Application of EbbHLH80, a transcription factor from Breviscapus breviscapus, in enhancing salt tolerance in tobacco

Abstract

The invention relates to an application of Erigeron breviscapus transcription factor EbbHLH80 in enhancing the salt tolerance of tobacco and a method for enhancing the salt tolerance of tobacco plants, belonging to the technical field of genetic engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of EbbHLH80, a transcription factor of Breviscapus breviscapus, in enhancing the salt tolerance of tobacco. Background Art

[0002] Erigeron chinensis ( Erigeron breviscapus ,Eb) is a plant of the Asteraceae family. Its rich flavonoids have a strong preventive and therapeutic effect on cardiovascular and cerebrovascular diseases. bHLH It is the second largest class of transcription factors in eukaryotes and is essential for plant growth and development.

[0003] Transcription factors typically regulate gene expression through cooperative mechanisms (Spitz et al., 2012). The most widely studied plant transcription factors include MYB, bHLH, ERF, and NAM. The bHLH protein family is one of the largest families of transcription factors (TFs). These transcription factors are widely distributed in plants, fungi, and animals. Conserved bHLH transcription factors contain two functionally distinct regions: a basic domain and a helix-loop-helix (HLH) domain. The HLH domain, consisting of 40–50 amino acid residues, is essential for dimer formation (Atchley et al., 1999). bHLHs are involved in regulating flavonoid biosynthesis and play a key role in ROS homeostasis under stress.

[0004] Excessive salt in the soil can lead to salt accumulation in the surface layer, forming saline-alkali soils, which can affect plant development and growth. Plants possess a comprehensive antioxidant system. Under adverse conditions, plants' ability to scavenge reactive oxygen species decreases, leading to a decrease in antioxidant capacity. Salt stress harms plants, including osmotic stress, ion toxicity, altered membrane permeability, and physiological and metabolic disorders (Liang et al., 2018; Zhao et al., 2021). Transcription factors have been reported to be involved in stress responses in plants. Their effects on salt stress have been extensively studied (Baillo et al., 2019; Warsi et al., 2021). Overexpression of VvMYBF1, cloned from grapevine, upregulates the expression levels of flavonoid and proline biosynthesis genes under salt stress (Wang et al., 2020). Knocking out OsNAC3 improves salt stress tolerance in rice (Zhang et al., 2021). Transcription factors influence the expression levels of several genes during their regulatory processes. The expression levels of some salt-stress-responsive genes in plants (such as SOD, POD, and proline) increase under salt stress (Wang et al., 2020). Salt stress may also affect the biosynthesis of phenylpropanoid flavonoids in buckwheat. Rutin content is significantly upregulated (Ma et al., 2019). The buckwheat cultivar "BeikaiT10" showed increased levels of quercetin, kaempferol, and sugars under NaCl treatment (Li et al., 2013). Salt stress has been shown to affect the biosynthesis of phenolic compounds in olives (Petridis et al., 2012). Studies have shown that salt damage severely affects phenylpropanoid biosynthesis, leading to changes in the production of several phenolic compounds, including increased production of chlorogenic acid, rutin, kaempferol, luteolin, and gallic acid.

[0005] In the present invention, we further studied the function of EbbHLH80 and found that EbbHLH80 may be a potential gene with salt tolerance. By exogenously adding different concentrations of NaCl to treat wild-type and EbbHLH80-OE tobacco seedlings, we experimentally verified that overexpression of EbbHLH80 in tobacco will improve the salt tolerance of transgenic tobacco. Summary of the Invention

[0006] In order to overcome the problems existing in the background technology, the present invention provides the use of EbbHLH80, a transcription factor of Breviscapus breviscapus, in enhancing the salt tolerance of tobacco.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The present invention provides application of Erigeron breviscapus transcription factor EbbHLH80 in enhancing salt tolerance of tobacco.

[0009] The present invention provides a method for enhancing tobacco salt tolerance using EbbHLH80, a transcription factor from Breviscapus breviscapus, comprising the following steps:

[0010] S1, the CDS of EbbHLH80 was cloned from leaves of 8-week-old Erigeron breviscapus. The CDS fragments of the target gene were recovered, ligated to T vectors after A addition, and transformed into DH5a competent cells. Positive clones were identified by colony PCR. Three positive clones were sequenced and the complete CDS nucleotide sequence of each gene was obtained by sequence assembly. The sequences of the primer pairs for cloning and colony PCR are shown in SEQ ID. 1-2.

[0011] S2: The full-length CDS of the EbbHLH80 gene was digested with BamHI and XbaI, and the 1896-bp CDS product was recovered by electrophoresis and ligated into the overexpression vector PC1300-35S, which had been digested with the same enzymes. Positive clones were identified by colony PCR, and three positive clones were sequenced. The sequences of the primer pairs for colony PCR are shown in SEQ IDs 1-2.

