CN116987164A

CN116987164A - Application of GmNAC46 gene in negative regulation of soybean salt stress response

Info

Publication number: CN116987164A
Application number: CN202310990652.3A
Authority: CN
Inventors: 倪志勇; 于月华; 杨梦诗
Original assignee: Xinjiang Agricultural University
Current assignee: Xinjiang Agricultural University
Priority date: 2023-08-07
Filing date: 2023-08-07
Publication date: 2023-11-03

Abstract

The invention provides an application of GmNAC46 gene in negative regulation of soybean salt stress response, and belongs to the technical field of gene regulation. The invention discovers that the GmNAC46 gene can be used as a negative regulatory factor to participate in the response of soybean roots to salt stress, and the salt tolerance detection discovers that the tolerance of the soybean roots to the salt stress is effectively reduced; the physiological data further show that the GmNAC46 gene is used for reducing the malondialdehyde content of soybeans after salt stress, improving the survival rate of the soybeans under salt stress and increasing the root length and root tip number of the soybeans under salt stress when improving the salt tolerance of the soybeans. The obtained product expands effective solving means for researching the mechanism of soybean root salt stress response molecules and cultivating new salt-tolerant soybean varieties through an RNAi method.

Description

Application of GmNAC46 gene in negative regulation of soybean salt stress response

Technical Field

The invention belongs to the technical field of gene regulation and control, and particularly relates to application of a GmNAC46 gene in negative regulation and control of soybean salt stress response.

Background

The soybean planting originates in China and has a long cultivation history in China. As an important grain and oil crop, soybean plays an extremely important role in agricultural production and planting in China. Meanwhile, soybean is also one of important sources of protein and edible oil in daily life, and is also a crop with more water demand and more sensitivity to water content in the growth and development process. In addition, soybean plays a role in the production of animal feeds and industrial products. In recent years, with the development of economy, the demand for soybeans is increasing, but the total amount of domestic soybeans is far from meeting the domestic demand due to the influence of factors such as reduced planting area and natural environment change. Research on stress resistance mechanism in the growth and development process of soybean, and further cultivation of stress-resistant high-yield varieties are the problems to be solved urgently.

Transcription factors are a class of proteins that bind to specific regions of a target gene promoter and thereby regulate transcription and translation of downstream genes. The transcription factors found in the current research include NAC, MYB and the like, and play an important role in the stress-resistance process of plants when the plants are subjected to various abiotic stresses in natural environments.

The NAC transcription factor family is a plant-specific transcription regulatory factor, and is named by NAM of petunia, ATAF1/2 of Arabidopsis thaliana and CUC2, and the common characteristics of NAC transcription factors are: the N-terminus is a highly conserved NAC domain consisting of about 150 amino acids, and the C-terminus is a diverse transcriptional structure activation domain. Many NAC genes have been cloned, such as 117 genes encoding NAC transcription factors in Arabidopsis, and 151 in rice. Many studies have shown that NAC family transcription factors can be involved not only in the growth and development of plants, such as root growth and plant senescence, through hormone signaling pathways, but also in the response of plants to biotic stress such as insect pests, weeds, etc., and in abiotic stress such as saline-alkali, drought environments, etc. The over-expression of the rice OsNAC2 can enhance the sensitivity of rice to ethylene, thereby affecting the germination and growth of seeds; the drought resistance of the transgenic rice can be obviously improved by over-expressing the rice stress response gene SNAC 1; overexpression of the OsNAC10 gene in rice roots can improve drought tolerance and yield of rice under drought conditions. NAC transcription factors not only regulate plant growth and development, but also respond to biotic and abiotic stresses to plants, but little research has been done on the mechanisms of soybean.

Disclosure of Invention

The invention provides an application of GmNAC46 gene in negative regulation of soybean salt stress response, which can inhibit GmNAC46 gene by RNAi method, and can improve salt tolerance of soybean, reduce malondialdehyde content of soybean after salt stress, improve survival rate of soybean under salt stress, and increase root length and root tip number of soybean under salt stress.

In order to achieve the above purpose, the invention provides an application of GmNAC46 gene in negative regulation of soybean salt stress response, and the nucleotide sequence of the GmNAC46 gene is shown as SEQ ID NO: 1.

Preferably, the amino acid sequence encoded by the above nucleotide is as set forth in SEQ ID NO: 2.

Preferably, in negatively regulating soybean salt stress response, root length and root tip number of soybean under salt stress can be increased.

