory cells. More than 300 genes were specifically expressed in glandular secretory cells, including cottonpol pathway synthase genes and some MVA pathway enzyme genes. Analysis of gland-specifically expressed genes revealed that an HSF-type transcription factor GoHSFA4a was specifically expressed in cotton glands ( Figure 1 A), suggesting that it is involved in regulating the biosynthesis of cotton phytoalexins.

[0105] b: GoHSFA4a is expressed in cotton glands

[0106] Using pGreenⅡ0800-LUC as a plant expression vector (backbone vector), the inventors constructed an expression vector for GUS driven by the GoHSFA4a promoter (the 2000 bp segment before the start codon of GoHSFA4a), carried out stable transformation of cotton, obtained transgenic plants, and after identifying positive plants, used X-Gluc dye to perform histochemical staining on pGoHSFA4a::GUS positive transgenic cotton leaves, and found that GUS spots only appeared in cotton glands ( Figure 1 B), demonstrating that GoHSFA4a is specifically expressed in cotton glands.

[0107] c: Construction of VIGS vector of GoHSFA4a gene

[0108] The gene-specific fragment (positions 786-1128 in SEQ ID NO: 1) was amplified by PCR, the forward primer introduced a BamHI restriction site, the reverse primer introduced an XbaI restriction site and loaded into the pTRV2 vector. The GoHSFA4a gene was connected to the pTRV2 vector by homologous recombination, and the expression was driven by the CaMV 35S promoter. The correctly sequenced vector was transferred into Agrobacterium GV3101 and cultured inverted at 28°C for 2 to 3 days.

[0109] d: Transfection of GoHSFA4a gene VIGS vector into cotton cotyledons

[0110] The plasmid with the correct sequencing was transferred into Agrobacterium GV3101 competent cells, and the Agrobacterium containing the pTRV1 vector was streaked, and the pTRV2 Agrobacterium (SU) containing the PDS gene was streaked. Single clones were picked for colony PCR verification, and the Agrobacterium clones with positive colony PCR were picked into 2mL liquid LB medium for selection of resistance, and cultured at 28°C overnight to an OD value of 2.5. At the same time, pTRV1 and SU Agrobacterium were shaken. The Agrobacterium in the small shake was transferred to 50mL of liquid LB medium for selection of resistance at a ratio of 1:100, and continued to be shaken and cultured at 28°C overnight. At room temperature, centrifuged at 8,000g for 10min, resuspended with an equal volume of resuspension solution (10mMMES, 10mM MgCl2, 150μM acetosyringone, pH=5.8), and the OD value was adjusted to about 1. It was placed at room temperature for at least 3h to activate the bacteria.

[0111] The Agrobacterium resuspension with different plasmids was mixed with the Agrobacterium resuspension with pTRV1 vector at a ratio of 1:1 (V / V) and injected from the back of the cotton cotyledon with a 1mL syringe for transfection. Observe whether the positive control turns yellow 2 to 3 weeks after injection, take the second true leaf, freeze it in liquid nitrogen, and store it at -70℃. The RNA of the second true leaf of the GoHSFA4a-inhibited plant was analyzed by qRT-PCR, and it was found that the expression level of GoHSFA4a was downregulated in the leaves of the VIGS plant ( Figure 1 F), but glandular development is not affected ( Figure 1 C).

[0112] e: HPLC detection of GoHSFA4a inhibiting the content of gossypol and other sesquiterpenoids in plant leaves

[0113] The plant material was ground into powder in liquid nitrogen, 1 mL of extract solution (acetonitrile: water: phosphoric acid = 80:20:0.1) was added to every 100 mg of powder, vortexed for 2 min, ultrasonicated for 15 min, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm PTFE filter.

[0114] High performance liquid chromatography (HPLC) was used to quantitatively analyze the cotton pol isomers in the sample using an Agilent C18 analytical column (150×2.1 mm, 2.7 μm). The mobile phase components were 0.1% formic acid water (A) and acetonitrile (B), the flow rate was 1 mL / min, the gradient was 16 min, and the detection was at 272 nm: 0-3 minutes, 20-70% B; 3-5 minutes, 70-80% B; 5-7 minutes, 80-84% B; 7-8 minutes, 84-100% B; 8-14 minutes, 100% B; 14-15 minutes, 100-20% B; 15-16 minutes, 20% B.

