Perforin CaMACPF6 and application thereof in construction of novel flood-resistant vegetable germplasm

Abstract

The invention discloses a Perforin CaMACPF6 and an application of the Perforin CaMACPF6 in construction of a novel flood-resistant vegetable germplasm. According to the invention, an Agrobacterium tumefaciens-mediated inflorescence dip dyeing method is utilized to construct an arabidopsis thaliana plant of the overexpressed perforation protein CaMACPF6 gene, and the obtained transgenic plant is cultured under a hypoxia stress condition to find that compared with a wild type control, the overexpressed perforation protein CaMACPF6 gene in the plant can significantly improve the hypoxia stress resistance of the plant. On the basis, the perforation protein CaMACPF6 can be used for constructing a crop variety resistant to hypoxia stress, so that the crop variety can resist waterlogging and hypoxia stress caused by other conditions, and the adverse effect of hypoxia stress on crop growth is relieved. The method is beneficial to the construction of hypoxia stress-resistant germplasm, and further reduces the loss of crops caused by flood disasters and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the perforating protein CaMACPF6 and its application in constructing new flood-resistant vegetable germplasm. Background Technology

[0002] Due to the impact of global climate change, floods are occurring more frequently and their affected areas are gradually expanding, severely damaging cash crops. According to statistics from the international disaster database (EM-DAT), in the past two decades, there have been 3,518 severe flood disasters globally, averaging 176 per year, affecting nearly 16% of the world's agricultural production areas and seriously threatening food security. In my country, the overall flood risk is also on the rise.

[0003] Flooding reduces oxygen levels around plant cells and closes stomata, inhibiting the respiratory chain in mitochondria and hindering aerobic respiration, causing hypoxia stress in plants. The environmental stress experienced by crops during floods leads to limited O2 and CO2 acquisition, accumulation of secondary metabolites, and enrichment of toxic substances in the soil, resulting in reduced crop yields, especially causing severe damage or even death to solanaceous crops such as peppers. Therefore, identifying genes related to plant hypoxia tolerance is of great significance for studying crop flood resistance mechanisms, breeding new flood-resistant crop germplasm, and developing suitable hydroponic vegetable varieties.

[0004] Currently, there are many reports on genes related to hypoxia tolerance in animals, but fewer in plants. There are currently no reports on the association between MACPF (Membrane Attack Complex and Perforin) protein and hypoxia stress tolerance. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides perforating protein CaMACPF6 and its application in constructing new flood-resistant vegetable germplasm.

[0006] The first objective of this invention is to provide the application of perforin CaMACPF6 in improving the tolerance of plants to hypoxia stress.

[0007] A second objective of this invention is to provide the application of the gene encoding the perforin CaMACPF6 in improving the tolerance of plants to hypoxia stress.

[0008] A third objective of this invention is to provide the application of a perforin CaMACPF6 expression promoter in improving plant tolerance to hypoxia stress.

[0009] A fourth objective of this invention is to provide the application of perforin CaMACPF6 in the construction of flood-tolerant plant lines.

[0010] A fifth objective of this invention is to provide the application of the gene encoding the perforin CaMACPF6 in the construction of flood-tolerant plant lines.

[0011] The sixth objective of this invention is to provide the application of a perforin CaMACPF6 expression promoter in the construction of flood-tolerant plant lines.

[0012] The seventh objective of this invention is to provide a method for constructing plant lines resistant to hypoxia stress.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention utilizes an Agrobacterium-mediated inflorescence staining method to construct an overexpression of perforation protein. CaMACPF6 Arabidopsis thaliana plants containing the gene (SEQ ID NO.1) were cultured under hypoxic stress. The results showed that, compared to the wild-type control, the transgenic Arabidopsis thaliana plants overexpressed perforin. CaMACPF6 Genes can significantly enhance a plant's tolerance to hypoxia stress. Therefore, this invention seeks protection for the use of perforin CaMACPF6 in improving plant tolerance to hypoxia stress.

[0015] Specifically, the amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.2.

[0016] The present invention also seeks protection for the use of the gene encoding the perforin CaMACPF6 in improving the tolerance of plants to hypoxia stress.

[0017] Optionally, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0018] Specifically, this invention improves the plant's tolerance to hypoxia stress by overexpressing the gene encoding the perforating protein CaMACPF6 in the plant. Therefore, this invention also claims protection for the use of CaMACPF6 expression promoters in improving plant tolerance to hypoxia stress.

[0019] Specifically, the expression promoter of perforin CaMACPF6 is a recombinant overexpression vector containing a gene encoding perforin CaMACPF6.

[0020] The application of the perforating protein CaMACPF6 described in this invention in constructing plant lines resistant to hypoxia stress should also be within the scope of protection of this invention.

[0021] Similarly, the gene encoding the perforating protein CaMACPF6 described in this invention, and the application of the expression promoter of the perforating protein CaMACPF6 in constructing plant lines resistant to hypoxia stress, should also be within the scope of protection of this invention.

[0022] Excessive irrigation and flooding can easily lead to insufficient oxygen supply to plant roots, resulting in hypoxia stress. The perforating protein CaMACPF6 described in this application can improve the plant's tolerance to hypoxia stress. Therefore, this invention also claims protection for the application of the perforating protein CaMACPF6 in constructing flood-tolerant plant lines.

[0023] The present invention also seeks protection for the use of the gene encoding the perforin CaMACPF6 in the construction of flood-tolerant plant lines.

[0024] The present invention also claims protection for the use of an expression promoter of perforin CaMACPF6 in the construction of flood-tolerant plant lines.

