Nitrate transporter gene and application thereof in regulation and control of salt resistance of corn

Abstract

The invention provides a nitrate transporter gene and application thereof in regulating and controlling salt resistance of corn. It is found that ZmNRT1.1 A positively regulates the salt tolerance of corn, the salt stress sensitive phenotype of ZmNRT1.1 A is caused by Cl-stress, an important corn salt-resistant molecular mechanism is disclosed, and an important theoretical basis and gene resources are provided for corn salt-resistant improvement.

Description

Technical Field

[0001] The present application belongs to the field of agricultural genetic engineering. Specifically, the present application provides a nitrate transporter gene and its application in regulating corn salt resistance. Background Art

[0003] The effects of salt stress on corn mainly include osmotic stress and ion (mainly Na + and Cl - ) toxicity (Munns and Tester, 2008). Therefore, enhancing the ability to regulate osmotic stress and improving the resistance to high concentrations of Na + and Cl - Tolerance is an important way to improve corn salt tolerance. Under salt stress, the decrease in soil water potential makes it difficult for plant roots to absorb water, inducing osmotic stress (vanZelm et al., 2020). Crops (including corn) can respond to osmotic stress by increasing the synthesis of osmotic regulating substances such as betaine, polyols, proline and quaternary amines (Liang et al., 2021), and can also enhance the plant's adaptability to osmotic stress by increasing ABA synthesis and activating the ABA signaling pathway (Deinlein U et al., 2014). In addition, crops under salt stress use a series of mechanisms to reduce salt ions (Na + and Cl - ) or segregate them to tissues or organelles with strong salt ion tolerance, thereby better adapting to the salt stress environment (Ismail and Horie, 2017). + Research on absorption and transport mechanisms has made progress in many aspects, such as: SOS signaling pathway in root Na + HKT1 and HAK family play a key role in the efflux of Na + Transporters are mainly involved in regulating Na + Long-distance transport (Yang and Guo, 2018; Zhang et al., 2018; Zhang et al., 2019). - In terms of the study of absorption and transport mechanisms, although some members of the anion transporter family such as NPF, SLAH1, CCCs, CLCs, DTX / MATE and ALMT have been found to have Cl - Transport activity, which may be involved in the Cl - The absorption and transport regulation of Cl in crops (including corn) is still unclear. - The molecular genetic basis of absorption and transport regulation remains to be studied. Summary of the invention

[0004] In order to explore the regulation of Cl - The inventors constructed a mutant material of ZmNRT1.1A and found that ZmNRT1.1A positively regulates the salt tolerance of corn, and its salt stress sensitive phenotype is caused by Cl - The relevant research results are expected to reveal the important molecular mechanism of corn salt resistance and provide an important theoretical basis and gene resources for the improvement of corn salt resistance.

[0005] On the one hand, the present application provides a nitrate transporter gene, which is from corn and has a nucleotide sequence of SEQ ID NO.1.

[0006] Furthermore, the nitrate transporter gene is located at 81,342,176-81,348,355 kb on chromosome 10 of the maize V5 reference genome.

[0007] On the other hand, the present application provides a corn nitrate transporter encoded by the above-mentioned nitrate transporter gene, and the amino acid sequence of the corn nitrate transporter is SEQ ID NO.2.

[0008] On the other hand, the present application provides the use of the above-mentioned nitrate transporter gene or corn nitrate transporter in regulating corn salt resistance.

[0009] Furthermore, in the application, the expression level of the nitrate transporter gene is knocked out or knocked down to reduce the salt resistance of corn.

[0010] Furthermore, the knockout is performed by CRISPR-Cas9 method.

[0011] Furthermore, the gRNA nucleotide sequence used in the CRISPR-Cas9 method is SEQ ID NO.3.

[0012] Furthermore, in the application, the expression level of the nitrate transporter gene is increased to improve the salt resistance of corn.

[0013] Furthermore, the salt resistance is Cl resistance - Stress performance.

[0014] On the other hand, the present application provides the use of the above-mentioned nitrate transporter gene or corn nitrate transporter in salt-resistant corn breeding.