[0115] HPLC detection revealed that in the strains where VIGS inhibited the expression of GoHSFA4a, the contents of major cotton phytoalexins such as gossypol, hemi-gossypol and noctuidin were significantly decreased. Figure 1 As shown in D. This suggests that GoHSFA4a can regulate the biosynthesis of sesquiterpenoid aldehydes such as cotton gossypol.

[0116] f: GC-MS detection of GoHSFA4a inhibiting the content of volatile monoterpenes and sesquiterpenes in plant leaves

[0117] Similarly, the plant material was ground into powder in liquid nitrogen, and 1 mL of n-hexane was added to every 100 mg of powder, vortexed for 2 min, sonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The content of volatile monoterpenes and sesquiterpenes in the samples was detected by gas chromatography (GC-MS). The gas chromatograph used an Agilent 6890 Series GC System coupled to an Agilent 5973 Network Mass Selective Detector, and the detection program was as follows: initial temperature 50 °C (5 min hold), increased to 180 °C at 10 °C / min, increased to 240 °C at 20 °C / min, and increased to 280 °C at 30 °C / min (5 min hold). The carrier gas (He) flow rate was 1 mL / min. Split injection (split ratio 5:2) was performed. Mass spectral data between m / z 50 and 550 were recorded.

[0118] The test results showed that GoHSFA4a inhibited the significant decrease in the content of volatile monoterpenes and sesquiterpenes in the leaves of plants ( Figure 1 E), indicating that GoHSFA4a not only regulates the biosynthesis of terpenoid phytoalexins such as gossypol in cotton, but also affects the accumulation of volatile monoterpenes and sesquiterpenes.

[0119] Analysis of glandular cotton single-cell transcriptome sequencing data and cotton VIGS (virus-induced gene silencing) experiments demonstrated that GoHSFA4a regulates the biosynthesis of gossypol terpenoid phytoalexins.

[0120] Example 2: Knockout of GoHSFA4a gene using CIRSPR-Cas9 technology

[0121] In order to improve the editing efficiency, the inventors designed two gRNAs for GoHSFA4a gene knockout, the sequences are:

[0122] gRNA1:TAGCCATTCCTTGAACCGGC (SEQ ID NO: 57);

[0123] gRNA2: GCGATTTCGGGACATCCTTA (SEQ ID NO: 58).

[0124] The two sgRNA sequences were cloned into the vector pRGEB32-GhU6.7-NPT2 and transformed into Agrobacterium LBA4404, which was used to infect cotton explants to obtain stably transformed transgenic seedlings. After obtaining the T0 generation transgenic lines, cotton DNA was extracted and sequenced to verify the gene editing target.

[0125] Cotton DNA extraction: Grind cotton leaves, add 800μL CTAB extraction solution, vortex to mix, and place in a 65℃ oven for 30min; add 800μL chloroform, mix by inversion, and centrifuge at 4℃, 12000rpm for 10min; take the supernatant to a new 1.5mL centrifuge tube, add an equal volume of chloroform, mix by inversion, and centrifuge again, repeat once; take the supernatant to a new centrifuge tube, add 1 / 2 volume of isopropanol, mix by inversion, let stand on ice for 1h, centrifuge at 4℃, 12000rpm for 10min, remove the supernatant, add the same volume of 75% ethanol as isopropanol to wash the precipitate, centrifuge at 4℃, 12000rpm for 10min, repeat once; dry the precipitate at 37℃ for 10min, dissolve it with 60μL water, then add RNaseⅠ to react at 37℃ for 30min to remove RNA, and then place the solution in a 65℃ oven to inactivate RNaseⅠ.