The invention provides a method for preparing salt stress resistant soybean, which comprises the following steps of carrying out gene RNAi inhibition on a coding region of GmNAC46 gene to cause the loss of GmNAC46 gene function, so as to obtain salt resistant soybean plants, wherein the nucleotide sequence of the salt resistant soybean plants is shown as SEQ ID NO: 1.

The invention provides an application of an RNAi vector for inhibiting GmNAC46 gene in preparing salt-tolerant soybeans, wherein the nucleotide sequence of the RNAi vector is shown as SEQ ID NO: 1.

Compared with the prior art, the invention has the advantages and positive effects that:

the invention discovers that the GmNAC46 gene can be used as a negative regulatory factor to participate in the response of soybean roots to salt stress, and the salt tolerance detection discovers that the tolerance of the soybean roots to the salt stress is effectively reduced; the physiological data further show that the GmNAC46 gene is used for reducing the malondialdehyde content of soybeans after salt stress, improving the survival rate of the soybeans under salt stress and increasing the root length and root tip number of the soybeans under salt stress when improving the salt tolerance of the soybeans. The obtained product expands effective solving means for researching the mechanism of soybean root salt stress response molecules and cultivating new salt-tolerant soybean varieties through an RNAi method.

Drawings

Fig. 1 is an amplification of GmNAC46 gene provided by an embodiment of the present invention, wherein: m: DL2000Marker;1: PCR amplification of GmNAC46 Gene

FIG. 2A is a tissue-specific expression of the GmNAC46 gene provided by an embodiment of the invention; FIG. 2B is a pattern of expression of GmNAC46 in 250mM NaCl stressed soybean roots;

fig. 3 shows amplification of GmNAC46 gene and PCR detection of GmNAC46-pEGAD bacterial liquid provided by the embodiment of the present invention, wherein: a: m: DL2000Marker;1: PCR (polymerase chain reaction) amplification of GmNAC46 genes; b: m: DL2000Marker;1-6: gmNAC46-pEGAD bacterial solution PCR;

FIG. 4 shows amplification of GmNAC46i gene and PCR detection of GmNAC46i-pCAMBIA3301 bacterial liquid provided by the embodiment of the invention, wherein: a: m: DL2000Marker;1: PCR (polymerase chain reaction) amplification of GmNAC46i gene; b: m: DL2000Marker;1-2: PCR (polymerase chain reaction) of GmNAC46i-pCAMBIA3301 bacterial liquid;

FIG. 5 shows PCR detection of an Agrobacterium tumefaciens solution of GmNAC46-pEGAD-K599 and GmNAC46i-pCAMBIA3301-K599 provided by the embodiments of the present invention, wherein: a: m: DL2000Marker;1-2: gmNAC46-pEGAD-K599 Agrobacterium tumefaciens bacterial liquid PCR; b: m: DL2000Marker;1-2: gmNAC46i-pCAMBIA3301-K599 Agrobacterium solution PCR;

FIG. 6 shows the detection of hairy roots of transgenic composite plants according to an embodiment of the present invention, wherein: a: m: DL2000Marker;1-3: detecting DNAPCR of the hairy roots of GmNAC 46; 4-6: detecting K599 hairy root DNAPCR; b: m: DL2000Marker;1-2: detecting K599 hairy root DNAPCR; 3-4: detecting DNAPCR of the hairy root of GmNAC46 i; c: the relative expression quantity of GmNAC46 gene is detected by qPCR of the transgenic plant;

FIG. 7 shows the phenotype and survival rate of a transgenic composite plant according to an embodiment of the present invention, wherein: a: the phenotype of the hydroponic plant before and after salt stress, and the white paper strip represents the length of 2 cm; b: the phenotype of the soil-cultivated plants before and after salt stress; c: survival rate of water-cultured plants after salt stress; d: survival rate of soil-cultivated plants after salt stress;

FIG. 8 shows malondialdehyde content determination of transgenic composite plants provided by an embodiment of the invention;

FIG. 9 is a diagram of a transgenic composite plant hairy root assay according to an embodiment of the present invention, wherein: a: root elongation; b: root tip number.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 GmNAC46 Gene cloning

1.1 extraction of RNA from Soy leaf blades and Synthesis of first strand cDNA

Leaves of soybeans in about 20d of hydroponic culture were placed in liquid nitrogen for quick-freezing and grinding, total RNA of leaf tissue was extracted with TRIzol kit (Invitrogen), RNA quality was detected by 1% agarose gel electrophoresis, RNA concentration was measured by Nanodrop One, and total RNA of leaf was reverse transcribed into cDNA first strand by First strand cDNA Synthesis kit (Thermo Fisher).

