Autophagy-related genes and application thereof in corn salt tolerance

Abstract

The application provides an autophagy-related gene ZmATG11a and application thereof in corn salt resistance, and the nucleotide sequence of the autophagy-related gene ZmATG11a is shown as SEQ ID NO. 1. The application discovers a new autophagy / salt resistance-related gene, and determines the correlation between autophagy and salt stress, thereby providing a new germplasm resource and theoretical basis for breeding salt-tolerant corn varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural genetic engineering technology. Specifically, this application provides an autophagy-related gene ZmATG11a and its application in maize salt tolerance. Background Technology

[0002] Abiotic stress has a very detrimental impact on crop yields. Due to the continuous increase in temperature worldwide, plants are increasingly subjected to adverse environmental stresses such as drought, floods, soil salinization, and extreme temperatures. Globally, of the 230 million hectares of arable land, approximately 20%-30% is suffering from salinity stress to varying degrees, and this proportion is continuously expanding, seriously affecting crop yields and the sustainable development of agriculture in my country.

[0003] Corn is one of my country's three major food crops. With high nutritional value, it is an excellent food and feed crop, playing a vital role in agricultural development. However, corn is a salt-sensitive crop. In recent years, factors such as increased extreme weather events, inappropriate irrigation methods, and improper farming practices have led to increasingly severe soil salinization in major corn-producing areas, becoming one of the main environmental factors contributing to declining corn yields. Therefore, in-depth analysis of the molecular mechanisms of corn salt tolerance and the cultivation of salt-tolerant corn varieties are of great significance.

[0004] Autophagy is a conserved process in eukaryotes where plants actively degrade cytoplasmic components such as proteins, macromolecular aggregates, and organelles into smaller molecules for reuse during growth, development, and environmental stress. In autophagy, substrates such as aggregated large proteins or damaged organelles are surrounded by double-membrane autophagic vesicles. With the participation of a series of autophagy-related genes, these vesicles eventually close, forming autophagosomes. The autophagosomes then transport the substances to be degraded into vacuoles for further degradation. The complete autophagy process requires the participation of a series of autophagy-related genes, which form different complexes involved in different stages of autophagy. These include the ATG1-ATG13 protein kinase complex, the phosphatidylinositol (PI3K) complex, the transmembrane protein ATG9 complex, and two ubiquitination-like binding systems: ATG5-ATG12 and ATG8-PE. Autophagy is relatively conserved in animals, plants, and microorganisms, and the process is generally similar, consisting of approximately six steps: 1. Induction of autophagy. 2. Lipid transport. 3. Vesicle nucleation. 4. Membrane elongation and closure of autophagic vesicles. 5. Autophagic vesicle transfer and fusion with the vacuolar membrane. 6. Degradation of target proteins. Increasing experimental results indicate that autophagy is involved in plant responses to abiotic stresses, enhancing plant tolerance. In Arabidopsis, drought stress promotes ATG18a expression and activates autophagy. Classical autophagy-deficient mutants atg5 and atg7, as well as downregulated ATG18a mutants, exhibit suppressed autophagy and decreased drought tolerance under drought stress. When plants are subjected to cold stress, ATG6a / c expression is downregulated in rice while ATG6 expression is upregulated in barley, indicating that ATG6 is involved in plant responses to cold stress.

[0005] Salt stress can also induce autophagy. In wheat, autophagy is inhibited and salt tolerance is reduced in ATG2 and ATG7 mutants. In rice, the atg10b mutant produces fewer autophagosomes during autophagy than the wild type, and the mutant is more sensitive to salt stress. In rapeseed, the roots of transgenic materials with reduced ATG8f expression have lower Na+ content. + The content decreases, destroying Na + / K + Homeostasis leads to decreased salt tolerance. In summary, autophagy is widely involved in salt stress response processes in different plants, but there are currently no reports on the regulation of salt stress response by autophagy in maize. In Arabidopsis, ATG11 is a component of the autophagy initiation complex and co-localizes with ATG1 and ATG13, helping the ATG1-ATG13 complex to anchor to the autophagosome membrane and exert its function. Similar to other classic autophagy mutants, ATG11 mutant plants age prematurely and are more sensitive to nitrogen and carbon nutrient deficiencies, meaning that ATG11 plays an important and indispensable role in autophagy in Arabidopsis. Summary of the Invention