[0126] CTAB extraction reagent: 2% CTAB, 0.1M Tris, 20mM EDTA, 1M NaCl (add 2% β-mercaptoethanol when used)

[0127] The gene editing site fragment was amplified by PCR and loaded into the full-strand gold pEASY-Blunt Zero vector. The target fragment was sequenced and two cotton lines with edited gene target sites were found ( Figure 2 A), the contents of terpenoid phytoalexins in sepals of gene-edited cotton and wild-type cotton of the same period were detected. Through liquid phase analysis, the inventors found that compared with wild-type cotton, the contents of gossypol, hemi-gossypolone and noctuidin, volatile monoterpenes and sesquiterpenes in sepals of gene-edited cotton were significantly reduced, and the reduction was more significant compared with the VIGS results ( Figure 2 B, C).

[0128] Example 3: GoHSFA4a regulates the expression of MVA pathway enzyme genes, cottonyphenol pathway synthase genes, and terpene synthase genes

[0129] a: q-PCR detection of GoHSFA4a inhibiting the expression of MVA pathway enzyme genes, cottonpol pathway enzyme genes and terpene synthase genes in plant leaves

[0130] 100 mg of cotton leaves were quickly ground into powder in liquid nitrogen, and the total RNA of the leaves was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANFEN, DP441), and reverse transcribed into cDNA for use as a q-PCR template. qPCR reaction system: 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 10 μL SYBR, 7 μL ddH2O, and a total volume of 20 μL. GhHIS3 (Gh_D03G0370) of upland cotton was used as an internal standard, and the gene expression level was expressed as (gene / HIS3 gene). The expression level changes of each gene in the leaves of GoHSFA4a-inhibited plants were calculated, with the expression level of GoHSFA4a in cotton leaves injected with the TRV2 empty vector as 1.

[0131] Figure 3 A, B and C show the relative expression levels of MVA pathway enzyme genes, cottonyphenol synthesis pathway enzyme genes and three terpene synthases GhTPS1 / 3 / 6, respectively. Compared with the control group, the expression levels of MVA pathway enzyme genes, cottonyphenol pathway enzyme genes and terpene synthase genes in the leaves of GoHSFA4a-inhibited plants were all decreased.

[0132] b: PCR amplification of target gene GoHSFA4a and construction of tobacco expression vector

[0133] High-fidelity enzyme HSDNA Polymerase was used to amplify the full-length cDNA fragment (1185 bp) of GoHSFA4a. The PCR reaction conditions were: 95°C, denaturation for 5 minutes; 95°C, denaturation for 30 seconds; 57°C, renaturation for 30 seconds; 72°C, extension for 2 minutes; repeat the second to fourth steps for 40 cycles; 72°C, 10 minutes; 4°C insulation. The GoHSFA4a PCR product was connected to the tobacco expression vector by homologous recombination. The 2,000 bp DNA fragment upstream of the start codon ATG of the cottonpol pathway enzyme gene and the MVA pathway enzyme gene was the promoter region and was connected to the pGreenII 0800-Luc vector plant expression vector.

[0134] c: transient transformation of tobacco

[0135] The expression vector plasmid of GoHSFA4a with correct sequencing, the promoter vector plasmid of cottonpol pathway enzyme gene and MVA pathway enzyme gene were respectively transferred into GV3101-p19 Agrobacterium strain and inverted at 28℃ for 2-3 days. The grown single clones were picked for colony PCR. The clones identified as positive by PCR were added to 2mL of resistant liquid LB and shaken slightly, and cultured at 28℃ overnight. At room temperature, centrifuged at 5,000g for 10min, resuspended with resuspension solution (10mM MES, 10mM MgCl2, 150μM acetosyringone, pH=5.8), and the OD value was adjusted to about 0.6. The cells were placed at room temperature for at least 3h to activate the bacteria. The Agrobacterium resuspension solution with the expression vector of GoHSFA4a was mixed with the Agrobacterium resuspension solution with the promoter vector plasmid of cottonpol pathway enzyme gene and MVA pathway enzyme gene at a ratio of 4:1, and injected from the back of tobacco true leaves with a 1mL syringe for transfection.

