Application of GhA01EP1 gene in promoting early maturity and high yield of plants

Abstract

The present invention belongs to the field of genetic engineering, and specifically relates to the application of the GhA01EP1 gene in promoting plant early maturity and high yield, including application in Arabidopsis and cotton. The present invention verifies that the effect of GhA01EP1 on the growth and development of Arabidopsis involves multiple metabolic pathways. It can be concluded that (1) GhA01EP1 promotes plant growth and early flowering by affecting the expression of genes related to circadian rhythm regulation, photoperiodic flowering, and hormone metabolism; (2) GhA01EP1 may increase the number of branches and thus increase yield by regulating genes related to branch development. The above metabolic pathways cooperate with each other to form a metabolic network, thereby synergistically improving the early maturity and high yield of plants, which provides ideas for improving comprehensive plant traits.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to an application of a GhA01EP1 gene in promoting early maturity and high yield of cotton. Background Art

[0002] Secretory proteins, as crucial vehicles for the exchange of information, substances, and energy between plants and the outside world, play a crucial role in regulating physiological activities. Research has shown that secretory proteins have important biological functions in extracellular and intracellular signaling, cellular responses to environmental stimuli, cell wall structure formation, pathogen defense, and intercellular interactions.

[0003] Currently, the study of the functions of secretory proteins has received widespread attention. For example, one of the most abundant structural proteins in plant cell walls, hydroxyproline-rich glycoproteins (HRGPs), plays a key role in cell wall-mediated signal cascades, stress tolerance and differentiation processes. AGPs are highly glycosylated members of the HRGPs superfamily and are present in many species. It is reported that AGPs have a fine spatiotemporal regulatory expression in plant organs (such as leaves, stems, roots, flowers and seeds). Moreover, AGPs are involved in a variety of developmental processes, such as seedling growth, cell division, mechanical damage, programmed cell death, and abiotic stress response mechanisms. In addition to cell wall structural proteins, the functions of other secretory proteins have also been analyzed by scholars.

[0004] Currently, little research exists on EP1-like proteins. Our previous studies have demonstrated that EP1-like proteins (GhA01EP1) in cotton are associated with drought resistance. Lei et al. found that PoEP1 in peony is associated with flowering time and can regulate early flowering. Both GhA01EP1 and PoEP1 encode epidermal glycoproteins containing two domains: B-lectin and Plant PAN / APPLE-like. B-lectin is a mannose-specific lectin widely distributed in higher plants that can specifically recognize a variety of carbohydrates and mediate diverse biological processes. Mannose-binding proteins (MBPs) are a class of plant lectins containing B-lectin domains. Through sugar binding, they participate in a variety of physiological processes, including cell-cell interactions and host-pathogen interactions. Plant PAN / APPLE-like domains are found in plant S-receptor protein kinases and secreted glycoproteins. This domain mediates protein-protein or protein-carbohydrate interactions to achieve diverse biological functions. Furthermore, S receptor protein kinases and S-site glycoproteins are involved in the self-compatibility response of Brassica sporophytes, which may be one of the many molecular mechanisms by which hermaphroditic flowering plants avoid self-fertilization. Engelen et al. cloned the EP1 gene in carrot, which is homologous to the S-locus glycoprotein (SLG) in Brassica, and speculated that carrot EP1 may simply participate in certain regulatory pathways that ultimately control water flow.

[0005] That is, current research mainly focuses on the role of the GhA01EP1 gene in cotton drought resistance, while there are few reports on the effects of this gene on cotton early maturity and high yield. Summary of the Invention

[0006] To address the problems existing in the above-mentioned prior art, the present invention provides the use of the GhA01EP1 gene in promoting early maturity and high yield in cotton. The effects of GhA01EP1 on plant growth and development involve multiple metabolic pathways. (1) GhA01EP1 promotes plant growth and early flowering by affecting the expression of genes related to circadian rhythm regulation, photoperiodic flowering, and hormone metabolism; (2) GhA01EP1 may increase the number of branches by regulating genes related to branch development, thereby improving yield.

[0007] The specific technical solution adopted in the present invention is:

[0008] Application of GhA01EP1 gene in promoting early maturity of plants.

[0009] Application of GhA01EP1 gene in promoting plant yield.

[0010] The applied plant is Arabidopsis thaliana.

[0011] The applied plant is cotton.

