CN118667828A - Autophagy-related gene and application thereof in salt resistance of corn - Google Patents

Abstract

The present application provides an autophagy-related gene ZmATG11a and its application in corn salt resistance, and its nucleotide sequence is shown in SEQ ID NO. 1. The present application discovers new autophagy/salt resistance-related genes and clarifies the relationship between autophagy and salt stress, providing new germplasm resources and theoretical basis for breeding salt-tolerant corn varieties.

Description

Autophagy-related genes and their application in salt resistance of maize

Technical Field

The present invention belongs to the technical field of agricultural genetic engineering. Specifically, the present application provides an autophagy-related gene ZmATG11a and its application in corn salt resistance.

Background Art

Abiotic stress has a very negative impact on crop yields. As temperatures continue to rise worldwide, plants are more frequently exposed to adverse environmental stresses such as drought, floods, soil salinization, and high and low temperatures. Globally, there are 230 million hectares of arable land available, and about 20%-30% of the arable land is suffering from salinity stress to varying degrees, and the proportion is constantly expanding, which has seriously affected crop yields and the sustainable development of my country's agriculture.

Corn is one of the three major grain crops in my country. It has high nutritional value and is an excellent food crop and feed crop. It plays an important role in agricultural development. Corn is a salt-sensitive crop. In recent years, due to factors such as increasing extreme weather, unreasonable irrigation methods and farming methods, the salinization of cultivated land in the main corn-producing areas has become increasingly serious, which has become one of the main environmental factors for the decline in corn yields. Therefore, it is of great significance to deeply analyze the molecular mechanism of corn salt tolerance and cultivate salt-tolerant corn varieties.

The autophagy process is a process that is conserved in the entire eukaryotes. During the growth and development and environmental stress, plants actively degrade cytoplasmic components such as proteins, macromolecular aggregates and organelles to generate small molecules for reuse. In the process of plant autophagy, substrates degraded by the autophagy process, such as aggregated macromolecular proteins or damaged organelles, are surrounded by double-layered autophagic vesicles. After the participation of a series of autophagy-related genes, the autophagic vesicles are finally closed and become autophagosomes. The autophagosomes transport the substances that need to be degraded to the vacuole for degradation. The complete autophagy process requires the participation of a series of autophagy-related genes. These genes form different complexes and participate in different stages of autophagy, including the ATG1-ATG13 protein kinase complex, the phosphatidylinositol (PI3K) complex, the transmembrane protein ATG9 complex, and the two ubiquitin-like binding systems ATG5-ATG12 and ATG8-PE. The process of autophagy is relatively conservative in animals, plants and microorganisms, and the process is roughly similar, which roughly includes six steps: 1. Induction of autophagy. 2. Delivery of lipids. 3. Vesicle nucleation. 4. Membrane extension and closure of autophagic vesicles. 5. Autophagic vesicle delivery and fusion with vacuole membrane. 6. Degradation of target protein. More and more experimental results show that autophagy is involved in the response of plants to abiotic stress and improves plant tolerance. In Arabidopsis, drought stress promotes the expression of ATG18a and the activation of autophagy in Arabidopsis. The classic autophagy-deficient mutants atg5 and atg7 and the down-regulated mutants of ATG18a have inhibited autophagy under drought stress and reduced drought tolerance. When plants are subjected to cold stress, the expression of ATG6a/c in rice is down-regulated, while the expression of ATG6 in barley is up-regulated, indicating that ATG6 is involved in the response of plants to cold stress.

Salt stress can also induce autophagy. In wheat, the autophagy process of ATG2 and ATG7 mutants is blocked and salt tolerance is reduced. In rice, the number of autophagosomes formed during autophagy in atg10b mutants is less than that of the wild type, and the mutant is more sensitive to salt stress. In rapeseed, the Na ⁺ content in the roots of transgenic materials with reduced ATG8f expression is reduced, which destroys Na ⁺ /K ⁺ homeostasis and leads to decreased salt tolerance. In summary, autophagy is widely involved in the salt stress response process in different plants, but there are no relevant reports on the regulation of autophagy in salt stress response in corn. 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 to play a role. Similar to other classic autophagy mutants, ATG11 mutant plants age prematurely and are more sensitive to the lack of nitrogen and carbon nutrients, which means that ATG11 in Arabidopsis plays an important and indispensable role in the autophagy process.

