Pen container tree SlbHLH1 gene and application thereof in improving plant salt-alkali stress tolerance

Abstract

The pen container tree SlbHLH1 gene and application thereof in improving plant salt-alkali stress tolerance capability belong to the technical field of genetic engineering. In order to improve the salt-alkali tolerance of the pen container tree, the bHLH transcription factor gene of the pen container tree is cloned, the pen container tree SlbHLH1 gene is transformed into wild-type Arabidopsis, the trans-SlbHLH1 gene Arabidopsis is subjected to exogenous salt solution and alkali solution stress treatment, the phenotype of the transgenic Arabidopsis is observed, and the physiological indexes of the transgenic Arabidopsis under salt and alkali stress are determined, and it is found that the overexpression of the SlbHLH1 gene can significantly promote the growth and development of the Arabidopsis under salt and alkali environments, improve the activities of SOD, POD and CAT in the Arabidopsis under salt and alkali stress, and reduce the content of malondialdehyde. The pen container tree SlbHLH1 gene provided by the application is expected to be used for cultivating the pen container tree with salt-alkali stress tolerance capability, and has important forestry application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a Sphaeropteris lepifera (Hook.) R.M. Tryon SlbHLH1 gene and application thereof in improving plant salt-alkali stress tolerance. BACKGROUND

[0002] Sphaeropteris lepifera (Hook.) R.M. Tryon, as a tree fern of the genus Sphaeropteris in Cyatheaceae, is known as the "living fossil" in the plant kingdom. This rare plant suffered heavy damage in the Quaternary glacial period, and at present, only a small amount of it is left in southern China and some parts of Southeast Asia. Due to its medicinal value, ornamental value and importance to ecological research, Sphaeropteris lepifera has attracted much attention from people, and has been listed as a national second-class protected plant and included in the international endangered species protection list by the World Conservation Union.

[0003] Although Sphaeropteris lepifera has a high requirement for the living environment and prefers a humid and warm environment, relatively little research has been conducted on its adaptability and stress resistance. In particular, no research report has been found on the salt-alkali tolerance gene of Sphaeropteris lepifera and the application of the gene in improving the salt-alkali stress tolerance of plants. Salt-alkali stress is one of the common abiotic stresses in agricultural and forestry production, and seriously affects the growth and yield of plants. Therefore, finding a gene that can improve the salt-alkali tolerance of plants is of great significance for the protection and utilization of this rare plant resource. SUMMARY

[0004] In order to improve the salt-alkali tolerance of Sphaeropteris lepifera and improve its adaptability and stress resistance, the present application successfully cloned a bHLH transcription factor gene that can improve the salt-alkali stress tolerance of Sphaeropteris lepifera, and the SlbHLH1 gene can significantly promote the growth and development of Sphaeropteris lepifera under salt-alkali stress, thereby providing a new strategy for the protection and sustainable utilization of this rare plant.

[0005] To solve the above technical problems and achieve the corresponding technical effects, the present application specifically provides the following technical solutions:

[0006] The first object of the present application is to provide a Sphaeropteris lepifera SlbHLH1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2.

[0007] The second object of the present application is to provide the application of overexpression of the above-mentioned Sphaeropteris lepifera SlbHLH1 gene in improving the salt-alkali stress tolerance of plants.

[0008] In an embodiment of the present application, the improvement of the salt-alkali stress tolerance of the plant refers to promoting the growth and development of the plant under salt-alkali stress, improving the activities of superoxide dismutase, peroxidase and catalase in the plant under salt-alkali stress, and reducing the content of malondialdehyde.

[0009] A third object of the present application is to provide a recombinant vector containing the above-mentioned Balfourodendron ruddae SlbHLH1 gene.

[0010] A fourth object of the present application is to provide the use of the above-mentioned recombinant vector in improving the salt-alkali stress tolerance of the plant.

[0011] In an embodiment of the present application, the improvement of the salt-alkali stress tolerance of the plant refers to promoting the growth and development of the plant under salt-alkali stress, improving the activities of superoxide dismutase, peroxidase and catalase in the plant under salt-alkali stress, and reducing the content of malondialdehyde.

[0012] A fifth object of the present application is to provide a recombinant bacterium containing the above-mentioned Balfourodendron ruddae SlbHLH1 gene or the above-mentioned recombinant vector.