[0006] To investigate the relationship between salt stress and autophagy in maize, this study identified the autophagy-related gene ZmATG11a or its encoded protein. By knocking out ZmATG11a, the phenotype of the knockout mutant was observed. Phenotypic observation revealed that the ZmATG11a mutant was more sensitive to salt stress than the wild type. Further research showed that the ZmATG11a mutant formed fewer autophagosomes under salt conditions than the wild type, thus exploring the relationship between autophagy and salt stress and providing a theoretical basis for further improving the salt tolerance of maize.

[0007] On the one hand, this application provides an autophagy-related gene ZmATG11a, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] On the other hand, this application provides the protein encoded by the autophagy-related gene ZmATG11a, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] On the other hand, this application provides the application of the aforementioned autophagy-related gene ZmATG11a or its encoded protein in regulating plant salt tolerance.

[0010] Furthermore, the plant in question is a grass belonging to the Poaceae family.

[0011] Furthermore, the plant in question is corn.

[0012] Furthermore, in this application, the expression of the autophagy-related gene ZmATG11a is increased to enhance the salt tolerance of the plant.

[0013] Furthermore, in this application, the expression of the autophagy-related gene ZmATG11a is reduced to decrease the salt tolerance of the plant.

[0014] Furthermore, the reduction of the expression of the autophagy-related gene ZmATG11a is achieved by knocking out the autophagy-related gene ZmATG11a using the CRISPR method.

[0015] Furthermore, the target sequence for knocking out the autophagy-related gene ZmATG11a using the CRISPR method is cgcgctgaaggcgctggta.

[0016] Furthermore, reducing the expression of the aforementioned autophagy-related gene ZmATG11a decreases the production of autophagosomes under salt stress.

[0017] This invention uncovers a new component involved in the autophagy process in response to salt stress, thereby improving the salt tolerance of plants, especially maize, and increasing final yield. It also provides a theoretical basis for studying plant salt tolerance responses and improving plant salt tolerance. Attached Figure Description

[0018] Figure 1 ZmATG11a crispr The sequence alignment diagram of the material is shown below; Part A is a schematic diagram of the ZmATG11a gene structure; Part B is a comparison of the nucleic acid knocked out by CRISPR-Cas9 with the original gene sequence; and Part C is a comparison of the amino acid sequence with the original gene sequence.

[0019] Figure 2 ZmATG11a crispr Phenotypic and physiological characteristics of the material after two weeks of growth under control and salt stress (100 mM NaCl); Part B represents biomass. Scale bar for Part A: 10 cm.

[0020] Figure 3 ZmATG11a crispr Results of autophagy detection in wild-type and mutant materials under salt stress. Part A shows the staining of autophagosomes in wild-type and mutant materials using the fluorescent dye monodansylcadaverine (MDC); Part B shows the count of autophagosomes. Scale bar for Part A: 20 μm. Detailed Implementation

[0021] The following embodiments are provided to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0022] Example 1: Screening of salt-tolerant genes in maize

[0023] Salt phenotype testing (100 mM NaCl) was conducted on over 1000 transgenic materials obtained from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University to screen for salt-tolerant genes. Ultimately, an autophagy-related gene, ZmATG11a, was identified. Compared to the wild type, ZmATG11a... crispr It exhibits a salt-sensitive phenotype.

[0024] The nucleotide sequence of the maize ZmATG11a gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. Its encoding in the maize genome database is GRMZM2G143445.