Summary of the invention

In order to study the relationship between salt stress and autophagy in corn, this study explored the maize autophagy-related gene ZmATG11a or its encoding protein. By knocking out the autophagy-related gene ZmATG11a and observing the phenotype of the knockout mutant, the phenotypic observation found that the ZmATG11a mutant was more sensitive to salt stress than the wild type. Further studies found that the number of autophagic bodies formed by the ZmATG11a mutant under salt was less than that of the wild type, thereby exploring the relationship between autophagy and salt stress, providing a theoretical basis for further improving the salt tolerance of corn.

On the one hand, the present application provides an autophagy-related gene ZmATG11a, and the nucleotide sequence of the autophagy-related gene ZmATG11a is shown in SEQ ID NO.1.

On the other hand, the present application provides a protein encoded by the above-mentioned autophagy-related gene ZmATG11a, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

On the other hand, the present application provides the use of the above-mentioned autophagy-related gene ZmATG11a or the protein encoded by it in regulating the salt resistance of plants.

Furthermore, the plant is a grass plant.

Furthermore, the plant is corn.

Furthermore, in the application, the expression of the autophagy-related gene ZmATG11a is increased to improve the salt resistance of the plant.

Furthermore, in the application, the expression of the autophagy-related gene ZmATG11a is reduced to reduce the salt resistance of the plant.

Furthermore, the reducing the expression of the autophagy-related gene ZmATG11a is performed by knocking out the autophagy-related gene ZmATG11a through a CRISPR method.

Furthermore, the target sequence of knocking out the autophagy-related gene ZmATG11a by the CRISPR method is cgcgctgaaggcgctggta.

Furthermore, reducing the expression of the autophagy-related gene ZmATG11a reduces the generation of autophagosomes under salt stress.

The present invention explores new components involved in the autophagy process in salt stress response, improves the salt tolerance of plants, especially corn, and increases the final yield. At the same time, it provides a theoretical basis for studying plant salt resistance response and improving plant salt resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the sequence alignment of ZmATG11a ^CRISPR materials; Part A is a schematic diagram of the ZmATG11a gene structure; Part B is a comparison of the nucleic acid after knockout using CRISPR-Cas9 with the original gene sequence; and Part C is a comparison of the amino acid sequence with the original gene sequence.

Figure 2 shows the phenotypic and physiological trait detection results of ZmATG11a ^crispr materials after two weeks of growth under control and salt stress (100 mM NaCl); Part B is biomass. Scale bar of Part A: 10 cm.

Figure 3 shows the results of autophagy detection of ZmATG11a ^CRISPR materials and wild-type materials under salt stress. Part A shows the autophagosomes of wild-type and mutant materials stained with fluorescent dye monodansylcadaverine (MDC); Part B shows the number of autophagosomes counted. Scale bar in Part A: 20 μm.

DETAILED DESCRIPTION

The following examples are provided to facilitate a better understanding of the present invention, but are not limited thereto. These examples are only used for illustrative purposes and in no way limit the scope of protection of the present invention.

Example 1 Screening of corn salt-resistant genes

More than 1,000 transgenic materials received from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University were tested for salt phenotype (100mM NaCl) to screen for salt-resistant genes. Finally, an autophagy-related gene ZmATG11a was found. Compared with the wild type, ZmATG11a ^crispr has a salt-sensitive phenotype.

The nucleotide sequence of the maize ZmATG11a gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 2. The code in the maize genome database is GRMZM2G143445.