1.2GmNAC46 Gene cloning

Primers were designed according to the Glyma.07G048000.1 gene sequence in the soybean genome data in the Phytozome V10.3 database using DNAMAN 8.0:

GmNAC46-F：5'-ATGGATGATGATATTGTAGGACTCGGTT-3'；

GmNAC46-R：5'-TTAATTTTCCCATCTCCATGAAGTGG-3'；

the Open Reading Frame (ORF) of the gene was PCR amplified using the soybean leaf cDNA first strand as a template and TransStart Taq DNA enzyme (Transgen).

PCR amplification system: 1. Mu.L of cDNA, 2. Mu.L of Primer-F, 2. Mu.L of Primer-R, 5. Mu.L of dNTPs, transStart Taq DNA polymerase 0.5.5. Mu.L, 5. Mu.L of 10 XPCR buffer, and 34.5. Mu.L of enzyme-free water.

The PCR procedure was: 94 ℃ for 5min;94 ℃ for 30s,60 ℃ for 45s and 72 ℃ for 90s, and 35 cycles are total; detection was performed by 1% agarose gel electrophoresis at 72℃for 10min. The PCR product is purified and recovered by using a common DNA product purification kit, and the specific steps are described in the specification. The recovered product (1218 bp in size, FIG. 1) was ligated to pMD18-T vector and transformed into E.coli DH 5. Alpha. Competent cells, and the positive clones obtained by bacterial liquid PCR detection were sent to Shanghai Bioengineering Co., ltd for sequencing. After sequencing, the correct bacterial liquid extracting plasmid is successfully cloned by sequencing verification according to the specification of a small extracting kit of the Tiangen plasmid, the nucleotide sequence of the GmNAC46 gene is shown as SEQ ID NO. 1, the coded amino acid sequence of the GmNAC46 gene is shown as SEQ ID NO. 2, and the plasmid is marked as GmNAC46-pMD18-T. As shown in FIG. 1, lane 1 is the result of PCR amplification of the GmNAC46 gene, indicating that the GmNAC46 gene has been successfully cloned and inserted into the pMD18-T vector.

Example 2 transcriptional level analysis of GmNAC46 in salt stressed soybean roots

To identify whether GmNAC46 is involved in soybean salt stress response. Extracting soybean roots, stems, leaves and cotyledon tissues of hydroponic 20d respectively, grinding and extracting RNA after quick freezing by liquid nitrogen, reversely transcribing into cDNA, and analyzing the expression quantity of GmNAC46 gene by adopting a qRT-PCR method by taking CYP2 as an internal reference gene. The qRT-PCR primers are as follows:

nGmNAC46-qF：5'-GATCTTTGCGATGTGGAACCTTG-3'；

nGmNAC46-qR：5'-AAGGTCCTAACTTCACGGTCTACTCCAG-3'。

the transcription level of GmNAC46 in soybean roots, stems, leaves and cotyledons was analyzed by using a real-time fluorescent quantitative PCR technique, and as shown in fig. 2A, gmNAC46 was expressed in all the above four tissues, and the expression level of GmNAC46 was highest in roots.

Since GmNAC46 is expressed in the highest amount in soybean roots, salt stress was selected on the roots to analyze the transcript levels of GmNAC46 in salt stressed soybean roots. Soybean roots 0h,0.5h,2h,6h,12h and 24h after 250mM NaCl stress. The soybean root RNA after stress is extracted, the expression quantity of the soybean root RNA under the salt stress is analyzed by using a real-time fluorescence quantitative PCR technology, and the result is shown in a figure 2B, and the relative expression quantity reaches the highest value at 12h after the relative expression quantity of the gene under the salt stress rises and slightly drops. These results indicate that GmNAC46 induces expression in response to salt stress.

EXAMPLE 3 construction of GmNAC46 overexpression vector

Analyzing enzyme cutting sites of pEGAD vector sequence and GmNAC46, selecting forward SmaI and reverse BamHI enzyme cutting sites, selecting a method for increasing corresponding enzyme cutting sites to amplify the sequence, wherein a GmNAC46 primer (both synthesized by Shanghai Biotechnology Co., ltd.) is as follows:

nGmNAC46-256F:5'-TTTCCCGGGATGGATGATGATATTGTAGGACTCGGTT-3'；

nGmNAC46-256R:5'-ATAGGATCCTTAATTTTCCCATCTCCATGAAGTGG-3'。

PCR reaction system: gmNAC120-pMD18-T plasmid 0.2. Mu.L, primer-F2. Mu.L, primer-R2. Mu.L, dNTPs 5. Mu.L, transStart Taq DNA polymerase 0.5.5. Mu.L, 10 XPCR buffer 5. Mu.L, and enzyme-free water 35.3. Mu.L.

