CN112778408B - Rubber tree transcription factor HbICE2, and coding gene and application thereof - Google Patents

Abstract

The present invention provides a Hevea transcription factor HbICE2, the amino acid sequence of which is shown in SEQ ID NO:1. The invention also provides the coding gene and application of the Hevea transcription factor HbICE2. The gene expression analysis of the present invention shows that HbICE2 gene is up-regulated in response to low temperature stress, and the expression level in rubber tree varieties with strong cold tolerance is significantly higher than that in varieties with weak cold tolerance. It is verified by transgenic that HbICE2 can enhance the cold tolerance and drought tolerance of transgenic plants This is of great importance for the understanding of the resistance mechanism of plants to low temperature stress, salt stress, drought stress, etc. Theoretical significance and practical application value, this gene can be used as an important genetic resource in rubber tree and other plant stress resistance genetic engineering.

Description

Rubber tree transcription factor HbICE2 and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to the rubber tree transcription factor HbICE2 and its coding gene and application.

Background technique

ICE (inducer of CBF expression) is a transcriptional activator induced by CBF. The structure of ICE in plants, in addition to the typical bHLH domain, also has an S-rich region, an NLS region and a transmembrane region, and the bHLH domain contains 19 conserved KMDRASILGDAI(D/E)YLKELL amino acids. Its genes are found in different plants such as tomatoes, cucumbers, tobacco, and bananas, which can improve the cold resistance of plants. Studies have shown that AtICE is a constitutively expressed gene, which encodes MYC-like transcription factors (MYC-like transcription factors) transcription activators containing bHLH. Very important transcriptional regulation.

Rubber tree (Hevea brasiliensis Muell. Arg.) is native to the Amazon River Basin in South America and is a typical tropical rainforest tree species. my country's rubber planting area is located on the northern edge of the tropics, which is a non-traditional rubber planting area and often encounters cold currents. Therefore, high-yield and cold-resistant new varieties are needed in production, but high-yield cold-resistant varieties are extremely scarce. This is due to the long cycle of conventional rubber tree breeding, few hybrid generations, and difficulty in consolidating high-yield and cold-resistant traits; the selection and breeding of rubber tree cold-resistant varieties relies on the identification of field and outpost nurseries, which not only has a long identification cycle and low efficiency, but also consumes a lot of manpower, material resources and land. resource. This project adopts the strategy of forward genetics research, using the established laboratory system for identification of cold resistance of rubber trees and rubber tree clones with known cold resistance as materials, from the aspects of molecular biology and genetic transformation, reveals the low temperature stress of rubber trees. The molecular mechanism of resistance, etc., identified the function of the rubber tree ICE2 gene, which has important theoretical and practical significance for the understanding of the resistance mechanism of plants to low temperature stress under non-dormant conditions and the directional improvement of low temperature stress resistance of high-yielding varieties through transgenic technology Value.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, to provide a rubber tree transcription factor HbICE2 and its coding gene and application.

A first aspect of the present invention provides a Hevea transcription factor HbICE2, the amino acid sequence of which is shown in SEQ ID NO:1.

Second aspect of the present invention provides a kind of Hevea transcription factor HbICE2 gene, its nucleotide sequence is as shown in SEQ ID NO:2.

The third aspect of the present invention is to provide a recombinant vector, which comprises the original vector and the Hevea transcription factor HbICE2 gene described in the second aspect of the present invention.

Wherein, the original vector can be a vector commonly used in the field of gene recombination, such as virus, plasmid and the like. The present invention is not limited thereto. In a specific embodiment of the present invention, the pCAMBIA1302 vector plasmid is used as the original vector, but it should be understood that other plasmids or viruses can also be used in the present invention.

Preferably, the original vector is a pCAMBIA1302 carrier plasmid, and the nucleotide sequence shown in SEQ ID NO: 2 is located between the Spe I and BstEII restriction endonuclease sites of the pCAMBIA1302 carrier plasmid.

The fourth aspect of the present invention provides the rubber tree transcription factor HbICE2 as described in the first aspect of the present invention, or the rubber tree transcription factor HbICE2 gene described in the second aspect of the present invention, or the gene described in the third aspect of the present invention Application of the recombinant vector in improving plant cold tolerance, and/or salt tolerance, and/or drought tolerance.

Wherein, the salt is sodium chloride.

A fifth aspect of the present invention provides a method for improving plant cold tolerance and/or salt tolerance and/or drought tolerance, characterized in that the rubber tree transcription factor HbICE2 described in the second aspect of the present invention Genes, or the recombinant vectors described in the third aspect of the present invention are transfected into plants.

The sixth aspect of the present invention is a pair of primers, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4.

The seventh aspect of the present invention provides a pair of primers, the nucleotide sequences of which are shown in SEQ ID NO:5 and SEQ ID NO:6.