SEQ ID NO.1

ATGAGTTCCGGGTCGGCGGTGACAGGCGGTGGTGCGGAGGAGGCGGCGGCGGTGCCG

CTGGGGCAGAAGCTGATTGTGCACGTGGCGGAGAACGGCCACACCTTGGAGTTCCAGT

GCGGCGGCGACACGCTCGTCGAGGCCATCCAGCACTCCATCCAGCTCCACTGCGAAATA

CCCCCTGCCGATCAGCTCCTCCTCTGCGGCAACATCTCCCTCGACGGCGCCAACGCGCT

CGCCACCTACAAGCTTCCGCGGGACGACCGTGAGGTCTTCCTCTACAACAAGGCCCGG

CTCCTTGCGGACTCCCGGCCCCCGGCGCCGGAGTCCCTCTACATCCCTGAGCCAAATATT

CCTCCGCCGCCCCGGCCCGCAGGGCTCACCACCTTCGGACGCATCTGCAGACCCCGCGCT

GAAGGCGCTGGTATCCTATGAAACAAGATTCAGATATCACTTCCAGGTCGCCAATGCGGT

GTATCAATCTAGTTTGGCAAAATTTGAGCTGTGCAGGCGGCTTCTGCGGGAGGGGCAGG

TCCAGGAGCGAGCGCTGGACACAGCAGGGAGCAACCTAGAGCACACATTCCGGAAACT

CTCACAGAGGTATTCAGAATTTTTGCGGTGCTTCACACAACAGCACCGTTCACATGTTG

AGATGCTGGCCAATTTCGAGAGAGATGTGCAGAAGCTGCGTGCTGTTAGGCTGCACCCA

GCCCTGCAAAGTGAGGGGCGGCATTGCTTGATGGACCTTCTCAAGGAAAATGACCTGA

GGAAATTGGCTGACGAATGCTTCTGCTCACATAAGAAGTTTGAGGTTAAGGTGTCACAG

CTGAAGGCAAACTTCTTGGAGCTGAAGAAGAGGGTGGAAGGCTTATTCCATGCCATGA

GCTCAGGTGGGTGCAAGGATGTTGAGAAGCTGATAAAGGAGCACCAGGGAGTCATTGG

TGACCAGAAGATCATGCAAGCTCTAAGCAAAGATGTGGACACCTCAAAGAAGCTTG

TTGATGACTGCTCAAGTTGCCAGCTATCTGCTTCTCTCCGTCCTCATGATGCAGTCTCAG

CGGTTGGCCGTATCTACGAAGTGCATGAAAAGGATAACTTGCCCAGTATACGGGATTTTG

ATCAGAGGCTTACAAAATTGCTTGAGAAATGCAAGGACAAGAAGAATGAAATGAATACT

TTGGTCCATGTTTGCATGCAAAGAGTAAAATCTTCTCAGATTAGTATCAAAGGCATGATG

AGTGAACTCGTTGCATTCCAAGAGGTGATGGGTCATCAAGAAGATTTTGATAATCTGAA

AATAGTCAGTGGCTTGGGTCATGCATATAGAGCTTGTGTCGCCGAGGTAGCCAGGAGGA

AATCGTATTTTAAGCTGTATACTGGATTGGCTGGAAAATATGCTGAAACGTTGGCAATCG

AGTGTCAAAACGAGAAAACAAGACGAGAGGATTTTCATAGGACATGGAGCAGGTACAT

TCCAGATGATGTCATGTGTTCCATGGGACTCTTTGATTCTCCAAGCCAGTGTGATGTAAA

AGTTGCTCCTTTTGATCTTGATCTTCTTCCCATTGATGTTGATGATGTGGAAAAGCTTGCT

CCCCAGTCTATACTGGGTTCTTTTTTAAAATCTGAGAGATCACAGCTAGCAAAGCCTTTG

CTAAGCAATTCTACCAGTGGAAATTTGAACAAATCTGAACAACATTCTCTGAGTGCTGAT

GATAAGATGGATTTCCAAGATTTTCTGGGGGGCTATGATACTATTGACATTGCAGGAACT

AGTAAGTTAGAAGTGGAAAATGCCAGGCTAAAAGCAGAACTTGCTTCTGCAATTGCAAT

TCTCTGCGGTGCTGGATATGGATATGAGTCTATTGACGAAGGGCAAATTGATGCTGTATT

GAAAAAAGCAAGGGAAAAAACTGCTGAGGCACTTGCTGCAAAGGATGAGTTTGCTTAC

CAGCTTCAGTCATTGCTCACTGCAAAGCAGGAAAAATGCTTGGCATATGAGAAGCGGAT

CCAGGATCTTGAGGAACGCTTAACCAACCAGTACATGCAAGGTCACATGGTGTCGGGAA