[0015] Beneficial effects of the present invention

[0016] The present invention provides a new corn salt resistance and its gene ZmNRT1.1A. Since corn salt resistance is a quantitative trait inheritance, phenotypic analysis is time-consuming and laborious, the chloride ion transporter of the present invention can be applied to corn salt resistance breeding, which can accelerate the corn salt resistance variety selection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a vector structure and sequence alignment diagram; Part A is a schematic diagram of the pBUE411-ZmNRT1.1A vector structure in the salt-resistant gene ZmNRT1.1A; Part B is a schematic diagram of the ZmNRT1.1A gene structure; Part C is a comparison of the ZmNRT1.1A mutant nucleic acid sequence after knockout using CRSPR-Cas9 with the wild-type nucleic acid sequence; Part D is a comparison of the ZmNRT1.1A mutant amino acid sequence after knockout using CRSPR-Cas9 with the wild-type amino acid sequence.

[0018] Figure 2 The growth of wild type and ZmNRT1.1A mutant is shown, where Part A is the phenotypic photos of wild type and ZmNRT1.1A mutant after 14 days of growth under control and salt stress conditions; Part B shows the biomass; Part C shows the percentage of biomass reduction. The scale bar in Part A is 10 cm.

[0019] Figure 3 The growth of wild type and ZmNRT1.1A mutant is shown; Parts AD are phenotypic photos of wild type and ZmNRT1.1A mutant after 14 days of growth under control, 100mM NaCl, 50mM Na2SO4, and 100mM KCl treatment conditions; Part E is the biomass of wild type and ZmNRT1.1A mutant under each treatment condition. The scale bar in Part AD is 10cm. DETAILED DESCRIPTION

[0020] Example 1 Cloning and salt-resistant phenotype analysis of maize salt-resistant gene ZmNRT1.1A

[0021] Ion stress caused by salt stress (mainly Na + and Cl - ) seriously limits the yield and quality of corn, so the identification of corn Na + and Cl - New genes related to transport regulation are of great significance for improving the salt tolerance of maize. ZmNRT1.1A, a member of the maize nitrate transporter NRT1 / PTR family, has Cl - The transport function may play an important role in the process of corn resisting salt stress.

[0022] In order to verify the function of ZmNRT1.1A, the inventors (1) used CRSPR-Cas9 technology to construct a gene knockout mutant of ZmNRT1.1A, such as Figure 1 (2) The PCR fragment containing the target sequence and the pBUE411 vector were digested and ligated with BsaI, and the ligation product was transformed into Escherichia coli. The colony PCR amplification was performed with universal primers FD3 / RD. The colonies with the target size bands detected by electrophoresis were positive clones. The corresponding bacterial liquid was sent to Beijing Liuhe BGI Biotechnology Co., Ltd. for sequencing to obtain the pBUE411-ZmNRT1.1A vector ( Figure 1 (A) pBUE411-ZmNRT1.1A was transferred into Agrobacterium EHA105; (3) transgenic plants were obtained by embryo infection; (4) transgenic positive plants were confirmed by PCR amplification and sequencing of gene fragments including the target site. Finally, two knockout materials of ZmNRT1.1A candidate genes were obtained (named ZmNRT1.1A crispr -1 and ZmNRT1.1A crispr -2)( Figure 1 BD part).

[0023] Wild-type and ZmNRT1.1A mutant seeds were planted under control and 100 mM NaCl conditions. After 14 days of growth, the salt phenotype of corn seedlings was recorded (e.g. Figure 2 At the same time, the biomass of wild-type and ZmNRT1.1A mutant plants was measured. The specific experimental process is as follows: (1) Take materials: Take the aboveground parts of the corresponding plants, weigh and record the fresh weight. (2) Calculate the biomass reduction percentage of the control and 100mM NaCl treatment conditions according to the formula: Biomass reduction percentage = (m 对照 -m 盐处理 ) / m 对照 × 100%. Figure 2 As shown, compared with the wild type, the salt resistance of the ZmNRT1.1A mutant was reduced, indicating that ZmNRT1.1A positively regulates maize salt tolerance.