[0025] SEQ ID NO.1

[0026] ATGAGTTCCGGGTCGGCGGTGACAGGCGGTGGTGCGGAGGAGGCGGCGGCGGTGCCG

[0027] CTGGGGCAGAAGCTGATTGTGCACGTGGCGGAGAACGGCCACACCTTGGAGTTCCAGT

[0028] GCGGCGGCGACACGCTCGTCGAGGCCATCCAGCACTCCATCCAGCTCCACTGCGAAATA

[0029] CCCCCTGCCGATCAGCTCCTCCTCTGCGGCAACATCTCCCTCGACGGCGCCAACGCGCT

[0030] CGCCACCTACAAGCTTCCGCGGGACGACCGTGAGGTCTTCCTCTACAACAAGGCCCGG

[0031] CTCCTTGCGGACTCCCGGCCCCCGGCGCCGGAGTCCCTCTACATCCCTGAGCCAAATATT

[0032] CCTCCGCCGCCCCGGCCGCAGGGCTCACCACCTTCGGACGCATCTGCAGACCCCGCGCT

[0033] GAAGGCGCTGGTATCCTATGAAACAAGATTCAGATATCACTTCCAGGTCGCCAATGCGGT

[0034] GTATCAATCTAGTTTGGCAAAATTTGAGCTGTGCAGGCGGCTTCTGCGGGAGGGGCAGG

[0035] TCCAGGAGCGAGCGCTGGACACAGCAGGGAGCAACCTAGAGCACACATTCCGGAAACT

[0036] CTCACAGAGGTATTCAGAATTTTTGCGGTGCTTCACACAACAGCACCGTTCACATGTTG

[0037] AGATGCTGGCCAATTTCGAGAGAGATGTGCAGAAGCTGCGTGCTGTTAGGCTGCACCCA

[0038] GCCCTGCAAAGTGAGGGGCGGCATTGCTTGATGGACCTTCTCAAGGAATTGACCTGA

[0039] GGAAATTGGCTGACGAATGCTTCTGCTCACATAAGAAGTTTGAGGTTAAGGTGTCACAG

[0040] CTGAAGGCAAACTTCTTGGAGCTGAAGAAGAGGGTGGAAGGCTTATTCCATGCCATGA

[0041] GCTCAGGTGGGTGCAAGGATGTTGAGAAGCTGATAAAGGAGCACCAGGGAGTCATTGG

[0042] TGACCAGAAGATCATCATGCAAGCTCTAAGCAAAGATGTGGACACCTCAAAGAAGCTTG

[0043] TTGATGACTGCTCAAGTTGCCAGCTATCTGCTTCTCTCCGTCCTCATGATGCAGTCTCAG

[0044] CGGTTGGCCGTATCTACGAAGTGCATGAAAAGGATAACTTGCCCAGTATACGGGATTTTG

[0045] ATCAGAGGCTTACAAAATTGCTTGAGAAATGCAAGGACAAGAAGAATGAAATGAAATGAAATACT

[0046] TTGGTCCATGTTTGCATGCAAAGAGTAAAATCTTCTCAGATTAGTATCAAAGGCATGATG

[0047] AGTGAACTCGTTGCATTCCAAGAGGTGATGGGTCATCAAGAAGATTTTGATAATCTGAA

[0048] AAAGTCAGTGGCTTGGGTCATGCATATAGAGCTTGTGTCGCCGAGGTAGCCAGGAGGA

[0049] AATCGTATTTTAAGCTGTATACTGGATTGGCTGGAAAATATGCTGAAACGTTGGCAATCG

[0050] AGTGTCAAAACGAGAAAACAAGACGAGAGGATTTTCATAGGACATGGAGCAGGTACAT

[0051] TCCAGATGATGTCATGTGTTCCATGGGACTCTTTGATTCTCCAAGCCAGTGTGATGTAAA

[0052] AGTTGCTCCTTTTGATCTTGATCTTCTTCCCATTGATGTTGATGATGTGGAAAAGCTTGCT

[0053] CCCCAGTCTATACTGGGTTCTTTTTTAAAATCTGAGAGATCACAGCTAGCAAAGCCTTTG

[0054] CTAAGCAATTCTACCAGTGGAAATTTGAACAAATCTGAACAACATTCTCTGAGTGCTGAT

[0055] GATAAGATGGATTTCCAAGATTTTCTGGGGGGCTATGATACTATTGACATTGCAGGAACT