[0013] A sixth object of the present application is to provide the use of the above-mentioned recombinant bacterium in improving the salt-alkali stress tolerance of the plant.

[0014] In an embodiment of the present application, the improvement of the salt-alkali stress tolerance of the plant refers to promoting the growth and development of the plant under salt-alkali stress, improving the activities of superoxide dismutase, peroxidase and catalase in the plant under salt-alkali stress, and reducing the content of malondialdehyde.

[0015] A seventh object of the present application is to provide a method for cultivating a plant with salt-alkali stress tolerance, which specifically constructs a transgenic plant overexpressing the SlbHLH1 gene by using the above-mentioned Balfourodendron ruddae SlbHLH1 gene.

[0016] The present application has the following beneficial effects:

[0017] The application successfully clones the bHLH transcription factor gene of Stylocarya gymnacarpa, transforms the SlbHLH1 gene of Stylocarya gymnacarpa into wild-type Arabidopsis by constructing a recombinant expression vector pBI121-SlbHLH1 containing the SlbHLH1 gene of Stylocarya gymnacarpa, and obtains T3 generation of Arabidopsis thaliana plants transformed with the SlbHLH1 gene through screening. The transgenic Arabidopsis thaliana is subjected to salt solution and alkali solution stress treatment, the phenotype of the transgenic Arabidopsis thaliana is observed, and the physiological indexes of the transgenic Arabidopsis thaliana under salt and alkali stress are measured, and it is found that the overexpression of the SlbHLH1 gene can significantly promote the growth and development of Arabidopsis thaliana under salt and alkali stress, improve the activities of superoxide dismutase, peroxidase and catalase in Arabidopsis thaliana under salt and alkali stress, and reduce the content of malondialdehyde. The SlbHLH1 gene of Stylocarya gymnacarpa provided by the application is expected to be used for cultivating Stylocarya gymnacarpa with salt and alkali stress resistance, and provides a new strategy for the protection and sustainable utilization of the rare plant Stylocarya gymnacarpa, and has important forestry application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is an agarose gel electrophoresis detection result graph of the PCR product of the SlbHLH1 gene of Stylocarya gymnacarpa, wherein M is Marker DL2000, 1 and 2 are both the PCR product of the SlbHLH1 gene of Stylocarya gymnacarpa;

[0019] Figure 2 It is a structural schematic diagram of the recombinant vector pBI121-SlbHLH1 containing the SlbHLH1 gene of Stylocarya gymnacarpa;

[0020] Figure 3 It is a result graph of identification of whether the T3 generation of Arabidopsis thaliana successfully introduces the SlbHLH1 gene, wherein M is Marker DL2000, lane 1 is a positive control, and lanes 2, 3, 4, 5, 6, 7 and 8 are respectively the genomic DNA amplification products of transgenic lines OE#1, OE#2, OE#3, OE#4, OE#5, OE#6, OE#7 and OE#8;

[0021] Figure 4 It is a phenotype graph of the transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after salt stress and alkali stress;

[0022] Figure 5 It is a detection result graph of the SOD activity in the transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana under salt and alkali stress and alkali stress, wherein N is a water control group, Salt is a salt stress treatment, and Alkali is an alkali stress treatment, and different letters represent significant differences;

[0023] Figure 6Figure of the detection results of the POD activity in the transgenic Arabidopsis and wild-type Arabidopsis under salt-alkali stress and alkali stress; wherein, N is the water control group, Salt is the salt stress treatment, Alkali is the alkali stress treatment, and different letters represent significant differences;

[0024] Figure 7 Figure of the detection results of the CAT activity in the transgenic Arabidopsis and wild-type Arabidopsis under salt-alkali stress and alkali stress; wherein, N is the water control group, Salt is the salt stress treatment, Alkali is the alkali stress treatment, and different letters represent significant differences;

[0025] Figure 8 Figure of the detection results of the MDA content in the transgenic Arabidopsis and wild-type Arabidopsis under salt-alkali stress and alkali stress; wherein, N is the water control group, Salt is the salt stress treatment, Alkali is the alkali stress treatment, and different letters represent significant differences. DETAILED DESCRIPTION

[0026] The application will be further described below in combination with specific embodiments and drawings. The following examples facilitate better understanding of the application, but are not used to limit the application. In the following examples, the experimental methods are conventional methods unless otherwise specified. The medicines, reagents and materials used in the following examples can be purchased through commercial channels unless otherwise specified.