PCR reaction procedure: 94 ℃ for 5min;94 ℃ for 30s,60 ℃ for 45s and 72 ℃ for 90s, and 35 cycles are total; 72 ℃ for 10min

After the PCR products were identified correctly by 1% agarose gel electrophoresis (FIG. 3 a), they were purified and recovered using the Tian Gen DNA product recovery kit, and simultaneously double-digested with pEGAD plasmid, enzyme-digested system:

GmNAC46 PCR product or pEGAD plasmid 10. Mu.L, sma I2. Mu.L, bamHI 2. Mu.L, 10 Xbuffer 5. Mu.L, no enzyme water 31. Mu.L, cleavage reaction conditions: and the enzyme digestion is carried out for 2.5 hours at 37 ℃.

After the cleavage, the cleavage product was purified and recovered using a Tiangen DNA product recovery kit, and ligated with T4 ligase.

The connection system is as follows: 10. Mu.L of GmNAC46 double cleavage product, 5. Mu.L of pEGAD plasmid double cleavage product, 1. Mu.L of T4 DNA ligation buffer, 2. Mu.L of T4 ligation buffer, and 2. Mu.L of enzyme-free water. Reaction conditions: and the connection is carried out for 3 to 4 hours at the temperature of 22 ℃.

Mixing 10 μl of the ligation product with 100 μl of DH5 α E.coli competent in ultraviolet ultra-clean bench, standing on ice for 30min, placing in a water bath at 42deg.C for 90s, rapidly placing in ice for 3min, adding 600 μl of antibiotic-free liquid LB, placing in a 37 ℃ and 220rpm/min incubator for 45min, centrifuging at 4000rpm/min in a centrifuge for 2min, absorbing 500 μl of supernatant, discarding the rest liquid, coating in solid culture medium containing Amp antibiotics, and placing in a 37 ℃ incubator for overnight culture.

The monoclonal bacterial plaque is selected, 800 mu L of liquid culture medium containing kanamycin antibiotics is added, the liquid culture medium is put into an incubator at 37 ℃ and 220rpm/min and is slightly shaken for 6-7 hours, 1 mu L of bacterial liquid is taken as a template, the primer is a universal primer of a carrier, and the reaction system and the reaction program are as described above. After the identity of the bacteria by PCR (FIG. 3 b), the positive clones were sent to Shanghai Biotechnology Co., ltd for sequencing.

Comparing the sequencing result with the gene sequence, extracting the plasmid by a plasmid small extraction kit after the comparison is correct, and referring to the instruction book of the kit for the steps. And (5) after the plasmid is successfully extracted, placing the plasmid into a temperature of minus 20 ℃ for preservation.

Sequencing results showed that: the construction of the plant expression vector for the overexpression of GmNAC46-pEGAD is completed.

EXAMPLE 4 construction of GmNAC46 inhibition vector

A fragment of 21bp in length was selected from the open reading frame sequence of the GmNAC46 gene as RNAi fragment. And analyzing the restriction enzyme sites of the GmNAC46-RNAi fragment and the pCAMBIA3301 vector, selecting forward Nco I and reverse Bgl II restriction enzyme sites, and selecting a method for adding corresponding restriction enzyme sites to artificially synthesize sequences.

GmNAC46i-F：

5'-CATGAGAGGAAATGCAGACTTCATTTTCAAGAGAAATGAAGTCTGCATTTCCTCT-3'；

GmNAC46i-R：

5'-GATCAGAGGAAATGCAGACTTCATTTCTCTTGAAAATGAAGTCTGCATTTCCTCT-3'。

Synthesizing genes by oligonucleotide annealing, and reacting the system: 10 XPCR buffer 5. Mu.L, gmNAC46 i-F10. Mu.L, gmNAC46 i-R10. Mu.L, 25. Mu.L of enzyme free water.

The reaction procedure: 95℃for 5min,75℃for 10min,65℃for 10min and 55℃for 10min; 10min at 4 ℃.

The oligonucleotides were annealed to synthesize GmNAC46-RNAi gene with cleavage sites, the size of amplified bands was expected as a result of agarose gel electrophoresis (FIG. 4 a), and the amplified bands were transformed by ligation with the pCAMBIA3301 plasmid recovered by cleavage (FIG. 4 b), and positive clones were identified and screened by bacterial liquid PCR (FIG. 4 b), and sequencing results showed that: the suppression vector GmNAC46i-pCAMBIA3301 has been successfully constructed.