GCAAAGGCATGTCTGATTCCCTGCTTTCTGCATTTAAAAGTAATGAGTGCAACCTGGATT

TATCTGAAGCAGGCAACCCCAAATACGTGATGAATCAAGTGTGGCCATGGATGAGGTC

TCTTCAACATCTGAACAGCCATCTAAACAAACAGAAGGTGGCGATGAGAATATGACTGA

CATTTCGGGTGCACTGAACTTGCAGTTGATCGATTCAGCAGCATGTACTAATCTGGATGC

TTTCATGACAGAACTGCCACGTGATAATGAACACAAGATTGTAAACATCAATAAGGAAG

GACACATGTTGACACAACTTACTATGGCTGATACTTCTGATGTTCCTATAGAAGATCCTCT

TAGCAACTTAAACTCAAGAACTGATGATCATCATGCCCTAGAGTTGAGGGATAAGGAGC

TCCTTGTGTCAGAGCTGCAAAACACGCTTGATCAAAAATCAAAACAGTTGGGTGAAAC

TGAAATTAAACTTAGTGCCATGATGGATGAGGTTAATTCTCTGAAGAAAGAACTTGAAC

AAACCCGGGGCCTTCTTGATGAATCTCAGATGAATTGTGCGCACCTTGAAAACTGTTTAC

ATGAAGCAAGAGAAGAGGCCCGAACAAACAAATGTTCAGCTGACAGAAGGGCTGTTG

AGTATGATGCTCTGCGGTCGTCTGCTTTGAGGATACATGGTTTGTTCGAAAGGCTAAATA

ACTGCATCACTGCACCAGGTGTGACTGGCTTTGCAGAGTCACTGCATTCTTTGGCTGCC

TCCTTGGCAAGCTCTGTAAAGAAGGATGAAGCTGATACCACTGTTCAGTTTCAACAATG

CATCAAGATCCTGGCGGACAAAGTTTATTTACTGACACGACAGAGTGCTGAGCTGCTAG

AACGCTATTCAGCTATGCAGGCAGTACATGGAGGTATCACAAAAGAGCTGGATGAGAAG

AAAGAGCTGATTAAGAATCTCTACAATAAACTTCAACAAGAAAAACAGGCCAGCAAAG

AGAAGATATCATTTGGTCGGTTTGAAGTCCATGAGCTTGCTGTCTTTTTCCGAAACCCTG

CTGGGCACTATGAGGCGATCAACCGGAACTGCTCAAACTATTATCTGTCTGAGGAATCTG

TTGCCTTATTCACGGAGCAACACTCGCAGCACCCAGTGTACATAATCGGGCAAATCGTTC

ATATTGAGCGGCGCGTAGCGCGTCCAGACCAGATGGGAGGAGCTCCACGCCCTGATAGC

AGTGGCGGCCATCGGTCGCCCGCATCCATGCTCAACCCCTACAACCTACCTGGGGGCTG

TGAGTACTTCGTGGTGACTGTTGCCATGCTGCCTGATGCTGCCAGTTAASEQ ID NO.2

MSSGSAVTGGGAEEAAAVPLGQKLIVHVAENGHTLEFQCGGDTLVEAIQHSIQLHCEIPPAD

QLLLCGNISLDGANALATYKLPRDDREFLYNKARLLADSRPPAPESLYIPEPNIPPPPRPQGS

PPSDASADPALKALVSYETRFRYHFQVANAVYQSSLAKFELCRRLLREGQVQERALDTAGS

NLEHTFRKLSQRYSEFLRCFTQQHRSHVEMLANFERDVQKLRAVRLHPALQSEGRHCLMD

LLKENDLRKLADECFCSHKKFEVKVSQLKANFLELKKRVEGLFHAMSSGGCKDVEKLIKE

HQGVIGDQKIIMQALSKDVDTSKKLVDDCSSCQLSASLRPHDAVSAVGRIYEVHEKDNLPSI

RDFDQRLTKLLEKCKDKKNEMNTLVHVCMQRVKSSQISIKGMMSELVAFQEVMGHQEDFD

NLKIVSGLGHAYRACVAEVARRKSYFKLYTGLAGKYAETLAIECQNEKTRREDFHRTWSRY

IPDDVMCSMGLFDSPSQCDVKVAPFDDLLPIDVDDVEKLAPQSILGSFLKSERSQLAKPLL

SNSTSGNLNKSEQHSLSADDKMDFQDFLGGYDTIDIAGTSKLEVENARLKAELASAIAILCG

AGYGYESIDEGQIDAVLKKAREKTAEALAAKDEFAYQLQSLLTAKQEKCLAYEKRIQDLEE

RLTNQYMQGHMVSGSKGMSDSLLSAFKSNECNLDLSEGRQPQIRDESSVAMDEVSSTSEQP