[0012] The nucleotide sequence of the GhA01EP1 gene is shown in SEQ ID NO. 1, and is as follows:

[0013]

[0014] The amino acid sequence of the GhA01EP1 gene is shown in SEQ ID NO. 2, and is as follows:

[0015] MLGCSISISSKLSYPTMSLHSSLTMSLLSFSFLLLFTFSAKAVVPPSETFRFVNDGEFGPFVVEYDANYRVISIANAPFQLAFYNTTPNAFTLALRMATTRSESLFRWVWEANR GNPVRENATFSLGTDGNLVLADADGRIAWQSNTANKGVVGFQLLPNGNMVLHDSNGKFIWQSFDHPTDTLLVGQSLRIGGATKLVSRASAQNNVDGAYSLVMEPKQLVLQYKGM NSPKPLVYFKSSVWPSTQDGTLQTVTLNVEETNDGFAYNVLLDYTVANSSIGTGNLILTRPKYNSTLSILRLGIDGNLRVFTYYDKVDSQAWEETFTLFSRDSIWGTECELPER CGNFGLCEENQCVACPSTNGLLGWSQNCQPKKVNCRPNGFSYYKLEGVDHFMSQYNEGEGIKESDCGRKCTSDCKCLGYFYHRETSKCWIANELKTLAKTSNSSHVGYIKAPNK.

[0016] The beneficial effects of the present invention are:

[0017] The research results of the present invention show that the effect of GhA01EP1 on plant growth and development involves multiple metabolic pathways.

[0018] (1) GhA01EP1 promotes plant growth and early flowering by affecting the expression of genes related to circadian rhythm regulation, photoperiodic flowering, and hormone metabolism;

[0019] (2) GhA01EP1 may increase the number of branches and thus improve yield by regulating genes related to branch development.

[0020] The above metabolic pathways cooperate with each other to form a metabolic network, thereby synergistically improving the early maturity and high yield of plants, which provides ideas for improving the comprehensive traits of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of flowering period between Col-0 and GhA01EP1 transgenic Arabidopsis under normal growth and drought stress;

[0022] Figure 2Comparison of branching between Col-0 and GhA01EP1 transgenic Arabidopsis;

[0023] Figure 3 GO annotation and KEGG analysis of DEGs in the CK-EP1 vs CK-Col comparison group;

[0024] Figure 4 For physiological index detection;

[0025] Figure 5 Co-IP verification of candidate proteins;

[0026] Figure 6 GST pull-down validation of candidate proteins; DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments:

[0028] The ORF sequence of GhA01EP1 was cloned from Ji 2658 using the homologous cloning method. The sequence is 1371bp long, encodes 456 amino acids, and is located on chromosome A01. GhA01EP1 is expressed in roots, stems, and leaves. Subcellular localization results show that GhA01EP1 is a secretory protein that can be transported to the cell wall. In order to further analyze the function of GhA01EP1, the present invention sequenced the transcriptomes of Col-0 and GhA01EP1-transgenic Arabidopsis, and compared and analyzed the differences between morphological and physiological indicators. At the same time, the interacting proteins of GhA01EP1 in Arabidopsis were screened in order to analyze the molecular function of GhA01EP1 in cotton.

[0029] 1. Effects of GhA01EP1 on the template plant Arabidopsis thaliana and its analysis

[0030] Effects of GhA01EP1 on Arabidopsis growth and development

[0031] To study the effect of GhA01EP1 (its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2) on the development of Arabidopsis, we selected the best-growing strains of GhA01EP1 transgenic Arabidopsis for further cultivation. Under normal growth conditions, GhA01EP1 transgenic Arabidopsis grew faster than Col-0 and flowered earlier ( Figure 1 ), and more branches than Col-0 in the later growth stage ( Figure 2 These results suggest that GhA01EP1 may regulate the expression of genes related to floral and branch development in Arabidopsis thaliana.

[0032] 1.2. GhA01EP1-transgenic Arabidopsis improves yield

[0033] To examine the effect of increased branch number on yield, seeds from Col-0 and GhA01EP1 transgenic Arabidopsis plants were harvested and weighed after they matured and dried. The weights of individual seeds from Col-0 and GhA01EP1 transgenic Arabidopsis plants showed extremely significant or significant differences. These results suggest that GhA01EP1 may improve yield in transgenic Arabidopsis by increasing branch number.

[0034] 1.3. Transcriptome analysis of Col-0 and GhA01EP1-transgenic Arabidopsis

[0035] To analyze the effects of GhA01EP1 on Arabidopsis growth, we sequenced the transcriptomes of two Arabidopsis plants subjected to 24 hours of drought stress (P), with water treatment serving as a control (CK). In the CK-EP1 vs. CK-Col comparison (EP1 represents transgenic Arabidopsis, Col represents wild-type Arabidopsis), 207 genes were upregulated and 21 genes were downregulated.