The eighth aspect of the present invention provides a pair of primers, the nucleotide sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

From the aspects of molecular biology and genetic transformation, the present invention reveals the molecular mechanism of rubber tree low temperature stress resistance, identifies the function of rubber tree HbICE2 gene, and gene expression analysis shows that HbICE2 gene responds to low temperature stress. The expression level in the medium was significantly higher than that of the varieties with weak cold tolerance. Through transgenic verification, HbICE2 can enhance the cold tolerance, drought tolerance and salt tolerance of transgenic plants, which is helpful for understanding the response of plants to low temperature stress, salt stress, The resistance mechanism of drought stress and the directional improvement of low temperature stress, salt stress, and drought stress in high-yielding varieties through transgenic technology have important theoretical significance and practical application value. This gene can be used as an important genetic resource in rubber trees and other plants. Applied in reverse genetic engineering.

Description of drawings

Figure 1 is the nucleotide sequence of the HbICE2 gene and its encoded amino acid sequence.

Fig. 2 is the phylogenetic tree analysis of HbICE2 gene.

Fig. 3 shows the expression analysis results of HbICE2 gene in cold-resistant varieties and non-cold-resistant varieties of rubber trees under low temperature conditions.

Figure 4 shows the cold tolerance identification results of Arabidopsis plants overexpressing the HbICE2 gene. In the figure, WT: control, 35::HbICE2: Arabidopsis transgenic for HbICE2.

Fig. 5 shows the salt tolerance identification results of Arabidopsis plants overexpressing the HbICE2 gene. In the figure, WT: control, 35::HbICE2: Arabidopsis transgenic for HbICE2.

Figure 6 shows the drought tolerance identification results of Arabidopsis plants overexpressing the HbICE2 gene. In the figure, WT: control, 35::HbICE2: Arabidopsis transgenic for HbICE2.

Detailed ways

Referring to the accompanying drawings, the present invention will be further described in conjunction with specific embodiments, so as to better understand the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

1. Gene acquisition

Utilize RT-PCR technology to clone and obtain transcription factor HbICE2 gene from the bark of Brasiliensis variety 93-114, and specific embodiment is as follows:

Refer to the operating instructions of Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit to extract the total RNA from rubber tree bark. The first strand of cDNA was synthesized according to the operation steps of the Ferments kit. According to the rubber tree genome database, the Unigene annotated as ICE was obtained, and the complete gene sequence was preliminarily identified by searching and comparing with the NCBI database.

Using the first strand of cDNA as a template, HbICE2-F (SEQ ID NO: 3): ATGCTATCTGGACTAAATGGCA and HbICE2-R (SEQ ID NO: 4): CTATATCATTCCATGGAAGCCTG as primers, the cDNA sequence of the transcription factor HbICE2 gene was obtained by PCR amplification reaction , amplification system: Pyrobest DNA Polymerase (5U·μL ^-1 ) 1μL, 10×Pyrobest Buffer II 5μL, dNTP Mixture (2.5mmol·L ^-1 ) 4μL, HbICE2-F (10μmol·L ^-1 ) 1μL, HbICE2- 1 μL of R (10 μmol·L ^-1 ), 1 μL of cDNA template, and 50 μL of sterilized water. The amplification program was: pre-denaturation at 95°C for 3 minutes; 32 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min; extension at 72°C for 10 min.

0.2% agarose gel electrophoresis to separate the PCR amplification products, recover and purify the target bands, clone them into the pEASY-Blunt Simple Cloning Vector vector, transform Escherichia coli competent cells DH5α, pick positive clones and send them to Invitrogen for sequencing. Using BLAST to search the GenBank database for homology analysis, the results showed that a complete reading frame in the ICE family gene was cloned (Figure 1). Using MEGA5.10 software to conduct multiple comparison analysis and construct a phylogenetic tree. The results showed that the cloned ICE gene belonged to the ICE family and was one of the five members of the family, so the gene was named HbICE2 (Fig. 2).

2. Analysis of gene expression pattern

In the long-term cold-resistant sentinel nursery and field practice, the cold-resistant varieties Hevea 93-114, Hevea GT1 and Hevea INA873 were identified, and the non-cold-resistant varieties Reken 501, Haiken 1 and Reken 515 were identified. Therefore, these six varieties were used as materials, treated at low temperature (4°C), and samples were collected at 0h, 4h, 8h and 24h respectively, with 5 plants as a mixed sample and 3 biological replicates. The expression pattern of the transcription factor HbICE2 gene in rubber tree was analyzed by fluorescence quantitative technique. HbICE2 gene fluorescent quantitative primer sequence, such as, HbICE2-Q-F (SEQ ID NO: 5): CGATCTTCCCTTTCCAACTACCT and HbICE2-Q-R (SEQ ID NO: 6): CAAAAATAGCGTATTCCCTGAGC, the primer sequence of the internal reference gene ACTIN7a (Gene Bank accession number: HQ260674), such as, ACTIN7a -F:GGCACTTTGGTACTCAAGTC and ACTIN7a-R:GAAGCATCCCAATCACTCTC. The CFX-384 fluorescent quantitative PCR instrument of BioRad Company was used for fluorescent quantitative PCR. The reaction program was: 95°C pre-denaturation for 30s, 94°C for 5s, 60°C for 20s, 72°C for 20s, and 45 cycles. After the amplification, the melting curve was drawn. Gradually raise the temperature from 50°C to 95°C at a heating rate of 0.2°C/s, and detect fluorescence signals throughout the process. The expression of HbICE2 in hardy and non-hardy varieties of rubber tree was detected respectively. Results The background expression of HbICE2 in rubber tree cold-tolerant cultivars 93-114, GT1 and INA873 was significantly higher than that in non-cold-tolerant cultivars Reken 501, Haiken 1 and Reken 515. Under low temperature stress, HbICE2 was up-regulated in both cold-tolerant and non-cold-resistant varieties, but the expression level in cold-resistant varieties 93-114, GT1 and INA873 was significantly higher than that in non-cold-resistant varieties Reken 501, Haiken 1 and Reken 515, especially when treated at low temperature for 4h and 8h, the difference is extremely significant. This indicates that the HbICE2 gene is related to the low temperature stress resistance of rubber trees (Fig. 3).

