Application of sodion transporter gene in regulation and control of salt tolerance of corn

Abstract

The invention provides an application of a sodion transporter gene in regulating and controlling salt tolerance of corn. The nucleotide sequence of the sodion transporter gene is SEQ ID NO.1. The sodion transporter gene can be used for regulating and controlling salt tolerance of corn. Through subcellular localization and in combination with defective yeast phenotypic complementation experiments, ZmHAK11 encodes a type of Na < + > transporter protein, is mainly localized on a plasma membrane, and has inward Na < + > transport activity. The ZmHAK11 under salt stress is also detected: the saline-alkaline tolerance of the corn is influenced by reducing the transport from the corn root to the overground part, which indicates that the corn ZmHAK11 gene or the encoded protein thereof can regulate and control the saline-alkaline tolerance of the corn in the seedling stage.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of a sodium ion transporter gene in regulating corn salt tolerance. Background Art

[0002] In saline-alkali soils, sodium ions (Na + ) is the most common and harmful cation, reducing Na + Entering the aboveground tissues is an important salt tolerance mechanism for most sweet soil plants (Yang et al., 2003; Munns and Tester, 2008). Corn (Zeamays ssp. mays) is one of the three major crops in my country. In recent years, my country's demand for corn-related products has continued to grow, and the gap in corn self-sufficiency has continued to expand. At the same time, the negative impact of environmental stresses such as salinity and alkali on my country's corn production has also shown an increasing trend, which has brought new challenges to the sustainable development of my country's corn production (Egamberdieva et al., 2019; Zhang et al., 2019). Existing studies have shown that the aboveground Na content of different corn varieties varies greatly. + There are significant differences in the content and salt tolerance of corn (Liang et al., 2024). + The salt-alkaline tolerance genes related to homeostasis maintenance were cloned and expressed in different Na + It plays a role in maintaining homeostasis and provides important genetic resources for corn salt-alkali tolerance. + QTL and GWAS analysis related to maize xylem vessel Na + Unloading of ZmNC1 / ZmHKT1 and Na + The selective HAK / KUP / KT family transporters ZmNC2 / ZmHAK4. These two proteins belong to different families and transport Na + transported to surrounding parenchyma cells to reduce Na + transport from roots to the aboveground parts, thereby reducing Na + The accumulation of ZmqKC3 / ZmHKT2 in the aboveground part promotes salt tolerance in maize (Zhang et al., 2018; Zhang et al., 2019). Studies have shown that ZmqKC3 / ZmHKT2 encodes an HKT family transporter that negatively regulates K in the aboveground part under salt stress. + content and salt tolerance of corn (Cao et al., 2019). For example, Cao et al. found that the degradation of ZmNSA1 can lead to the degradation of plasma membrane H + -ATPase (MHA2 and MHA4) transcription levels increased, thereby enhancing the plasma membrane H+ -ATPase function, promoting root H + Efflux and transmembrane H + Gradient maintenance, thus promoting Na + Homeostasis and salt-alkali tolerance in maize (Cao et al., 2020). Among them, ZmHAK4 is a new type of Na + Transport protein.

[0003] The HAK gene family contains many members in most higher plants, but only a few have been identified as sodium ion selective transporters. Therefore, the identification of new sodium ion selective transporters in the HAK transporter family is of great significance for the discovery of sodium ion transporters in plants, especially crops, and the study of salt-alkali tolerance mechanisms. Phylogenetic analysis divides HAK family transporters into four categories (I-IV). The ZmHAK11 involved in this patent is a newly identified sodium ion selective transporter belonging to Class III of the HAK family by the applicant. The identification and functional analysis of ZmHAK11 can provide a new method for enhancing Na + It provides important theoretical and material support for improving the salt tolerance of corn through steady-state regulation. Summary of the invention

[0004] On the one hand, the present application provides an application of a sodium ion transporter gene in regulating corn salt tolerance, wherein the nucleotide sequence of the sodium ion transporter gene is SEQ ID NO.1.

[0005] Furthermore, in the application, the sodium ion transporter gene is knocked out or the expression level of the sodium ion transporter gene is knocked down to reduce the salt tolerance of corn.

[0006] Furthermore, the CRISPR method is used to knock out the sodium ion transporter gene or knock down the expression level of the sodium ion transporter gene.

[0007] Furthermore, the knockout target of the CRISPR method is SEQ ID NO.3.

[0008] Furthermore, in the application, a sodium ion transporter gene is overexpressed to improve the salt tolerance of corn.

[0009] On the other hand, the present application provides an application of a sodium ion transporter in regulating corn salt tolerance, wherein the amino acid sequence of the sodium ion transporter is SEQ ID NO.2.