[0036] GO annotation and KEGG analysis were performed on the differentially expressed genes (DEGs). In the CK-EP1 vs CK-Col comparison group, DEGs were mainly involved in responses to stimulus, stress, hormone, as well as photosynthesis, light harvesting in photosystem II and other biological processes ( Figure 3 A); the metabolic processes involved mainly include circadian rhythm, photosynthesis, plant hormone signal transduction and MAPK signaling pathway, terpenoid biosynthesis (carotenoid, diterpenoid, terpenoid backbone biosynthesis), amino acid metabolism (tryptophan, cysteine ​​and methionine metabolism), etc. ( Figure 3 C).

[0037] A search and analysis of the functions of DEGs in the CK-EP1 vs. CK-Col comparison group on the UniProt website revealed a large number of DEGs related to floral development, including pollen tube development, pollen secondary cell wall development, tapetum development, pollen maturation and dehiscence, and maturation of male and female gametophytes. Furthermore, a number of genes are involved in circadian rhythm regulation, controlling the photoperiodic flowering response. Genes that respond to hormone metabolism are also numerous, including auxin, cytokinin, jasmonic acid, salicylic acid, and ethylene. DEGs were also found to be associated with carbon and nitrogen allocation and lateral branch development. The expression levels of detoxification-related glyoxalase I (Gly I) and antioxidant-related genes were also increased, and some DEGs conferred broad-spectrum resistance.

[0038] Comparison of physiological parameters between Col-0 and GhA01EP1 transgenic Arabidopsis

[0039] In addition, since DEGs involved in carbon and nitrogen distribution were found, the starch and protein contents of Arabidopsis seeds were measured. The results showed that the protein content in transgenic GhA01EP1 Arabidopsis was significantly higher than that in Col-0, while the starch content was significantly lower than that in Col-0.

[0040] 1.5. Discovery of GhA01EP1-interacting proteins in GhA01EP1-transgenic Arabidopsis

[0041] In order to further analyze the effect of GhA01EP1 on Arabidopsis development, GST Pull-down technology was used to explore the interacting proteins of GhA01EP1 in Arabidopsis. After protein spectrum analysis, a total of 486 proteins were obtained, 78 of which were highly reliable and analyzed, and one candidate protein SCPL35 (Q9LEY1) was selected for verification. The selected candidate protein Q9LEY1 was verified by co-immunoprecipitation CO-IP and GST Pull-down technology. Figure 5 ) and GST Pull-down( Figure 6 ) Experimental results showed that SCPL35 interacts with GhA01EP1.

[0042] SCPL35 is a serine carboxypeptidase-like enzyme. UniProt annotation indicates that it is a secreted protein involved in proteolysis, but its specific metabolic processes are poorly understood. STRING Version 12.0 analysis revealed that SCPL35 interacts with 10 proteins, one of which, CEP1 (Q9FGR9), is involved in programmed cell death in the tapetum during anther development, leading to tapetal cell degeneration and functional pollen formation. This further suggests that GhA01EP1 is involved in floral development in GhA01EP1-transgenic Arabidopsis plants.

[0043] 2. Analysis of the mechanism of GhA01EP1 in early maturity and high yield

[0044] 2.1. Mechanisms of GhA01EP1 involvement in early maturity and rapid growth

[0045] GhA01EP1 upregulates the expression of genes involved in circadian rhythm regulation, photoperiodic flowering response, and floral development, including LNK1 (AT5G64170), GIGANTEA (AT1G22770), APRR5 (AT5G24470), APRR7 (AT5G02810), and TEM2 (AT1G68840). LNK1 activates clock-controlled genes and is a transcriptional coactivator required for the expression of the clock genes PRR5 and TOC1. GIGANTEA proteins are involved in regulating circadian rhythms, photoperiodic flowering, and phytochrome B signaling. APRR5 and APRR7 belong to the same family of proteins and are involved in both positive and negative feedback loops of the circadian clock. APRR7 is also involved in regulating oxidative stress responses and stomatal conductance. TEM2, as a direct repressor of the flowering gene FT, balances the expression of other flowering-related genes, ensuring strict regulation of flowering time. The earlier flowering time suggests differences in the expression of genes involved in floral development. In GhA01EP1-transgenic Arabidopsis, many DEGs (AT5G50800, AT5G46795, AT2G21650, AT1G72290, AT1G54560, AT3G61910, AT5G07530, AT1G18280, AT3G13890, and AT2G44810) are involved in floral development, primarily involving the formation of male and female gametophytes, pollen tube and anther development, pollen and stigma recognition, and seed and ovule development. The upregulation of these DEGs may be responsible for the earlier flowering of GhA01EP1-transgenic Arabidopsis compared to Col-0, consistent with the findings of Lei et al. (2014), which showed that PoEP1 in tree peony can regulate early flowering.