[0136] d: Dual-LUC analysis

[0137] Use a punch to remove the injection site of the tobacco leaf and place it in a 2 mL centrifuge tube, add steel balls and crush it with a grinder, add 200 μL of lysis solution to the powder, vortex for 1 min, and centrifuge at 4°C, 12,000 rpm for 1 min; take a 1.5 mL centrifuge tube and add 40 μL of LARII, then add 8 μL of the supernatant from the previous step and mix well, and measure the LUC enzyme activity with a Promega GloMax 20 / 20 luminescence detector; add 40 μL Stop&Glo reagent, mix well and measure the REN enzyme activity; calculate the ratio of LUC enzyme activity to REN enzyme activity, the LUC value of the empty vector is defined as unit 1, and draw a graph for comparison.

[0138] The analysis results showed that GoHSFA4a could activate some MVA pathway enzyme genes ( Figure 3 D) and cottonpol biosynthesis pathway enzyme genes ( Figure 3 E).

[0139] Example 4: GoPGF regulates GoHSFA4a gene expression

[0140] GoPGF has been reported to regulate the development of cotton glands. In this example, whether it is associated with GoHSFA4a was studied.

[0141] The GenBank accession number of GoPGF is KP072743.

[0142] The GoPGF constructed above was connected to the TRV2 vector for VIGS. After silencing GoPGF, total RNA was extracted from cotton leaves and used as a q-PCR template after reverse transcription to detect the expression of GoHSFA4a in GoPGF-VIGS cotton leaves (the detailed steps are as described above), and it was found that it was significantly downregulated ( Figure 4 A).

[0143] The constructed vectors were co-transformed into tobacco and Dual-LUC analysis was performed (the detailed steps were as described above).

[0144] The results showed that GoPGF could directly activate GoHSFA4a ( Figure 4 B). GoPGF belongs to the bHLH protein family and is reported to usually bind to the G-box (CACGTG) or E-box (usually CANNTG) in the promoter region of the target gene (Hao et al., 2021). Analysis of the promoter region of GoHSFA4a revealed that it contains multiple E-boxes and one G-box ( Figure 4 C), suggesting that GoPGF may activate GoHSFA4a expression by binding to the G-box or E-box in the promoter region of GoHSFA4a.

[0145] Example 5: GoPGF binds to the G-box in the promoter region of GoHSFA4a

[0146] a: PCR amplification of GoPGF to construct a prokaryotic expression vector

[0147] High-fidelity enzyme HSDNA Polymerase was used to amplify the full-length cDNA fragment (1428 bp) of GoPGF. The PCR reaction conditions were: 95°C, denaturation for 5 minutes; 95°C, denaturation for 30 seconds; 57°C, renaturation for 30 seconds; 72°C, extension for 1 minute and 30 seconds; repeat the second to fourth steps for 40 cycles; 72°C, 10 minutes; 4°C insulation. The pET-32a vector was double-digested with BamHI / SacI, and then connected to the pET-32a prokaryotic expression vector by homologous recombination.

[0148] b: Prokaryotic expression

[0149] The GoHSFA4a prokaryotic expression vector plasmid with correct sequencing was transferred into the BL21 strain, spread on the Amp-resistant LB medium, and inverted and cultured at 37°C overnight. Amp-resistant LB was added to a 96-well plate, and a single clone was picked for colony PCR. The clones identified as positive by PCR were added to 2mL of resistant liquid LB (100μg / mL Amp) and cultured at 37°C overnight. Transferred to 500mL LB at a ratio of 1:100, cultured at 37°C until OD600 was in the range of 0.6-0.8, added IPTG to a final concentration of 0.5mM / L, transferred to 18°C ​​to induce protein expression, and cultured for about 20h.