[0012] In step S3, the positive plasmid was transformed into Agrobacterium tumefaciens GV3101, which was then used to transform the tobacco variety Yunyan 87 using the leaf disc method. Regenerated shoots and healthy, resistant shoots were selected on selective and rooting media, and well-developed rooted plants were transferred to soil to obtain salt-tolerant tobacco plants.

[0013] Furthermore, in step S3, the selective culture medium is: MS medium + 0.01 mg / L NAA + 1 mg / L 6-BA, and then 50 mgL-1 kanamycin and 250 mgL-1 carbenicillin are added; the rooting medium is: 1 / 2 MS medium + 50 mgL-1 kanamycin and 250 mgL-1 carbenicillin.

[0014] Beneficial effects of the present invention: The present invention provides an application and method of using the EbbHLH80 transcription factor of Breviscapus breviscapus to enhance the salt tolerance of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the growth status of wild type (WT) and EbbHLH80-OE (OE1, OE5, and OE9) after exogenous addition of different concentrations of salt in Example 2 of the present invention. The scale bar in the figure is 1 cm. The four figures on the top are actual experimental pictures, and the circular figure on the bottom is used to indicate the specific type of tobacco in each direction of the four actual pictures;

[0016] Figure 2 WT and EbbHLH80Schematic diagram of root growth of -OE on 0 mM NaCl and 50 mM NaCl media. The scale bar is 1 cm and the error bars are standard deviations. **P<0.01, *P<0.05.

[0017] Figure 3 The present invention contains 0mM NaCl and 300mM NaCl WT and EbbHLH80 - DAB staining histochemical localization of H2O2 accumulation in OE seedlings. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by technicians.

[0019] Example 1. Plant materials and production conditions

[0020] 1. The present invention uses tobacco obtained from Yunnan Agricultural University ( Nicotiana tabacum L.) (cultivar Yunyan87, wild type as control, WT) and Erigeron breviscapus obtained from Longjin Biotechnology Co., Ltd., Xuanwei City, Yunnan Province ( Erigeron breviscapus ) seeds were used for subsequent research.

[0021] 2. Construction of stable transgenic tobacco lines:

[0022] S1. The CDS of EbbHLH80 was cloned from leaves of 8-week-old Erigeron breviscapus. The CDS fragments of the target gene were recovered, ligated to T vectors after A addition, and transformed into DH5a competent cells. Positive clones were identified by colony PCR. Three positive clones were sequenced and the complete CDS nucleotide sequence of each gene was obtained by sequence assembly. The sequences of the primer pairs for cloning and colony PCR are shown in SEQ ID 1-2 and Table 1 below.

[0023] S2, the full-length CDS containing the EbbHLH80 gene was digested with the endonucleases BamHI and XbaI, and the 1896-bp CDS product was recovered by electrophoresis and ligated into the overexpression vector PC1300-35S that had been digested with the same enzymes. Positive clones were identified by colony PCR, and three positive clones were sequenced. The sequences of the primer pairs for colony PCR are shown in SEQ IDs 1-2 and in Table 1 below.

[0024] In step S3, positive plasmids were transformed into Agrobacterium tumefaciens GV3101, which was then used to transform tobacco cultivar Yunyan 87 using the leaf disc method. Regenerated and healthy resistant shoots were selected on selective medium (MS medium + 0.01 mg / L NAA + 1 mg / L 6-BA, 50 mg / L kanamycin and 250 mg / L carbenicillin) and rooting medium (1 / 2 MS medium + 50 mg / L kanamycin and 250 mg / L carbenicillin). Well-developed rooted plants were transferred to soil to obtain salt-tolerant tobacco plants. Tobacco plants were grown in a growth chamber at 25 ± 2°C, 65-70% relative humidity, and a 16 / 8 hour photoperiod.

[0025] Table 1. Clones EbbHLH80 Primers

[0026]

[0027] 3. Wild-type tobacco Yunyan87 (WT) and overexpression EbbHLH80 Tobacco with the gene overexpressed was grown in a greenhouse at Yunnan Agricultural University. EbbHLH80 After screening positive plants from the tobacco plants carrying the gene, T2 generation plants were obtained.

[0028] Example 2: Salt tolerance experiment of transgenic tobacco

[0029] 1. For research EbbHLH80 To investigate the role of exogenous NaCl in salt tolerance, WT and transgenic tobacco seedlings were treated with different concentrations of NaCl (0, 50, 100, and 150 mM) for 4 weeks.

[0030] As attached Figure 1 As shown, when grown without NaCl, the wild type (WT) and EbbHLH80 No significant differences in seedling growth conditions were observed among the OEs (OE1, OE5, and OE9).

[0031] As attached Figure 1 As shown in the figure, the effect of salt stress on tobacco phenotype is that tobacco seedlings grown in 50, 100 and 150 mM NaCl grow slower. The growth of tobacco is significantly inhibited under salt stress, and the leaves turn yellow. The higher the exogenous salt concentration, the deeper the yellowing of the leaves. However, transgenic tobacco shows better growth than WT under salt stress. When 50 mM NaCl is added exogenously EbbHLH80 -OE (OE1, OE5 and OE9) also had better growth conditions.