[0027] The plant seeds involved in the application are as follows:

[0028] The wild-type Arabidopsis seeds (Arabidopsis thaliana) are preserved by the laboratory.

[0029] The vectors involved in the application are as follows:

[0030] The pBI121 expression vector can be purchased through a commercial channel and is preserved by the laboratory.

[0031] The strains involved in the application are as follows:

[0032] Agrobacterium tumefaciens LBA4404 is recorded in the following literature: Chen Zhongjian, Liu Bing, Wang Hongbin, Wang Jinfa, Liu Liang. (2003) Expression of gusA in rice callus mediated by rice repetitive sequence RRD3 deletion. Tropical and Subtropical Botany, II (2): 127-131, and is preserved by the laboratory.

[0033] Escherichia coli DH5α is preserved by the laboratory.

[0034] The reagents involved in the application are as follows:

[0035] T4 DNA ligase, BamH I, Xba I were purchased from Bosheng Engineering Co., Ltd. of Dalian; TIANprep Rapid Mini Plasmid Kit was purchased from Tiangen Bioscience Co., Ltd.; Gel Extraction Kit and 2x E-Taq PCR Master Mix were purchased from Hangzhou Xinjing Biological Technology Co., Ltd.; Plant DNA extraction kit was purchased from SIMGEN company; Universal Plant RNA Extraction Kit (DNase I) was purchased from Kangwei Century Company; HiFiScript cDNA Synthesis Kit was purchased from Kangwei Century Company.

[0036] Example 1: Cloning of SlbHLH1 gene of Stychnospsis suaveolens

[0037] (1) Extraction of Stychnospsis suaveolens RNA

[0038] The gametophyte of Stychnospsis suaveolens stored at -80℃ was taken out and placed in a mortar in an ice box. After adding liquid nitrogen, it was quickly ground into white powder. The universal plant RNA extraction kit (DNase I) was used to extract RNA according to the instructions, and agarose gel electrophoresis was used for detection. The RNA with clear bands and correct positions was stored in a -80℃ refrigerator to prevent degradation.

[0039] (2) Reverse transcription to obtain cDNA

[0040] The RNA obtained in step (1) was taken out and placed in an ice box. The HiFiScript cDNA Synthesis Kit was used for reverse transcription. The reaction system of reverse transcription was as follows: 4 mmol / L dNTP Mix 4 μL, 5x RT Buffer 4 μL, Primer Mix 2 μL, RNA 3 μL, 0.1 M DTT 2 μL, 200 U / μL HiFiScript 1 μL, 3-TY(dT) 1 μL, RNase-Free Water 3 μL. The reaction conditions of reverse transcription were 42℃ incubation for 40 min and 85℃ incubation for 5 min. The product obtained by reverse transcription was stored in a -20℃ refrigerator for subsequent experiments.

[0041] (3) Cloning of target gene

[0042] According to the genomic sequence information of the penicula quinquefolia provided by the gene bank, specific primers were designed at both ends of the target gene sequence and were entrusted to a biological company for synthesis, and the primers SlbHLH1-F and SlbHLH1-R for cloning the SlbHLH1 gene were obtained (the nucleotide sequences are shown in Table 1). The penicula quinquefolia DNA obtained in step (2) was taken as a template, and PCR amplification was performed using the above primers, and the amplification system was as follows: 2x Taq MasterMix (Dye) 12.5 μL, SlbHLH1-F 1 μL, SlbHLH1-R 1 μL, DNA template 1 μL, ddH2O 10.5 μL. The obtained PCR product was subjected to agarose gel electrophoresis to separate the target band (see Figure 1 ), and the target fragment was purified using a gel recovery kit. The purified product was ligated with a pMD19T vector to obtain a recombinant plasmid pBI121-SlbHLH1, which was transformed into DH5α competent cells using a heat stress method, and after obtaining positive strains, the plasmid was extracted and sent to a sequencing company for sequencing. The sequencing result showed that the penicula quinquefolia SlbHLH1 cDNA was 1122 bp (the nucleotide sequence is shown as SEQ ID NO. 1), and encoded 373 amino acids (the amino acid sequence is shown as SEQ ID NO. 2).