[0010] Furthermore, in the application, the expression level of the sodium ion transporter is reduced to reduce the salt tolerance of corn.

[0011] Furthermore, in the application, the expression level of the sodium ion transporter is increased to improve the salt tolerance of corn.

[0012] Furthermore, the regulation is to regulate the Na content of corn under salt stress conditions. + content.

[0013] Furthermore, the regulation is to regulate the Na content of corn under salt stress conditions. + Transport from roots to aboveground parts.

[0014] On the other hand, the present application provides the application of the above-mentioned application in corn breeding.

[0015] The present invention clarifies that ZmHAK11 encodes a Na + Transporter, mainly located on the cytoplasmic membrane, with an inward Na + The role of ZmHAK11 under salt stress was also tested: it affected the salt-alkali tolerance of corn by reducing the transport from corn roots to the aboveground parts, indicating that the corn ZmHAK11 gene or its encoded protein can regulate the salt-alkali tolerance of corn seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of constructing ZmHAK11 knockout mutant using CRISPR-Cas9 technology.

[0017] Figure 2 Figure 2 Phenotypes, biomass, and shoot Na+ content of wild type and ZmHAK11 knockout mutant under control and salt stress conditions. + Part A: Phenotypes of hak11-1, hak11-2 and wild type after 14 days of growth under control and salt stress conditions. Part BC: Sprout biomass (Part B) and aboveground Na content of different genotypes under control and salt stress conditions. + Content (Part C).

[0018] Figure 3 Schematic diagram of the ZmHAK11 gene structure.

[0019] Figure 4 The results of subcellular localization of ZmHAK11. The fusion proteins ZmHAK11-GFP and CBL1-OFP were co-expressed in maize protoplasts.

[0020] Figure 5 The Na expression of ZmHAK11 in the defective yeast system + , K + Ion transport activity assay. Part A is for Na + The efflux-deficient yeast strain ant5 was transformed with plasmids expressing different proteins and grown in AP medium supplemented with different concentrations of NaCl. +Plasmids expressing different proteins were transferred into the absorption-trapped yeast strains trk1 and trk2, and the strains were grown in AP medium supplemented with different concentrations of KCl.

[0021] Figure 6 Figure 2 Na in roots (Part A) and xylem sap (Part B) of wild type and ZmHAK11 knockout mutant under control and salt stress conditions. + Content detection. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1 Construction of genetic material and functional analysis of maize salt-alkali tolerance gene ZmHAK11

[0024] In order to study the biological function of ZmHAK11, the gene knockout mutant of ZmHAK11 was first obtained by CRISPR / Cas9 technology. The inventors (1) designed a CRSPR-Cas9 knockout target, and the target sequence was:

[0025] GACAAACCGGGGAAGCATG (SEQ ID NO.: 3); (2) The PCR fragment containing the target sequence and the pBUE411 vector were digested and ligated with BsaI, the ligation product was transferred into Escherichia coli, and colony PCR amplification was performed with universal primers FD3 / RD. The colonies with the target size bands were detected by electrophoresis as positive clones, and the corresponding bacterial liquid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the correct pBUE411-ZmHAK11 vector, and pBUE411-ZmHAK11 was transferred into Agrobacterium EHA105; (3) Transgenic plants were obtained by young embryo infection; (4) Transgenic positive plants were determined by PCR amplification and sequencing of the gene fragment including the target site, and finally two independent transgenic strains of ZmHAK11 (named hak11-1 and hak11-2) were obtained. In the two mutants, ZmHAK11 had one insertion and four deletions, respectively, resulting in a frameshift mutation and loss of ZmHAK11 function (e.g. Figure 1 The wild type and mutant were germinated under control and 100 mM NaCl conditions, and the salt tolerance phenotype was observed and measured. The results showed that under control conditions, the wild type and mutant grew similarly, while the mutant was dwarfed under salt treatment (as shown in Figure 2). Figure 2 The phenotypic tests of these materials showed that under the control conditions, the aboveground biomass, Na + There was no significant difference in the content; under salt stress conditions, the biomass of the hak11 mutant was reduced compared with the wild type, and Na+ The content ratio increased (such as Figure 2 BC part), indicating that ZmHAK11 positively regulates maize salt tolerance.

[0026] Example 2 ZmHAK11 encodes a cytoplasmic membrane localized Na + Transporter

[0027] The gene structure of ZmHAK11 was predicted by Gramene website. The full length of the gene (from the start codon to the stop codon) is 4402 bp, including 8 exons and 7 introns. The full length of the coding region sequence is 2430 bp. The gene structure is as follows Figure 3 shown.