[0046] Summary: GhA01EP1 can regulate the up-regulation of genes related to circadian rhythm regulation, photoperiodic flowering response and flower development, leading to early flowering in plants.

[0047] GhA01EP1 also regulates the expression of multiple hormone metabolism-related genes, achieving early maturity and rapid growth of plants. Plant hormones play a vital role in regulating plant growth and development and adversity response. Jasmonate is a general term for jasmonic acid (JA) and its derivatives methyl jasmonate (MeJA), jasmonic acid isoleucine (JA-Ile), etc., which are widely used to increase yield and improve quality, resist cold and frost, and resist insect pests. In the present invention, AOC1 (AT3G25760) and DAD1 (AT2G44810) are upregulated and participate in the synthesis of jasmonic acid. AOC1 participates in the synthesis of jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA), and DAD1 encodes a phospholipase that catalyzes the initial step of jasmonic acid biosynthesis, which synchronizes the pollen maturation, anther dehiscence and flower opening of Arabidopsis. However, high concentrations of jasmonic acid are harmful to plants and can cause leaf deformity, drying and falling off, and in severe cases, the entire plant to die. Many genes in the CYP94 subfamily of the cytochrome P450 family oxidize JA-Ile to 12OH-JA-Ile, thereby regulating the concentration of jasmonate. In the present invention, the expression of two genes, CYP94B1 (AT5G63450) and CYP94B3 (AT3G48520), was upregulated in GhA01EP1-transgenic Arabidopsis thaliana and involved in the oxidative decomposition of JA-Ile.

[0048] In addition to jasmonate, genes related to auxin metabolism were also upregulated. YUC5 (AT5G43890) encodes a monooxygenase involved in auxin synthesis. The transcription factor MYB73 (AT4G37260) responds to auxin and activates the transcription of the auxin-responsive gene IAA19. Furthermore, two DEGs (AT3G53250 and AT4G22620) belong to the auxin-responsive protein family. These auxin metabolism-related DEGs promoted the growth of GhA01EP1-transgenic Arabidopsis. Furthermore, DEGs responsive to cytokinin signaling (salicylic acid signaling (AT5G47240)) were upregulated, while DEGs involved in ethylene synthesis (AT4G37770 and AT2G22810) were downregulated. In summary, GhA01EP1 achieves rapid growth in transgenic Arabidopsis by regulating multiple hormone metabolism-related genes.

[0049] Summary: GhA01EP1 upregulates the expression of genes involved in jasmonic acid, auxin, cytokinin signaling, and salicylic acid signaling metabolism, hormones that promote plant growth. It also downregulates the expression of genes involved in ethylene synthesis, leading to reduced ethylene production.

[0050] 2.2. Analysis of the mechanism of GhA01EP1 involvement in high productivity

[0051] The present invention found that GhA01EP1 can affect the regulatory expression of DEGs related to lateral branch development in Arabidopsis. COL12 (AT3G21880) is an upregulated transcription factor. Ordonez-Herrera et al. found that overexpression of COL12 increases the number of rosette branches, reduces inflorescence height, and also regulates flowering time. CCD8 (AT4G32810) and CCD7 (AT2G44990) encode carotenoid cleavage dioxygenases and jointly participate in the biosynthesis of strigolactones. Strigolactone is a hormone that inhibits tillering and branch branching through a MAX-dependent pathway. The max3 and max4 mutants in Arabidopsis are caused by damage to AtCCD7 and AtCCD8, and both mutants show a significant increase in lateral branches. In the present invention, the down-regulation of both CCD7 and CCD8, combined with the up-regulation of COL12, can explain why transgenic GhA01EP1 Arabidopsis has more branches than Col-0, thereby leading to an increase in yield.

[0052] Summary: GhA01EP1 can affect the regulatory expression of DEGs related to lateral branch development in Arabidopsis, leading to increased branching and higher yield in Arabidopsis.

[0053] The results of this study show that the effects of GhA01EP1 on plant growth and development involve multiple metabolic pathways. (1) GhA01EP1 promotes plant growth and early flowering by affecting the expression of genes related to circadian rhythm regulation, photoperiodic flowering, and hormone metabolism; (2) GhA01EP1 may increase branch number and thus improve yield by regulating genes related to branch development.

Claims

1. The application of the GhA01EP1 gene in promoting plant yield is characterized by: The high yield refers to increasing the weight of plant seeds.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the GhA01EP1 gene is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The amino acid sequence of the GhA01EP1 gene is shown in SEQ ID NO.2.

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