[0043] Table 1 sequence information of the upper and lower primers for cloning the SlbHLH1 gene

[0044]

[0045] SEQ ID NO. 1:

[0046]

[0047] SEQ ID NO.2:

[0048] MSGNLLLANLVSQGEASSCPQRHSNYIFEDDAMCEEASKLMMWPSWPDLRPPKAPPYDFLPNLLSSSCIAPHHDPELELFRRLVSGGEASCLASHEVGLQGNAFMPLQQQLVANQSTHLGPNKPIPKRGRECGPPNEAKEPNTENFDSSKRQKSLPIKKEEPTASAEDDKGRINTPSGGQARPKSSGIKAKTPTKPAEAHTKSPTKPAEARIKAPAKSAEDPKTDFIHVRARRGQATDSHSLAERVRREKISQRMRFLQDLVPGCSKITGKAMMLDEIINYVQSLQHQVEFLAMKLAAVSPKLDLNFDNFMGEEFATTLGEVNGACHDMVTNSVETQSPSYHIQLQNLCPFDNLRPCDEDLSFISDVSTTSLI

[0049] Example 2: Construction of a plant expression vector containing the SlbHLH1 gene

[0050] The recombinant plasmid pBI121-SlbHLH1 and pBI121 obtained in Example 1 were digested with restriction enzymes BamH I and Xba I, respectively, to obtain a sequence fragment of the SlbHLH1 gene and a digestion product of the pBI121 plasmid. Then the sequence fragment of the SlbHLH1 gene and the digestion product of the pBI121 plasmid were ligated. The ligation reaction system was: 4 μL of the digestion product of the pBI121 plasmid, 3 μL of the sequence fragment of the SlbHLH1 gene, 1 μL of 10×T4 DNA ligation buffer, and 1 μL of T4 DNA ligase. The ligation reaction temperature was 16°C, and the reaction time was 12 h. After ligation, the recombinant vector pBI121-SlbHLH1 was obtained. The structure of the recombinant vector pBI121-SlbHLH1 is shown in FIG. 2. Figure 2 .

[0051] Example 3: Construction of a SlbHLH1 gene overexpressing Arabidopsis thaliana

[0052] The recombinant vector pBI121-SlbHLH1 obtained in Example 2 was transformed into the competent cells of Agrobacterium tumefaciens LBA4404. The pBI121-SlbHLH1 transformed Agrobacterium LBA4404 was cultured in YEB medium at 28°C for 2 days. After centrifugation to remove the supernatant, the cells were resuspended with a 5% sucrose solution. Subsequently, the OD600 of the resuspended cells was adjusted to 0.1.600nm The value is adjusted to 0.6-1.0 to obtain a resuspension, and the inflorescence infection method is used to use the above-mentioned resuspension to infect Arabidopsis thaliana whose buds are about to open to obtain Arabidopsis thaliana carrying the SlbHLH1 gene. The T0 generation Arabidopsis thaliana that is transformed with the SlbHLH1 gene is cultured using the soil culture method, and the T1 generation Arabidopsis thaliana seeds are collected. After the positive transgenic Arabidopsis seeds to be obtained have passed the dormant period, they are germinated using a 1 / 2 culture medium containing Kn, and the operation is repeated until the T3 generation Arabidopsis plants are obtained. PCR is used to identify whether the T3 generation Arabidopsis thaliana has been successfully transformed with the SlbHLH1 gene. Specifically, T3 generation Arabidopsis leaves are taken, and the genomic DNA of each strain is extracted by the CTAB method. The genomic DNA of each T3 generation Arabidopsis thaliana is PCR amplified using the primers SlbHLH1-F and SlbHLH1-R in Example 1 (amplification system isogenic cloning), and then the PCR products are detected by agarose gel electrophoresis to screen positive plants (see Figure 3 ).Depend on Figure 3 It can be seen that the transgenic lines OE#3, OE#5 and OE#7 are positive plants, and the above three positive lines will be used for subsequent research.