[0028] In order to clone the full-length coding sequence of ZmHAK11, 10-day-old maize inbred line B73 seedlings were used as materials, and total RNA was extracted using the plant total RNA extraction kit (Cat.# DP432) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and cDNA was obtained by reverse transcription using M-MLV reverse transcriptase of Promega (Beijing) Biotechnology Co., Ltd., and PCR amplification was performed using ZmHAK11-specific primers to obtain a product consistent with the expected size (2430 bp). PCR amplification was performed using ZmHAK11-specific primers (forward primer ZmHAK11-gene-F ATGAGCTCAATGGCATCGCT (SEQ ID NO.4); reverse primer ZmHAK11-gene-RTCAGATGTAGAATATCTGCC (SEQ ID NO.5)). The PCR amplification system was 50 μl, including: 2×SuperMultiplex PCR Mix 25 μl; 10 μM Primer ZmHAK11-gene-F 2 μl; 10 μM Primer ZmHAK11-gene-R 2 μl; DNA 1 μl, ddH2O 20 μl. The PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, 35 cycles from denaturation to extension, and final extension at 72°C for 5 min.

[0029] The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and a 2430 bp ZmHAK11 exon sequence was obtained. The nucleotide sequence is shown in SEQ ID No. 1, and the corresponding protein sequence is shown in SEQ ID No. 2:

[0030] SEQ ID No.1:

[0031] ATGAGCTCAATGGCATCGCTGTCAGAAAGTGAGCTGACAAACCGGGGAAGCATGTGGG

[0032] AGCTAGATCAGAACCTTGATCAACCCATGGATGAGGAGGCAGGCAGGCTGAAGAATATG

[0033] TACAGAGAAAAGAAGTTCTCATCAGCTTTGTTACTACGGTTGGCATTTCAGAGCCTTGG

[0034] GGTGGTCTTTGGTGACTTGGGCACGTCACCTTTATATGTGTTCTATAATATATTTCCTCGTG

[0035] GAATAGGTGAGGGTGAGGATGAGGATGTTATTGGAGCTCTTTCCTTGATTATTTACACCC

[0036] TCACTTTCATACCGCTTCTGAAGTACGTTTTTGTTGTCTTGAGGGCAAATGACAACGGTC

[0037] AAGGCGGTACATTTGCTCTTTATTCCCTGTTATGCCGTCATGCAAAGATCAACACCATACC

[0038] TAATCAACACAGGACCGATGAGGAATTAACGACGTATAGCCGGCAAACATATGAAGAGA

[0039] ATTCAGTTGCAGCAAAAATAAAGAGATGGATAGAGTCACATGCATATAAGAGAAACATT

[0040] CTTCTTGTTCTTGTTTTAATTGGTACTTGTACAGCAATTGGAGATGGAATCCTTACGCCTG

[0041] CTATATCTGTTCTTTCTGCATCAGGTGGCATTAGGGTCCAGAATCAGAATATGAGCACCGA

[0042] TGTTGTTGTGCTCGTCGCCGTGGTCATCTTAATTGGATTATTTAGCATGCAACACTATGGA

[0043] ACAGATAAAGTGGGACTGCTCTTTGCACCGATAGTTCTCCTATGGTTCATCTTAATTGGA

[0044] AGCGTCGGAGCTATAAACATACACAAGTATAATAACTCTGTGCTAAGAGCATACAACCCG

[0045] ATATCTATATACCGTTTTTTCCAACGGAGAAGAAATTATGACATATGGACGTCTCTTGGAG

[0046] GAATCATGCTTAGCATCACGGGAACCGAAGCGTTATTTGCTGATCTCTGTCACTTTCCTG

[0047] TATTGGCAATTCAGATTGCTTTCACCTTGATTGTGTTTCCATGCCTTCTACTGGCGTACAC

[0048] AGGACAGGCTGCCTACATAATTTCCAACAAAACACATGTGGCTGACGCCTTCTATCGATC

[0049] AATTCCAGATGCCATATACTGGCCCGCCTTTGTCATAGCAACTGCTGCAGCAATTGTTGC

[0050] AAGTCAAGCTACCATATCTGCAACCTACTCAATCATAAAGCAAGCTCTTGCACTTGGTTG

[0051] CTTCCCCCGCGTGAAGATAGTCCACACCTCGAAGAAATTCCTTGGGCAGATTTACATGCC

[0052] TGACATCAATTGGATCCTTCTGGTTCTCTGCATTGCCGTGACTGCAGGATTCAAGAACCA

[0053] GAGCCAGATAGGAAATGCATATGGTACTGCTGTGGTTATAGTGATGCTAGTTACCACATT

[0054] CCTGATGGTGCCAATAATGCTGCTGGTATGGAAGAGCCACTGGATCCTCGTGGTGACCTT

[0055] CATCGTGCTGTCGCTCATGGTGGAGGTTCCCTACTTCGTGGCCTGCATACTGAAGATCGA

[0056] CCAGGGTGGCTGGGTCCCACTCGTCGTGGCGACGGCCTTCTTCCTCATCATGTACGTGT

[0057] GGCACTTCTGCACGGTGAAGCGGTACGAGTTCGAGATGCACAGCAAGGTGTCCATGGC

[0058] GTGGATCCTGGGGCTGGGCCCGAGCCTGGGCCTGGTGAGGGTCCCAGGGATCGGCTTC

[0059] GTGTACACGGAGCTGGCGAGCGGCGTGCCGCACATCTTCTCCCACTTCATCACCAACCT

[0060] CCCCGCGATCCACTCGGTGGTGGTGTTCGTCTGCGTCAAGTACCTCCCCGTGTACACCG

[0061] TGCCGACGGAGGAGCGGTTCCTGGTGCGGAGGATCGGGCCCAGGAGCTTCCACATGTT

[0062] CCGGTGCGTGGCGAGGTACGGGTACAAGGACCTGCACAAGAGGGACGAGGACTTCGA

[0063] GAAGGTGCTGTTCGACTGCGTCCTCTTCTTCGTCCGGCTGGAGAGCATGATGGAGGGCT

[0064] ACTCCGACTCGGATGAGTTCAGCGTGCCGGAGCAGGCGCCCGGCATCGGCCGCGCGGC

[0065] GTTCTTGAGCGTGGGCGAGCGGACCTGCGCGACTGTGTGCTCCAGCGGTGATCTGAGCT

[0066] TCTCGTCACAGGACTCGGTCGTGCCCGCCGCCGCGCAGTCGTCCCGAGCGCCGACGGG

[0067] GCTGCGGGTGCTGCACTACTCGGCGTCGGCGTCGGCGGCGGGGCAGGGCAGCAGCGGC

[0068] GGAACGGTGGGCGACGAGCTGGAGTTCCTGAACCGGTGCAAGGACGCCGGCGTGGTG

[0069] CACATCCTCGGGAACACCATCGTGCGCGCGCGGCGGGATTCGGGCGTCGTGAAGAAGC

[0070] TCGCCGTTGACTACATGTACGCTTTCATGAGGAGGGTGTGCAGGGAGAACAGCGTCATCTTCAACGTGCCCCATGAGAGCCTGCTCAATGTCGGGCAGATATTCTACATCTGA;

[0071] SEQ ID No.2:

[0072] MSSMASLSESELTNRGSMWELDQNLDQPMDEEAGRLKNMYREKKFSSALLLRLAFQSLG

[0073] VVFGDLGTSPLYVFYNIFPRGIGEGEDEDVIGALSLIIYTLTFIPLLKYVFVVLRANDNGQGG

[0074] TFALYSLLCRHAKINTIPNQHRTDEELTTYSRQTYEENSVAAKIKRWIESHAYKRNILLVLVLI

[0075] GTCTAIGDGILTPAISVLSASGGIRVQNQNMSTDVVVLVAVVILIGLFSMQHYGTDKVGLLFA

[0076] PIVLLWFILIGSVGAINIHKYNNSVLRAYNPISIYRFFQRRRNYDIWTSLGGIMLSITGTEALFA

[0077] DLCHFPVLAIQIAFTLIVFPCLLLAYTGQAAYIISNKTHVADAFYRSIPDAIYWPAFVIATAAAI

[0078] VASQATISATYSIIKQALALGCFPRVKIVHTSKKFLGQIYMPDINWILLVLCIAVTAGFKNQSQ

[0079] IGNAYGTAVVIVMLVTTFLMVPIMLLVWKSHWILVVTFIVLSLMVEVPYFVACILKIDQGGW

[0080] VPLVVATAFFLIMYVWHFCTVKRYEFEMHSKVSMAWILGLGPSLGLVRVPGIGFVYTELAS

[0081] GVPHIFSHFITNLPAIHSVVVFVCVKYLPVYTVPTEERFLVRRIGPRSFHMFRCVARYGYKDL

[0082] HKRDEDFEKVLFDCVLFFVRLESMMEGYSDSDEFSVPEQAPGIGRAAFLSVGERTCATVCS

[0083] SGDLSFSSQDSVVPAAAQSSRAPTGLRVLHYSASASAAGQGSSGGTVGDELEFLNRCKDAG

[0084] VVHILGNTIVRARRDSGVVKKLAVDYMYAFMRRVCRENSVIFNVPHESLLNVGQIFYI

[0085] To further verify whether ZmHAK11 has Na +Transport function, first by (1) HindIII and SpeI containing HindIII and SpeI restriction sites of ZmHAK11 fragment and pCAMBIAsuper1300-GFP vector double restriction digestion, enzyme fragments connected by T4 ligase, transferred into Escherichia coli, colony PCR amplification screening of the target band of the positive clone; (2) the corresponding bacterial liquid sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and finally obtain the correct pCAMBIAsuper1300-GFP-ZmHAK11; (3) pCAMBIAsuper1300-GFP-ZmHAK11 and CBL1-OFP were transferred into corn protoplasts. (4) Culture for 16-24 hours. (5) Use laser confocal microscopy to observe the protoplast fluorescence expression. The results are shown in Figure 4 As shown, ZmHAK11 is localized on the plasma membrane.

[0086] At the same time, the defective yeast phenotype complementation experimental system confirmed that ZmHAK11 has the function of inward transport of Na + activity. The specific experimental process is as follows: (1) Use SpeI and HindIII to double-digest the ZmHAK11 recovery fragment and p416 vector containing SpeI and HindIII restriction sites, connect the restriction fragments with T4 ligase, and transfer them into Escherichia coli. Screen the positive clones with the target bands by colony PCR amplification; (2) Send the corresponding bacterial solution to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and finally obtain the correct p416-ZmHAK11; (3) Transfer the yeast plasmids expressing different proteins into the sodium efflux defective yeast ant5 and the potassium absorption defective yeast trk1 trk2, respectively, and culture them at 30°C for 2 days; (4) Pick the corresponding yeast single clone and shake the bacteria, dilute them into different concentration gradients, and dot them on the culture medium with different concentrations of NaCl or KCl; (5) After culturing at 30°C for 2 days, observe the growth of the yeast clone. The results showed that compared with the yeast clone transferred with the empty vector, the growth of the ant5 yeast clone transferred with ZmHAK11 was more obviously inhibited as the salt concentration increased (such as Figure 5 The trk1 trk2 yeast clones transformed with ZmHAK11 showed the same growth phenotype as the yeast clones transformed with the empty vector, and both could not grow on a medium with a low concentration of KCl (e.g. Figure 5 B), indicating that ZmHAK11 has an inward-facing Na + transport activity but not K + Transport activity.

[0087] Example 3 ZmHAK11 regulates Na + Root to aerial transport

[0088] To further explore the regulation of Na by ZmHAK11+ The molecular mechanism of Na transport was investigated by measuring the Na + The results showed that under control conditions, Na content in root and xylem sap of wild type and hak11 mutant was + There was no significant difference in Na content; under salt stress conditions, compared with the wild type, the Na + The content of Na in xylem sap decreases. + Increased levels (such as Figure 6 AB part), combined Figure 1 The results of part C showed that ZmHAK11 regulates maize Na + Salt tolerance in maize is achieved through root-to-shoot transport.

Claims

1. An application of a sodium ion transporter gene in regulating corn salt tolerance, wherein the nucleotide sequence of the sodium ion transporter gene is SEQ ID NO.

1.

2. The use according to claim 1, wherein the sodium ion transporter gene is knocked out or the expression level of the sodium ion transporter gene is knocked down to reduce the salt tolerance of corn.

3. The use according to claim 2, using a CRISPR method to knock out the sodium ion transporter gene or to knock down the expression level of the sodium ion transporter gene.

4. The use according to claim 3, wherein the knockout target of the CRISPR method is SEQ ID NO.

3.

5. The use according to claim 1, wherein the sodium ion transporter gene is overexpressed to improve the salt tolerance of corn.

6. Application of a sodium ion transporter in regulating corn salt tolerance, wherein the amino acid sequence of the sodium ion transporter is SEQ ID NO.

2.

7. The use according to claim 6, wherein the expression level of the sodium ion transporter is reduced to reduce the salt tolerance of corn, or the expression level of the sodium ion transporter is increased to increase the salt tolerance of corn.

8. The use according to any one of claims 1 to 7, wherein the regulation is to regulate the Na content of corn under salt stress conditions. + content.

9. The use according to any one of claims 1 to 8, wherein the regulation is to regulate the Na content of corn under salt stress conditions. + Transport from roots to aboveground parts.

10. Use of the method according to any one of claims 1 to 8 in corn breeding.

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