[0032] Second, in order to study whether salt stress affects root length, the present invention detected WT and EbbHLH80 -OE root growth on 0 mM NaCl and 50 mM NaCl media.

[0033] The results are as attached Figure 2 As shown in Figures E and H, after 3 weeks of growth on MS plates containing 50 mM NaCl, the root length of WT seedlings was EbbHLH80 -OE root growth was more severely inhibited, while EbbHLH80 -The root length of OE tobacco was longer than that of WT.

[0034] 3. To investigate the differences in root length between WT and EbbHLH80-OE tobacco seedlings in the presence of NaCl, we analyzed the fresh weight, dry weight, and root length of seedlings grown in medium supplemented with 0 mM NaCl and 50 mM NaCl.

[0035] The results are as follows Figure 2 As shown in BD and FH, WT and EbbHLH80 There were no differences in fresh weight, dry weight and root length between WT and OE. However, on the medium containing 50 mM NaCl, EbbHLH80 -There were significant differences in fresh weight, dry weight and root length among OEs. EbbHLH80 -OE showed faster germination than WT in the presence of 50 mM NaCl.

[0036] 4. Determination of H2O2 content in tobacco seedlings after salt stress. Seedlings grown for 2 weeks on 1 / 2 MS medium (0.7% agar) were treated with 300mM NaCl for 1 hour, and the control was treated with the same volume of water. EbbHLH80 -OE seedlings were immersed in 1 mg / mL DAB staining solution and stained in the dark at room temperature for 6 hours. After rinsing in pure water, the stained seedlings were destained by immersion in 95% ethanol at 40°C for 12 hours. After destaining, the seedlings were thoroughly washed and placed in DAB sample storage solution for 30 minutes before being photographed.

[0037] The photo results are as attached Figure 3 As shown: Under adverse conditions, reactive oxygen species can be produced and accumulated in large quantities in plants, causing varying degrees of toxicity to plant cells. Under abiotic stress, plant cells respond to stress through H2O2 signaling (Chen et al., 2022). After 24 h of treatment with 300 mM NaCl, we used 3,3'-diaminobenzidine (DAB) staining to detect hydrogen peroxide levels. It was observed that after 300 mM NaCl treatment, the levels of hydrogen peroxide in WT and EbbHLH80-OE seedlings had different H2O2 accumulation patterns. DAB staining experiments showed that after high concentration NaCl treatment, EbbHLH80 -OE tobacco seedlings contain less H2O2, specifically: Figure 3 The results show that H2O2 accumulated significantly in WT seedlings, while H2O2 accumulated less in OE1, OE5 and OE9 seedlings. EbbHLH80 Reduced salt stress-induced damage in transgenic tobacco seedlings.

[0038] In summary, EbbHLH80 -OE tobacco seedlings (OE1, OE5, and OE9) showed significantly higher salt tolerance compared with WT tobacco seedlings.

[0039] References:

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[0058] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Application of EbbHLH80, a transcription factor from Breviscapus breviscapus, in enhancing salt tolerance in tobacco, characterized in that: The EbbHLH80 is a 1896 bp CDS product obtained by PCR cloning from Erigeron breviscapus leaves using the primer pairs shown in SEQ ID. 1-2.

2. A method for enhancing tobacco salt tolerance using the EbbHLH80 transcription factor, characterized in that The following steps are involved: S1, clone the CDS of EbbHLH80 from 8-week-old Erigeron breviscapus leaves, recover the CDS fragments of the target gene, add A, ligate to T vectors, transform into DH5a competent cells, identify positive clones by colony PCR, sequence three positive clones, and obtain the complete CDS nucleotide sequence of each gene by sequence assembly; the sequences of the primer pairs for cloning and colony PCR are shown in SEQ ID.1-2; S2, the full-length CDS containing the EbbHLH80 gene was digested with endonucleases BamHI and XbaI and cloned from the T-vector. The 1896-bp CDS product was recovered by electrophoresis and ligated into the overexpression vector PC1300-35S that had been digested with the same enzymes. Positive clones were identified by colony PCR, and three positive clones were sequenced. The sequences of the primer pairs for colony PCR are shown in SEQ IDs 1-2. S3, the positive plasmid was transformed into Agrobacterium tumefaciens GV3101, which was then used to transform the tobacco variety Yunyan 87 using the leaf disc method; regenerated buds and healthy resistant buds were selected on selective culture medium and rooting medium, and the well-developed rooted plants were transferred to soil to obtain salt-tolerant tobacco plants.

3. The method according to claim 2, characterized in that The selective culture medium in step S3 is: MS medium + 0.01 mg / L NAA + 1 mg / L 6-BA, and then 50 mg / L-1 kanamycin and 250 mg / L-1 carbenicillin are added; the rooting culture medium is: 1 / 2 MS medium + 50 mg / L-1 kanamycin and 250 mg / L-1 carbenicillin.

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