[0024] Example 2 Functional analysis of maize salt resistance gene ZmNRT1.1A

[0025] ZmNRT1.1A belongs to the maize nitrate transporter NRT1 / PTR family and has Cl - In order to verify the salt stress-sensitive phenotype of the ZmNRT1.1A mutant and its relationship with Cl -Stress-related, the inventors planted wild-type and ZmNRT1.1A mutant seeds under control, 100 mM NaCl, 50 mM Na2SO4 and 100 mM KCl treatment conditions, and took photos of the maize seedlings after 14 days of growth (such as Figure 3 At the same time, the biomass of wild-type and ZmNRT1.1A mutant plants was measured using the same method as in Example 1. Figure 3 As shown in the figure, under the control and 50 mM Na2SO4 treatment conditions, the wild type and the ZmNRT1.1A mutant grew in the same way; under the 100 mM NaCl and 100 mM KCl treatment conditions, the salt resistance of ZmNRT1.1A decreased significantly, indicating that the salt stress sensitive phenotype of the ZmNRT1.1A mutant is caused by Cl - Caused by coercion.

[0026] Example 3 Related sequences

[0027] SEQ ID NO.1

[0028] ATGGTCGGACTCCTCCCCGAGACCAATGCCGCGGCGGAGACGGACGTCCTCCTCGACGCCTGGGACTTCAAGGGCCGGCC

[0029] GGCCCCGCGCGCCACCACCGGCCGCTGGGGCGCCGCCGCCATGATCCTAGTGGCGGAGCTGAACGAGCGGCTGACGACGC

[0030] TGGGCATCGCCGTGAACCTGGTGACGTACCTGACGGGCACCATGCACCTGGGCAACGCCGAGTCCGCCAACGTCGTCACC

[0031] AACTTCATGGGCACCTCCTTCATGCTCTGCCTCCTCGGCGGCTTCGTCGCCGACTCCTTCCTCGGCCGCTACCTCACCAT

[0032] CGCCATCTTCACCGCCGTCCAGGCCTCGGGCGTGACGATCCTGACGATCTCGACGGCGGCGCCGGGGCTACGGCCGGCGT

[0033] CCTGCTCCGCGACCGGCGGAGGCGTCGTCGGGGAGTGCGCGCGGGCGTCGGGGGCGCAGCTGGGGGTGCTGTACCTGGCG

[0034] CTGTACCTGACGGCGCTGGGCACGGGTGGGCTAAAGTCGAGCGTGTCGGGGTTCGGGTCGGACCAGTTCGACGAGTCGGA

[0035] CGGCGGGGAGAAGCGGCAGATGATGCGCTTCTTCAACTGGTTCTTCTTCTTCATCTCGCTGGGGTCGCTGCTGGCCGTCA

[0036] CCGTGCTGGTGTACGTCCAGGACAACCTGGGCAGGCGCTGGGGCTACGGCGCCTGCGCCTGCGCCATCGCCGCGGGCCTC

[0037] CTCGTCTTCCTGGCCGGCACACGCAGGTACCGCTTCAAGAAGCTGGCCGGCAGCCCCCTCACGCAGATCGCCGCCGTCGT

[0038] CGTCGCCGCCTGGCGCAAGCGCCGCCTCCCTCTCCCCGCCGACCCCGCCATGCTCTACGACGTCGACGTCGGCAAGGCCG

[0039] CCGCCGTCGAGGATGGGTCCTCCAGCAAGAAGAGCAAGCGCAAGGAGCGCCTCCCCCACACCGACCAGTTCCGCTTCCTG

[0040] GACCACGCGGCGATCAACGAGGATCCGGCGGCGGGGGCGAGCAGCAGCAGCAAGTGGCGGCTGGCGACGCTGACGGACGT

[0041] GGAGGAGGTGAAGACGGTGGCGCGGATGCTGCCGATCTGGGCGACCACGATCATGTTCTGGACGGTGTACGCGCAGATGA

[0042] CCACCTTCTCGGTGTCGCAGGCCACCACCATGGACCGCCGCGTCGGGGGCTCGTTCCAGATCCCCGCGGGCTCCCTCACC

[0043] GTCTTCTTCGTCGGCTCCATCCTGCTCACCGTGCCCGTCTACGACCGCCTGGTGGTGCCCGTCGCGCGCCGCGTCAGCGG

[0044] CAACCCGCACGGCCTCACCCCGCTGCAGCGGATCGCCGTCGGCCTCGCGCTCTCCGTCGTCGCCATGGCGGGCGCCGCGC

[0045] TCACGGAGGTCCGCCGCCTCCGCGTCGCGCGCGATTCCTCCGAGTCCGCCTCCGGAGGCGTCGTGCCCATGTCCGTGTTC