SKQTEGGDENMTDISGALNLQLIDSAACTNLDAFMTELPRDNEHKIVNINKEGHMLTQLTM

ADTSDVPIEDPLSNLNSRTDDHHALELRDKELLVSELQNTLDQKSKQLGETEIKLSAMMDE

VNSLKKELEQTRGLLDESQMNCAHLENCLHEAREEARTNKCSADRRAVEYDALRSSALRI

HGLFERLNNCITAPGVTGFAESLHSLAASLASSVKKDEADTTVQFQQCIKILADKVYLLTRQ

SAELLERYSAMQAVHGGITKELDEKKELIKNLYNKLQQEKQASKEKISFGRFEVHELAVFFR

NPAGHYEAINRNCSNYYLSEESVALFTEQHSQHPVYIIGQIVHIERRVARPDQMGGAPRPDSS

GGHRSPASMLNPYNLPGGCEYFVVTVAMLPDAAS

When conducting functional analysis of ZmATG11a, mutant materials were first constructed and the salt phenotype of the mutants was tested.

Example 2 Construction of ZmATG11a ^crispr transgenic material

The method for constructing ZmATG11a ^crispr transgenic material comprises the following steps:

According to the coding region sequence of ZmATG11a gene, the website http://omap.org/crispr/CRISPRsearch.html was used to design

Screen candidate target sequences, preferably located in the conserved functional region of CDS or the upstream region of the gene.

The website http://www.rgenome.net/cas-offinder/ was used to evaluate the off-target situation and further screen the optimal target sequence. The target sequence is SEQ ID NO.3: cgcgctgaaggcgctggta.

After the target is determined, specific amplification primers are designed according to the target sequence, the target gene sequence is amplified using the genomic DNA of the receptor material as a template, the PCR fragment is recovered and digested, and it is digested and ligated with the pBUE411 vector. The ligated product is transformed into Escherichia coli, and colony PCR amplification is performed using the universal primers FD3/RD. Sequencing is then performed to verify whether the amplified fragment is correct.

The correct vector pBUE411-ZmATG11a was constructed to transform Agrobacterium tumefaciens strain EHA105. The immature embryos 14 days after pollination were infected with Agrobacterium. After differentiation, greening, leaf growth and rooting, the mutant positive seedlings were identified. After breeding, two homozygous knockout materials (ZmATG11a ^crispr -1 and ZmATG11a ^crispr -2) that can be used for experiments were obtained (Part B of Figure 1).