[0056] AGTAAGTTAGAAGTGGAAAATGCCAGGCTAAAAGCAGAACTTGCTTCTGCAATTGCAAT

[0057] TCTCTGCGGTGCTGGATATGGATATGAGTCTATTGACGAAGGGCAAATTGATGCTGTATT

[0058] GAAAAAAGCAAGGGAAAAAACTGCTGAGGCACTTGCTGCAAAGGATGAGTTTGCTTAC

[0059] CAGCTTCAGTCATTGCTCACTGCAAAGCAGGAAAAATGCTTGGCATATGAGAAGCGGAT

[0060] CCAGGATCTTGAGGAACGCTTAACCAACCAGTACATGCAAGGTCACATGGTGTCGGGAA

[0061] GCAAAGGCATGTCTGATTCCCTGCTTTCTGCATTTAAAAGTAATGAGTGCAACCTGGATT

[0062] TATCTGAAGGCAGGCAACCCCAAATACGTGATGAATCAAGTGTGGCCATGGATGAGGTC

[0063] TCTTCAACATCTGAACAGCCATCTAAACAAACAGAAGGTGGCGATGAGAATATGACTGA

[0064] CATTTCGGGTGCACTGAACTTGCAGTTGATCGATTCAGCAGCATGTACTAATCTGGATGC

[0065] TTTCATGACAGAACTGCCACGTGATAATGAACACAAGATTGTAAACATCAATAAGGAAG

[0066] GACACATGTTGACACAACTTACTATGGCTGATACTTCTGATGTTCCTATAGAAGATCCTCT

[0067] TAGCAACTTAAACTCAAGAACTGATGATCATCATGCCCTAGAGTTGAGGGATAAGGAGC

[0068] TCCTTGTGTCAGAGCTGCAAAACACGCTTGATCAAAAATCAAAACAGTTGGGTGAAAC

[0069] TGAAATTAAACTTAGTGCCATGATGGATGAGGTTAATTCTCTGAAGAAAGAACTTGAAC

[0070] AAACCCGGGGCCTTCTTGATGAATCTCAGATGAATTGTGCGCACCTTGAAAACTGTTTAC

[0071] ATGAAGCAAGAGAAGAGGCCCGAACAAACAAATGTTCAGCTGACAGAAGGGCTGTTG

[0072] AGTATGATGCTCTGCGGTCGTCTGCTTTGAGGATACATGGTTTGTTCGAAAGGCTAAATA

[0073] ACTGCATCACTGCACCAGGTGTGACTGGCTTTGCAGAGTCACTGCATTCTTTGGCTGCC

[0074] TCCTTGGCAAGCTCTGTAAAGAAGGATGAAGCTGATACCACTGTTCAGTTTCAACAATG

[0075] CATCAAGATCCTGGCGGACAAAGTTTATTTACTGACACGACAGAGTGCTGAGCTGCTAG

[0076] AACGCTATTCAGCTATGCAGGCAGTACATGGAGGTATCACAAAAGAGCTGGATGAGAAG

[0077] AAAGAGCTGATTAAGAATCTCTACAATAAACTTCAACAAGAAAAACAGGCCAGCAAAG

[0078] AGAAGATATCATTTGGTCGGTTTGAAGTCCATGAGCTTGCTGTCTTTTTCCGAAACCCTG

[0079] CTGGGCACTATGAGGCGATCAACCGGAACTGCTCAAACTATTATCTGTCTGAGGAATCTG

[0080] TTGCCTTATTCACGGAGCAACACTCGCAGCACCCAGTGTACATAATCGGGCAAATCGTTC

[0081] ATATTGAGCGGCGCGTAGCGCGTCCAGACCAGATGGGAGGAGCTCCACGCCCTGATAGC

[0082] AGTGGCGGCCATCGGTCGCCCGCATCCATGCTCAACCCCTACAACCTACCTGGGGGCTG

[0083] TGAGTACTTCGTGGTGACTGTTGCCATGCTGCCTGATGCTGCCAGTTAASEQ ID NO.2

[0084] MSSGSAVTGGGAEEAAAVPLGQKLIVHVAENGHTLEFQCGGDTLVEAIQHSIQLHCEIPPAD