3. Genetic Transformation

Using the sequence-verified plasmid as a template, HbICE2-TR-F (SEQ ID NO:7): G ACTAGT ATGCTATCTGGACTAAATGG (the underline is the Spe I restriction site) and HbICE2-TR-R (SEQ ID NO:8): G GGTGACC CTATATCATTCCATGGAAGC (the underline is the BstEⅡ restriction site) is used as a primer to amplify the ORF of the HbICE2 gene, and the amplified product is double-digested and connected to the expression vector pCAMBIA1302 after the same restriction digestion, and the target gene is identified by sequencing The integrity and the correctness of the connection sequence were used to transform the plasmid carrying the HbICE2 gene ORF into the GV3101 strain by heat shock. Infect the inflorescence of Arabidopsis thaliana through the Agrobacterium-mediated method, transform the HbICE2 gene into the seeds to be formed for overexpression, and then identify the transgenic positive plants, and screen the T3 positive plants containing the target gene and cold resistance Sexual identification analysis, using normal plants without transgene as control (WT), the results showed that the transgenic Arabidopsis lines of HbICE2 cultured for 4 weeks were treated at low temperature -5℃ for 15h, and then normal recovery cultured for 2 weeks, overexpression Arabidopsis lines with HbICE2 had a survival rate of more than 60%, compared to only 11% for control WT (Fig. 4). This shows that HbICE2 can improve the ability of transgenic plant lines to resist low temperature stress. It may have a great regulatory effect on the low temperature stress resistance of rubber trees.

Taking the normal plants without transgene as the control (WT), the transgenic HbICE2 Arabidopsis lines and WT plants cultivated for 4 weeks were sprayed with 300mM sodium chloride solution, once every 5 days. After 2 weeks of stress culture, the results showed that the leaves of the control WT plants were dehydrated and dry, some plants were dead, and the survival rate was 54%. The survival rate was 100% (Fig. 5). It shows that HbICE2 can improve the ability of transgenic plant lines to resist high-salt stress.

Taking the normal plants without transgene as the control (WT), the transgenic Arabidopsis plants of HbICE2 were cultured under normal conditions for 4 weeks, and then the watering was stopped, that is, the drought treatment was performed for 2 weeks. The results showed that the leaves of WT plants lost water and withered. The old leaves of the HbICE2 plants were withered, but the young leaves were still growing normally, and the plants had bolted, bloomed and produced pods. Then watered the drought-treated WT and transgenic HbICE2 Arabidopsis plants, and cultured them normally for 8 days. The results showed that the survival rate of WT was 63%, while the transgenic HbICE2 Arabidopsis plants did not die completely, and all of them had flowered As a result, the survival rate was 100% (Fig. 6). It can be seen that HbICE2 can improve the ability of transgenic plant lines to resist drought stress.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

sequence listing

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Claims (5)

1. Application of rubber tree transcription factor HbICE2 or rubber tree transcription factor HbICE2 gene or recombinant vector in improving cold resistance, salt resistance and/or drought resistance of plants;

the amino acid sequence of the rubber tree transcription factor HbICE2 is shown as SEQ ID NO. 1;

the nucleotide sequence of the rubber tree transcription factor HbICE2 gene is shown as SEQ ID NO. 2;

the recombinant vector comprises a raw vector and the rubber tree transcription factor HbICE2 gene.

2. The use according to claim 1, wherein the original vector is a pCAMBIA1302 vector plasmid and the nucleotide sequence of SEQ ID NO. 2 is located between the Spe I and BstE II restriction enzyme sites of the pCAMBIA1302 vector plasmid.

3. Use according to claim 1 or 2, characterized in that the salt is sodium chloride.

4. A method for improving cold resistance, and/or salt resistance, and/or drought resistance of a plant, characterized in that a rubber tree transcription factor HbICE2 gene, or a recombinant vector, is transfected into the plant;

the nucleotide sequence of the rubber tree transcription factor HbICE2 gene is shown as SEQ ID NO. 2;

the recombinant vector comprises a raw vector and the rubber tree transcription factor HbICE2 gene.

5. The method of claim 4, wherein the original vector is a pCAMBIA1302 vector plasmid and the nucleotide sequence of SEQ ID NO. 2 is located between the Spe I and BstE II restriction enzyme sites of the pCAMBIA1302 vector plasmid.

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