Example 5 function of GmNAC46 under salt stress

5.1 transformation of Soy hairy roots

The plasmids of the overexpression vector GmNAC46-pEGAD and the inhibition vector GmNAC46i-pCAMBIA3301 which are successfully sequenced are extracted, K599 competent cells are transformed, and bacterial liquid PCR is used for identifying and screening positive clones (figure 5) for generating hairy roots in the subsequent transformation of soybeans.

Molecular level identification is carried out on a soybean plant transformed with a complex of the over-expressed GmNAC46, K599 and GmNAC46i vectors, the DNA of the complex soybean hairy roots is extracted as a template, PCR identification is carried out by using a vector universal primer, and the gel electrophoresis result is that: the K599 control group plants did not amplify bands, and the target bands were amplified from both the GmNAC46 and GmNAC46i composite plants, indicating that the target genes had been successfully transformed into soybean hairy roots.

RNA of transgenic soybean hairy roots is extracted, and the relative expression amount of GmNAC46 genes in the composite plants of the over-expressed GmNAC46, K599 and GmNAC46i is measured, so that the results show that: the relative expression level of the GmNAC46 gene in the overexpressed GmNAC46 plants was higher than that in the control plants, and the relative expression level of the GmNAC46 gene in the GmNAC46i composite plants was lower than that in the control plants (fig. 6).

5.2 identification of salt tolerance of Complex soybean

To study the phenotype and survival rate of GmNAC46 and GmNAC46 i-transformed soybean under salt stress, complex plants with more consistent growth vigor around 20d after transformation were selected, and stressed for 24h with 250mM NaCl, and as a result, the following findings: after the same time of salt stress treatment, the leaf wilting degree of the complex plant of GmNAC46i is lower, the survival rate is 65.72%, the survival rate of the control plant is 52.65%, the leaf wilting degree of the complex plant over-expressing GmNAC46 is highest, and the survival rate is 36.11%.

Culturing the composite soybean plants transferred into soil culture for 20d, pouring 250mM NaCl stress liquid for one week, recording the phenotype difference before and after salt stress and the survival rate of the plants after stress, and finding out the analysis result: the soil culture trend was similar to that of water culture, leaf wilting of the complex plants overexpressing GmNAC46 was higher, survival was 32.1%, survival of control group plants was 42.43%, leaf wilting of the complex plants of GmNAC46i was lower, and survival was 56.11% (fig. 7).

These results demonstrate that over-expression of GmNAC46 reduces salt tolerance of the composite plant and that GmNAC46i improves salt tolerance of the composite plant.

5.3 physical index before and after salt stress of the compound soybean and root morphology statistics

Hairy roots of GmNAC46, K599 and GmNAC46 i-transformed soybean plants before and after 250mM NaCl stress treatment are taken for MDA index measurement. The results show that: the MDA content of the hairy roots of the GmNAC46, K599 and GmNAC46i composite soybean plants before stress is not obviously different; after salt stress, the MDA content in the overexpressing GmNAC46 composite plants was higher than the K599 control group, and the MDA content in the GmNAC46i composite plants was lower than the K599 control group (fig. 8); after salt stress, the root elongation and root tip number of the overexpressing GmNAC46 composite plants were both lower than those of the K599 control group, and the GmNAC46i plants were both higher than those of the K599 control group (fig. 9).

These results indicate that over-expression of GmNAC46 reduces salt tolerance of the composite plant, increases the damage degree of the composite plant under salt stress, and the root system growth rate under salt stress is lower than that of the K599 control group, while inhibition of GmNAC46 improves salt tolerance of the composite plant, reduces the damage degree of the composite plant under salt stress, and the root system growth rate under salt stress is higher than that of the K599 control group.

Claims

Application of GmNAC46 gene in negative regulation of soybean salt stress response is characterized in that the nucleotide sequence is shown in SEQ ID NO: 1.
2. The use according to claim 1, wherein the amino acid sequence encoded by said nucleotide is as set forth in SEQ ID NO: 2.
3. The use according to claim 1, wherein the root length and root tip number of soybeans under salt stress can be increased in negative regulation of soybean salt stress response.
4. A method for preparing salt stress resistant soybean, which is characterized in that the coding region of GmNAC46 gene is subjected to gene RNAi inhibition, so that the GmNAC46 gene is lost in function, and a salt resistant soybean plant is obtained, wherein the nucleotide sequence of the salt resistant soybean plant is shown as SEQ ID NO: 1.
5. The application of an RNAi vector for inhibiting GmNAC46 gene in preparing salt-tolerant soybeans is characterized in that the nucleotide sequence of the RNAi vector is shown as SEQ ID NO: 1.