[0085] QLLLCGNISLDGANALATYKLPRDDREVFLYNKARLLADSRPPAPESLYIPEPNIPPPPRPQGS

[0086] PPSDASADPALKALVSYETRFRYHFQVANAVYQSSLAKFELCRRLLREGQVQERALDTAGS

[0087] NLEHTFRKLSQRYSEFLRCFTQQHRSHVEMLANFERDVQKLRAVRLHPALQSEGRHCLMD

[0088] LLKENDLRKLADECFCSHKKFEVKVSQLKANFLELKKRVEGLFHAMSSGGCKDVEKLIKE

[0089] HQGVIDGDQKIIMQALSKDVDTSKKLVDDCSSCQLSASLRPHDAVSAVGRIYEVHEKDNLPSI

[0090] RDFDQRLTKLLEKCKDKKNEMNTLVHVCMQRVKSSQISIKGMSELVAFQEVMGHQEDFD

[0091] NLKIVSGLGHAYRACVAEVARRKSYFKLYTGLAGKYAETLAIECQNEKTRREDFHRTWSRY

[0092] IPDDVMCSMGLFDSPSQCDVKVAPFDLDLLPIDVDDVEKLAPQSILGSFLKSERSQLAKPLL

[0093] SNSTSGNLNKSEQHSLSADDKMDFQDFLGGYDTIDIAGTSKLEVENARLKAELASAIAILCG

[0094] AGYGYESIDEGQIDAVLKKAREKTAEALAAKDEFAYQLQSLLTAKQEKCLAYEKRIQDLEE

[0095] RLTNQYMQGHMVSGSKGMSDSLLSAFKSNECNLDLSEGRQPQIRDESSVAMDEVSSTSEQP

[0096] SKQTEGGDENMTDISGALNLQLIDSAACTNLDAFMTELPRDNEHKIVNINKEGHMLTQLTM

[0097] ADTSDVPIEDPLSNLNSRTDDHHALELRDKELLVSELQNTLDQKSKQLGETEIKLSAMMDE

[0098] VNSLKKELEQTRGLLDESQMNCAHLENCLHEAREEARTNKCSADRRAVEYDALRSSALRI

[0099] HGLFERLNNCITAPGVTGFAESLHSLAASLASSVKKDEADTTVQFQQCIKILADKVYLLTRQ

[0100] SAELLERYSAMQAVHGGITKELDEKKELIKNLYNKLQQEKQASKEKISFGRFEVHELAVFFR

[0101] NPAGHYEAINRNCSNYYLSEESVALFTEQHSQHPVYIIGQIVHIERRVARPDQMGGAPRPDSS

[0102] GGHRSPASMLNPYNLPGGCEYFVVTVAMLPDAAS

[0103] When performing functional analysis on ZmATG11a, mutant materials were first constructed and the mutant salt phenotype was tested.

[0104] Example 2 ZmATG11a crispr Construction of genetically modified materials

[0105] Building ZmATG11a crispr The method for using genetically modified materials includes the following steps:

[0106] Based on the coding region sequence of the ZmATG11a gene, and using the website http: / / omap.org / crispr / CRISPRsearch.html, a design was created.

[0107] Screen candidate target sequences, preferably located in conserved functional regions of CDS or upstream regions of genes.

[0108] Off-target effects were assessed using the website http: / / www.rgenome.net / cas-offinder / to further screen for the optimal target sequence. The target sequence is SEQ ID NO.3: cgcgctgaaggcgctggta.