[0150] c: Purification of prokaryotic expressed proteins

[0151] Take the bacterial liquid and resuspend it in 15mL Lysis buffer by centrifugation. The precipitate is broken by one shot Cell DisrupterSystem with a breaking pressure of 20-25kpsi. Centrifuge at 4℃, 13,500g, and take the supernatant for protein purification. Use a crusher (pre-cool the corresponding accessories in advance) to break at low temperature, and transfer the supernatant to an 8mL tube. Centrifuge at 4℃, 13,500g for 20min. At the same time, install a 2mL Ni-NAT agrose column and equilibrate the Ni column with 20mL Lysis buffer. Add the supernatant from the previous step to the equilibrated 1mL Ni-NAT agrose column and flow through. Use 20mL Wash buffer to wash the Ni-NATagrose. Use 3mL elution buffer to elute the GoPGF protein from the nickel column, and use the Bradford method to quantify the protein concentration with bovine serum albumin as a reference.

[0152] Buffer:

[0153] Lysis buffer: 50 mM Tris-HCl, 300 mM NaCl

[0154] Wash buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole

[0155] Elution buffer: 50 mM Tris-HCl, 300 mM NaCl, 200 mM Imidazole

[0156] d: MST

[0157] The purified GoPGF protein was labeled using Protein Labeling Kit RED-NHS2nd Generation (MO-LO11, Nanotempertech), and the ligand was a DNA fragment containing the G-box in the promoter region of GoHSFA4a. The operation was performed according to the instructions of MSTMonolith NT.115 to detect the interaction between the protein and the ligand. The fitting analysis of the detection results showed that the GoPGF protein bound to the DNA fragment containing the G-box in the promoter region of GoHSFA4a ( Figure 4 D).

[0158] e: EMSA

[0159] 5×EMSA buffer: 25% glycerol, Tris 100mM, BSA 0.2mg / mL, pH = 7.9. Filter through a 0.45μm filter membrane and store in aliquots at -20℃.

[0160] 5×binding buffer (160μL): 5×EMSA buffer (141.6μL), 0.5M DTT (1.6μL), 1MMgCl2 (8μL), H2O (8.8μL)

[0161] Non-denaturing protein gel (10 μL for making 1.5 mm thick non-denaturing gel): 10×TBE 0.5 mL, 50% glycerol 0.5 mL, 40% acrylamide (29:1) 1.125 mL, 30% APS 23.35 μL, TEMED 3.5 μL, H2O 7.875 mL

[0162] Binding system (20 μL): 5×EMSA binding buffer 4 μL, salmon sperm DNA (1 μg / μL) 2 μL, DNA-cy5 (0.4 nmol, 30 bp 3'cy5 labeled probe containing the G-box in the promoter region of GoHSFA4a), GoPGF protein (3 ng), add water to 20 μL, mix well, and incubate at room temperature in the dark for 30 min.

[0163] After the non-denaturing gel solidified, pre-cooled 1×TBE was added to the electrophoresis tank, and the reaction sample was added after pre-running at 180V for 60 minutes without adding samples, and electrophoresis was performed at 100V for 60 minutes. After the electrophoresis, the non-denaturing gel and the gel plate were developed using the FLA-9000 instrument. The development results showed that the G-box DNA fragment in the promoter region of GoHSFA4a labeled with cy5 was bound to GoPGF, that is, the corresponding lane had two bands, the bound probe and the free probe; when the competitive probe without cy5 label was added to the binding system, the bound probe band weakened, and weakened with the increase of the competitive probe concentration; GoPGF could not bind to the DNA fragment without G-box ( Figure 4 E).

[0164] The above results prove that GoPGF can directly bind to the G-box in the promoter region of GoHSFA4a.

[0165] Example 6: Yeast one-hybrid verification of the interaction between GoPGF and the G-box in the promoter region of GoHSFA4a

[0166] a: Construction of yeast expression vector

[0167] High-fidelity enzyme HSDNA Polymerase was used to amplify the full-length cDNA fragment (1428 bp) of GoPGF. The PCR reaction conditions were: 95°C, denaturation for 5 minutes; 95°C, denaturation for 30 seconds; 57°C, renaturation for 30 seconds; 72°C, extension for 1 minute and 30 seconds; repeat the second to fourth steps for 40 cycles; 72°C, 10 minutes; 4°C insulation. The homologous recombination method was used to construct the yeast expression vector pGADT7 Rec2.