[0053] Example 4: Application of the SlbHLH1 gene of the Chinese hollyhock tree in improving the ability of plants to tolerate salt and alkali stress

[0054] To detect the tolerance of T3 generation transgenic SlbHLH1 Arabidopsis to saline-alkali stress, T3 generation transgenic SlbHLH1 Arabidopsis (OE) seeds and wild-type Arabidopsis (WT) seeds were cultured in sterile soil for 50 days. The Arabidopsis leaves were sprayed with nutrient solutions containing 200 mmol / L NaCl (Salt) and 100 mmol / L NaHCO3 (Alkali), respectively. The control group was sprayed with the same volume of water. After 3 days of stress, the phenotypes of the transgenic Arabidopsis and wild-type Arabidopsis were observed, and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the content of malondialdehyde (MDA) in the transgenic and wild-type Arabidopsis were detected (three biological replicates were performed for each sample).

[0055] We observed that after 3 days of salt or alkali stress treatment, the phenotypes of the T3 generation of Arabidopsis thaliana transgenic with SlbHLH1 gene were significantly different from those of wild-type Arabidopsis. The growth of wild-type Arabidopsis was significantly inhibited after salt or alkali stress, while salt or alkali stress had no significant effect on transgenic Arabidopsis. Transgenic Arabidopsis grew well (see Figure 4 ).

[0056] The results of the detection of the superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity and malondialdehyde (MDA) content of the T3 generation of the transgenic Arabidopsis thaliana with the SlbHLH1 gene and the wild-type Arabidopsis thaliana after the salt stress or alkali stress treatment for 3 days are shown in Tables 1 to 4, respectively. Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown in the above results, under the salt stress or alkali stress, the SOD, POD and CAT activities of the transgenic Arabidopsis thaliana are significantly higher than those of the wild-type Arabidopsis thaliana, and the MDA content of the transgenic Arabidopsis thaliana is significantly lower than that of the wild-type Arabidopsis thaliana; when not stressed, the SOD and CAT activities of the transgenic Arabidopsis thaliana are higher than those of the wild-type Arabidopsis thaliana, and the POD activity and MDA content have no obvious difference with those of the wild-type Arabidopsis thaliana. The results further indicate that the Arabidopsis thaliana with the overexpressed SlbHLH1 gene responds to the salt stress and alkali stress, and the overexpression of the SlbHLH1 gene can improve the salt and alkali tolerance of the plant.

[0057] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the present application, and all the modifications or replacements should be included in the scope of the claims of the present application.

Claims

1. A Pen Holder Tree SlbHLH1 A gene characterized by The pen holder tree SlbHLH1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. Overexpression of the pen holder tree described in claim 1 SlbHLH1 Application of genes in improving the tolerance of Arabidopsis to salt and alkali stress.

3. The use according to claim 2, characterized in that Improving the salt-alkali stress tolerance of Arabidopsis thaliana refers to promoting the growth and development of Arabidopsis thaliana under salt-alkali stress, increasing the activities of superoxide dismutase, peroxidase and catalase in Arabidopsis thaliana under salt-alkali stress, and reducing the content of malondialdehyde.

4. A pen holder tree containing the pen holder tree according to claim 1 SlbHLH1 Recombinant gene vector.

5. Use of the recombinant vector according to claim 4 in improving the salt-alkali stress resistance of Arabidopsis thaliana.

6. The use according to claim 5, characterized in that Improving the salt-alkali stress tolerance of Arabidopsis thaliana refers to promoting the growth and development of Arabidopsis thaliana under salt-alkali stress, increasing the activities of superoxide dismutase, peroxidase and catalase in Arabidopsis thaliana under salt-alkali stress, and reducing the content of malondialdehyde.

7. A pen holder tree containing the pen holder tree according to claim 1 SlbHLH1 A recombinant bacterium containing the gene or the recombinant vector according to claim 4.

8. Use of the recombinant bacteria according to claim 7 in improving the salt-alkali stress tolerance of Arabidopsis thaliana.

9. The use according to claim 8, characterized in that Improving the salt-alkali stress tolerance of Arabidopsis thaliana refers to promoting the growth and development of Arabidopsis thaliana under salt-alkali stress, increasing the activities of superoxide dismutase, peroxidase and catalase in Arabidopsis thaliana under salt-alkali stress, and reducing the content of malondialdehyde.

10. A method for cultivating Arabidopsis thaliana with salt-alkali stress tolerance, characterized in that: Utilize the pen holder tree described in claim 1 SlbHLH1 Gene construct overexpression SlbHLH1 Genes of transgenic Arabidopsis thaliana.

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