[0109] After identifying the target site, specific amplification primers were designed based on the target sequence. The target gene sequence was amplified using the genomic DNA of the recipient material as a template. The PCR fragment was recovered and digested with enzymes, ligated with the pBUE411 vector, and the ligation product was transformed into E. coli. Colony PCR amplification was performed using the universal primers FD3 / RD, and sequencing was used to verify the correctness of the amplified fragment.

[0110] The correct vector pBUE411-ZmATG11a was constructed and transformed into Agrobacterium strain EHA105. The embryos, 14 days after Agrobacterium infection and pollination, underwent differentiation, greening, leaf growth, and rooting processes to obtain mutant-positive seedlings. Two homozygous knockout materials (ZmATG11a) were obtained after propagation and experimentation. crispr -1 and ZmATG11a crispr -2)( Figure 1 Part B).

[0111] Example 3 ZmATG11a crispr Salt phenotype test of transgenic materials

[0112] ZmATG11a crispr Phenotypic characteristics of transgenic and wild-type ND101 materials were observed two weeks after germination under normal and 100 mM NaCl conditions, respectively. Figure 2 As shown in Part A, ZmATG11a crispr The transgenic material and the wild-type material grew identically under normal conditions, but under salt stress, ZmATG11a... crispr The transgenic material exhibited a salt-sensitive phenotype compared to the wild-type material. Biomass measurements revealed that, compared to the wild-type, ZmATG11a... crispr The biomass of the transgenic material under salt conditions was significantly lower than that of the wild type, and the rate of biomass decline was significantly higher than that of the wild type. Figure 2 (B / C section).

[0113] Example 4: Detection of ZmATG11a crispr Autophagy in wild type under salt stress

[0114] To investigate whether ZmATG11a is involved in the regulation of autophagy under salt conditions, autophagosomes in wild-type and mutant materials were stained with the fluorescent dye monodansylcadaverine (MDC), and observed using confocal microscopy. The specific steps are as follows:

[0115] ZmATG11a crispr Mutant and wild-type materials germinated vermiculite in 4 days under normal conditions.

[0116] After germination, the roots of both materials were gently washed with clean water, and then the roots were immersed in clean water with 1 μM Concanamycin A (a type of H2O) added. + -ATPase inhibitor, which prevents the degradation of autophagosomes (facilitating microscopic observation) and 100mM NaCl with 1μM Concanamycin A were incubated in the dark for 12h.

[0117] Configure the MDC staining PBS buffer for use the next day:

[0118]

[0119] Add 0.05 mM MDC dye to PBS buffer, place the treated seedlings in the dye, and shake on a shaker at the lowest speed for 10 minutes in the dark. After staining, aspirate the staining solution and wash three times with PBS buffer in the dark.

[0120] After washing, samples were taken from the root tip and fluorescence was observed using a confocal microscope with the DAPI channel.

[0121] like Figure 3 As shown in Part A, under normal circumstances, ZmATG11a crispr The mutant and wild-type materials showed a low number of autophagosomes, with autophagosomes being almost invisible. After 12 hours of salt treatment, the number of autophagosomes in wild-type cells increased significantly, but the number of autophagosomes in ZmATG11a cells remained low. crispr The number of autophagosomes in the mutant cells also increased slightly, but the increase was much smaller than that in the wild-type cells. This indicates that mutants of autophagy-related genes have defects in the autophagy process under salt stress, further demonstrating that salt stress induces autophagy and that autophagy plays an important role in plant salt stress responses.

[0122] Finally, it should be noted that the above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of an autophagy-related gene ZmATG11a in regulating salt tolerance in maize, wherein the nucleotide sequence of the autophagy-related gene ZmATG11a is SEQ ID NO.1; in the application, the autophagy-related gene ZmATG11a is knocked out using the CRISPR method to reduce its expression and thus reduce maize salt tolerance; the knockout target of the CRISPR method is cgcgctgaaggcgctggta.

2. The application according to claim 1, wherein the autophagy-related gene ZmATG11a is knocked out using the CRISPR method to reduce its expression and decrease the production of autophagosomes under salt stress.

Images