[0168] A 6× DNA fragment containing the G-box of the GoHSFA4a promoter region was synthesized and loaded into pHis2.1.

[0169] b: Yeast transformation

[0170] The constructed GoPGF-pGADT7 Rec2 vector was co-transformed with the pHis2.1 vector (GoHSFA4a-pHis2.1) containing the DNA fragment of the G-box of the GoHSFA4a promoter region and the pHis2.1 empty vector into the yeast AH109 strain: 2-5 μg of each plasmid, salmon sperm DNA (10 μg), 500 μL of PEG / LiAc, and 100 μL of AH109 strain. After mixing, the mixture was incubated at 30°C for 30 min, 42°C for 15 min, and centrifuged at 5,000 rpm for 2 min. The precipitate was washed with 400 μL of sterile water, centrifuged again to remove the supernatant, and the cells were resuspended with 50 μL of sterile water, spread on SD-LT medium, and inverted at 28°C for 48-72 hours.

[0171] Pick up the clones grown in SD-LT medium, add them to SD-LT liquid medium, and culture them overnight at 28°C. The next day, dilute the yeast liquid transformed with different plasmids to OD600≈1, spread them on SD-LT and SD-LTH medium respectively, and culture them upside down at 28°C for 3 days.

[0172] The results showed that on SD-LT medium, yeast clones transformed with GoPGF-pGADT7 Rec2 and GoHSFA4a-pHis2.1 vectors and yeast in the control group grew normally, while on SD-LTH medium, only yeast clones transformed with GoPGF-pGADT7 Rec2 and GoHSFA4a-pHis2.1 vectors grew normally ( Figure 4 F), demonstrating that GoPGF interacts with the G-box in the promoter region of GoHSFA4a.

[0173] Example 7: GoHSFA4a expression is induced by pests and diseases

[0174] Infection with Verticillium dahliae: Divide cotton seedlings that have just grown for about 9 days (cotyledons are stretched) into two groups, clean the roots of cotton, and transfer them to hydroponic solution for five days; at the same time, pick the Verticillium dahliae strain, add it to Cha's medium, and place it in a 25°C shaker for 5 days. Immerse the roots of cotton seedlings in the five-day-old Verticillium dahliae bacterial solution, infect for 3 hours, and re-transfer to hydroponic solution for growth. Cotton begins to show symptoms such as leaf wilt after about 20 days. Take the true leaves of cotton that have not been infected and infected to extract total RNA, reverse transcribe it into cDNA as a q-PCR template, and detect the expression of GoHSFA4a.

[0175] Feeding by cotton bollworm larvae: Take cotton seedlings (two true leaves) that are about three weeks old, place the same number of fourth-instar larvae of cotton bollworm on each true leaf of cotton, remove the true leaves after the cotton bollworms have fed for 24 hours, grind them in liquid nitrogen, extract total RNA, reverse transcribe it into cDNA and use it as a q-PCR template to detect the expression level of GoHSFA4a.

[0176] q-PCR results showed that the expression level of GoHSFA4a increased significantly in cotton leaves infected by Verticillium dahliae and fed by cotton bollworm ( Figure 4 G, H), indicating that GoHSFA4a expression is induced by infection with Verticillium dahliae and feeding by cotton bollworm, and is directly related to cotton's resistance to diseases and insect pests.

[0177] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of HSFA4a or its upregulator for: Regulating the phytoalexin level of cotton plants or regulating the level of volatile monoterpenes and sesquiterpenes; Preparation of a formulation for regulating the level of phytoalexins in cotton plants or regulating the level of volatile monoterpenes and sesquiterpenes; or, As a molecular marker for identifying phytoalexin levels, volatile monoterpenes and sesquiterpenes or insect resistance in cotton plants.