Example 3 Salt phenotype test of ZmATG11a ^crispr transgenic material

The ZmATG11a ^crispr transgenic material and the wild-type ND101 material were germinated under normal conditions and 100mM NaCl conditions for two weeks and then the phenotypes were observed. As shown in Part A of Figure 2, the ZmATG11a ^crispr transgenic material and the wild-type material grew in the same way under normal conditions, but under salt stress conditions, the ZmATG11a ^crispr transgenic material showed a salt-sensitive phenotype relative to the wild-type material. The biomass was measured and it was found that the biomass of the ZmATG11a ^crispr transgenic material under salt was significantly lower than that of the wild-type, and the biomass decline rate was significantly higher than that of the wild-type (Part BC of Figure 2).

Example 4 Detection of autophagy in ZmATG11a ^crispr and wild type under salt stress

In order to detect whether ZmATG11a is involved in the regulation of autophagy under salt, the autophagosomes of wild-type and mutant materials were stained with the fluorescent dye monodansylcadaverine (MDC), and the autophagosomes were observed using a confocal microscope. The specific steps are as follows:

ZmATG11a ^crispr mutant materials and wild-type materials were germinated in vermiculite under normal conditions for 4 days.

After germination, the roots of the two materials were gently washed with clean water, and the roots were immersed in clean water plus 1 μM Concanamycin A (a H ⁺ -ATPase inhibitor that prevents the degradation of autophagosomes to facilitate microscopic observation) and 100 mM NaCl plus 1 μM Concanamycin A, respectively, and cultured in the dark for 12 h.

Prepare the MDC staining PBS buffer for the next day:

0.05 mM MDC dye was added to PBS buffer, and the treated seedlings were placed in the dye and shaken at the lowest speed for 10 min in a light-proof shaker. After staining, the staining solution was aspirated and the seedlings were washed three times with PBS buffer in the dark.

After washing, the root tip was sampled and the fluorescence was observed using the DAPI channel using a confocal microscope.

As shown in part A of Figure 3, under normal circumstances, the number of autophagosomes in the ZmATG11a ^crispr mutant material and the wild-type material is small and almost invisible; after 12 hours of salt treatment, the number of autophagosomes in the wild-type cells increased significantly, but the number of autophagosomes in the ZmATG11a ^crispr mutant material cells also increased slightly, but the increase was far less than that in the wild-type cells. This shows that under salt stress, mutants of autophagy-related genes will have defects in the autophagy process, further indicating that salt stress can cause the autophagy process and that the autophagy process plays an important role in the plant salt stress response.

Finally, it is noted that the above-described embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The autophagy-related gene ZmATG a is characterized in that the nucleotide sequence of the autophagy-related gene ZmATG a is shown as SEQ ID NO. 1.

2. The protein encoded by autophagy-related gene ZmATG a according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.

3. Use of the autophagy-related gene ZmATG a according to claim 1 or the protein according to claim 2 for modulating salt resistance in a plant.

4. The use according to claim 3, wherein the plant is a gramineous plant.

5. The use according to claim 4, wherein the plant is maize.

6. Use according to any one of claims 3-5, wherein the expression of the autophagy-related gene ZmATG a according to claim 1 is increased to increase the salt resistance of a plant.

7. Use according to any one of claims 3-5, wherein the expression of the autophagy-related gene ZmATG a according to claim 1 is reduced to reduce the salt resistance of a plant.

8. The use of claim 7, wherein said reducing the expression of the autophagy-related gene ZmATG a of claim 1 is performed by CRISPR method knockout of the autophagy-related gene ZmATG a of claim 1.

9. The use according to claim 7, wherein the target sequence in the autophagy-related gene ZmATG a according to claim 1 is … … by CRISPR method knockout.

10. The use according to claim 8, wherein said decreasing the expression of autophagy-related gene ZmATG a according to claim 1 decreases the production of autophagosomes under salt stress.