[0046] TGGCTCATCCCGCAGTTCTTCCTCGTGGGGGCGGGCGAGGCGTTCACGTACATCGGCCAGCTCGACTTCTTCCTGCGCGA

[0047] GTGCCCCAAGGGGATGAAGACCATGAGCACGGGGCTGTTCCTCAGCACCCTGTCGCTGGGATTCTTCGTCAGCTCCGCGC

[0048] TCGTCGCCGCCGTGCACAGGGTCACGGGCGACCGCCACCCCTGGATCGCCAACGACCTCAACAAGGGCCGCCTCGACAAC

[0049] TTCTACTGGCTGCTCGCCGCCGTCTGCCTCGCCAACCTACTAGTCTACCTCGTCGCCGCCCGCTGGTACAAGTACAAGGC

[0050] GGGCCGCCCCGGCGCCGACGGCAGCGTCAACGGCGTCGAGATGGCCGACGAGCCCACGCTCCACTGASEQ ID NO.2

[0051] MVGLLPETNAAAETDVLLDAWDFKGRPAPRATTGRWGAAAMILVAELNERLTTLGIAVNLVTYLTGTMHLGNAESANVVT

[0052] NFMGTSFMCLLLGGFVADSFLGRYLTIAIFTAVQASGVTILTISTAAPGLRPASCSATGGGVVGECARASGAQLGVLYLA

[0053] LYLTALGTGGLKSSVSGFGSDQFDESDGGEKRQMMRFFNWFFFFISLGSLLAVTVLVYVQDNLGRRWGYGACACAIAAGL

[0054] LVFLAGTRRYRFKKLAGSPLTQIAAVVVAAWRKRRLPLPADPAMLYDVDVGKAAAVEDGSSKKSKRKERLPHTDQFRFL

[0055] DHAAINEDPAAGASSSSKWRLATLTDVEEVKTVARMLPIWATTIMFWTVYAQMTTFSVSQATTMDRRVGGSFQIPAGSLT

[0056] VFFVGSILLTVPVYDRLVVPVARRVSGNPHGLTPLQRIAVGLALSVVAMAGAALTEVRRLRVARDSSESASGGVVPMSVF

[0057] WLIPQFFLVGAGEAFTYIGQLDFFLRECPKGMKTMSTGLFLSTLSLGFFVSSALVAAVHRVTGDRHPWIANDLNKGRLDN

[0058] FYWLLAAVCLANLLVYLVAARWYKYKAGRPGADGSVNGVEMADEPTLH

[0059] SEQ ID NO.3

[0060] CTCCCCGAGACCAATGCCG.

Claims

1. A nitrate transporter gene, characterized in that The nitrate transporter gene is from corn, and its nucleotide sequence is SEQ ID NO.

1.

2. The nitrate transporter gene according to claim 1, wherein the nitrate transporter gene is located at 81,342,176-81,348,355 kb on chromosome 10 of the maize V5 reference genome.

3. The corn nitrate transporter encoded by the nitrate transporter gene according to claim 1, characterized in that ,, the amino acid sequence of the corn nitrate transporter is SEQ ID NO.

2.

4. Use of a nitrate transporter gene according to claim 1 or a nitrate transporter according to claim 2 in regulating salt resistance of corn.

5. The use according to claim 4, wherein the expression level of the nitrate transporter gene is knocked out or knocked down to reduce the salt resistance of corn.

6. The use according to claim 5, wherein the knockout is performed by the CRISPR-Cas9 method.

7. The use according to claim 6, wherein the gRNA nucleotide sequence used in the CRISPR-Cas9 method is SEQ ID NO.

3.

8. The use according to claim 4, wherein the expression level of the nitrate transporter gene is increased to improve the salt resistance of corn.

9. The use according to any one of claims 4 to 8, wherein the salt resistance is Cl resistance. - Stress performance.

10. Use of a nitrate transporter gene according to claim 1 or a nitrate transporter according to claim 2 in salt-resistant corn breeding.

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