2. The use according to claim 1, characterized in that The up-regulator includes: a polynucleotide or expression construct encoding HSFA4a, a gene editing agent that promotes HSFA4a gene expression, an up-regulator that promotes the driving ability of the HSFA4a gene promoter, an up-regulator that interacts with the HSFA4a protein to increase its expression or activity, a down-regulator of HSFA4a gene-specific microRNA, a chemical up-regulator of HSFA4a, or a combination thereof.

3. The use according to claim 1, characterized in that The plant protection agents include: gossypol, hemi-gossypol, noctuidin; or The volatile monoterpenes and sesquiterpenes include: α-pinene, β-pinene, β-myrcene, trans-β-ocimene, β-caryophyllene, and α-humulene.

4. A method for regulating the level of phytoalexin or volatile monoterpenes and sesquiterpenes in cotton plants, comprising: Upregulating the expression or activity of HSFA4a in a cotton plant; preferably, the cotton plant is a plant with insufficient expression or activity of HSFA4a.

5. The method according to claim 4, characterized in that The upregulation of the expression or activity of HSFA4a includes: administering an upregulator of HSFA4a, thereby increasing its expression or activity; preferably, the upregulator includes: a polynucleotide or expression construct encoding HSFA4a, an upregulator that promotes the driving ability of the HSFA4a gene promoter, an upregulator that interacts with the HSFA4a protein to increase its expression or activity, a downregulator of HSFA4a gene-specific microRNA, a chemical upregulator of HSFA4a, or a combination thereof.

6. The use according to claim 1 or the method according to claim 4, characterized in that: The HSFA4a is selected from the following group: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2; (b) HSFA4a derivatives having the polypeptide functions of (a) or (b), or active fragments thereof, formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in (a); (c) a HSFA4a derivative having a sequence identity of ≥ 80% with the amino acid sequence shown in SEQ ID NO: 2, or an active fragment thereof; or (d) A polypeptide formed by adding a tag sequence or an enzyme cleavage site sequence to the N- or C-terminus of the polypeptide with the amino acid sequence shown in SEQ ID NO: 2, or by adding a signal peptide sequence to its N-terminus.

7. The use according to claim 1 or the method according to claim 4, characterized in that: The expression of HSFA4a is regulated by upstream PGF; preferably, the PGF binds to the G-box in the promoter region of HSFA4a; and / or The HSFA4a is induced by pests and diseases to increase its expression.

8. A cotton plant cell, tissue or organ, wherein the cotton plant is a cotton plant expressing HSFA4a, wherein the cell, tissue or organ contains an exogenous HSFA4a up-regulator, wherein the up-regulator comprises: A polynucleotide or expression construct encoding HSFA4a, an upregulator that promotes the driving ability of the HSFA4a gene promoter, an upregulator that interacts with the HSFA4a protein to increase its expression or activity, a downregulator of HSFA4a gene-specific microRNA, a chemical upregulator of HSFA4a, or a combination thereof.

9. A method for identifying phytoalexins, volatile monoterpenes or sesquiterpenes, or insect resistance in cotton plants, the method comprising: Identifying and testing the expression or activity of HSFA4a in cotton plants; If the expression or activity of HSFA4a in the test cotton plant is high or normal, then the level of phytoalexin, volatile monoterpenes or sesquiterpenes is normal, or the plant has insect resistance; If the HSFA4a expression or activity of the tested cotton plant is low, then its phytoalexin, volatile monoterpene or sesquiterpene level is low, or its insect resistance is poor; preferably, it is identified as a cotton plant that needs to be regulated by HSFA4a or its up-regulator to restore the HSFA4a expression or activity to normal.

10. A method for screening a potential substance having a high level of phytoalexin, volatile monoterpene or sesquiterpene, or having insect resistance, the method comprising: (1) treating an expression system expressing HSFA4a with a candidate substance; and (2) detecting the expression or activity of HSFA4a in the system; if the candidate substance statistically increases the expression or activity of HSFA4a, it indicates that the candidate substance is a potential substance for regulating the level of phytoalexins, volatile monoterpenes or sesquiterpenes in cotton plants to maintain the level at a normal level or promoting the increase thereof.

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