[0025] Specifically, the amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.2.

[0026] Optionally, the nucleotide sequence of the gene encoding the perforin CaMACPF6 is shown in SEQ ID NO.1.

[0027] In a specific embodiment of the present invention, the plant is Arabidopsis thaliana. Meanwhile, the perforin CaMACPF6 described in this application is derived from chili peppers, therefore the plant can also be chili peppers.

[0028] The present invention also provides a method for constructing plant lines resistant to hypoxia stress, the method comprising: increasing the expression level of perforating protein CaMACPF6 in plants by transgenic modification or by using an expression promoter; the amino acid sequence of the perforating protein CaMACPF6 is shown in SEQ ID NO.2.

[0029] Specifically, the method involves transferring a recombinant overexpression vector containing a gene encoding the perforin CaMACPF6 into plant lines via Agrobacterium-mediated inflorescence staining.

[0030] Alternatively, the plant may be Arabidopsis thaliana or pepper.

[0031] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes Agrobacterium-mediated inflorescence staining to construct an overexpression of perforation protein. CaMACPF6 Transgenic Arabidopsis plants were cultured under hypoxic stress, and it was found that, compared with the wild-type control, the transgenic plants overexpressed perforin. CaMACPF6The gene can significantly enhance the crop's tolerance to hypoxia stress. Based on this, the perforin CaMACPF6 can be used to construct hypoxia-tolerant crop varieties, enabling them to withstand waterlogging and other conditions causing hypoxia stress, thus mitigating the adverse effects of hypoxia stress on crop growth. This invention is beneficial for constructing hypoxia-tolerant germplasm, thereby reducing losses to crops caused by floods and other disasters, and has significant economic value and application prospects. Attached Figure Description

[0032] Figure 1 To construct the obtained overexpressed perforated protein CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 )middle, CaMACPF6 The relative expression level of genes.

[0033] Figure 2 Wild-type (WT) Arabidopsis thaliana and negative control ( erfⅦ Arabidopsis thaliana and overexpression of perforin CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 The germination rates of the seeds of Arabidopsis thaliana after hypoxia treatment were compared. Figure a shows the plate plots of the germination of each Arabidopsis thaliana seed after 11 days of treatment with 3% hypoxia. Figure b shows the statistical results of the germination rate of each Arabidopsis thaliana seed after 11 days of treatment with 3% hypoxia. The horizontal axis represents the sample name and the vertical axis represents the germination rate. Scale bar = 10 mm.

[0034] Figure 3 Wild-type (WT) Arabidopsis thaliana and negative control ( erfⅦ Arabidopsis thaliana and overexpression of perforin CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 The graph shows a comparison of root lengths of the seeds after hypoxia treatment; graph a shows the root lengths of each Arabidopsis thaliana after 11 days of treatment with 3% hypoxia; graph b shows the statistical results of root lengths of each Arabidopsis thaliana after 11 days of treatment with 3% hypoxia. The horizontal axis represents the sample name, and the vertical axis represents the root length; scale bar = 10 mm. P <0.05. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] The wild-type (WT) Arabidopsis thaliana used in the embodiments of this invention ( Arabidopsis thaliana The sample was identified as Columbia (Col-0) ecotype; the negative control used was... erfⅦ ) References for Arabidopsis thaliana: Marín-de la Rosa, N., Sotillo, B., Miskolczi, P., Gibbs, DJ, Vicente, J., Carbonero, P., Oñate-Sánchez, L., Holdsworth, MJ, Bhalerao, R., Alabadí, D., and Blázquez, MA (2014). Large-Scale Identification of Gibberellin-Related TranscriptionFactors Defines Group VII ETHYLENE RESPONSE FACTORS as Functional DELLAPartners. Plant Physiology 166, 1022-1032. Example 1: Overexpression of perforin CaMACPF6 Construction of genes in Arabidopsis thaliana This invention utilizes T-DNA insertion technology and Agrobacterium-mediated plant transgenic technology to construct a gene that overexpresses perforin. CaMACPF6 Arabidopsis thaliana plants with genetic information for research CaMACPF6 The regulatory effect on the plant's tolerance to hypoxia stress. Among these effects, overexpression of perforin... CaMACPF6 The process of constructing the Arabidopsis gene is as follows: 1. Overexpression vector ( pEGOEP35S-CaMACPF6-H-EGFP Construction of ) Total RNA was extracted from chili peppers and reverse transcribed into cDNA. Using the resulting cDNA as a template, primer pair CaMACPF6CDS-F (ACTAGGGTCTCGCACCATGGAAGAAAAAACAGCA) was used. GCTTTTCATACAGC) and CaMACPF6CDS-R (ACTAGGGTCTCTCGCCATAATTT (AACAAGGAATACTTCAATCTCAAAACAATTCTCCC) Amplification of total cDNA from chili peppers CaMACPF6 ( Capana10g001546 The CDS of the perforating protein described in this invention. CaMACPF6 The gene has the following nucleotide sequence (SEQ ID NO.1), which is 1758 bp in length.

[0038] The perforating protein CaMACPF6 The amino acid sequence of the gene-encoded perforin CaMACPF6 is shown below (SEQ ID NO.2), and its length is 585 aa.

[0039] MEEKTAAFHTAINAVQALGRGFDVNYDTRLLYCKGVAGSRVVEIDEEHKRDLCLYDNIVLPNVSRDINNFQEQGGRDGSSVCNYNEMVEYFNRKANISGHSPLGSFNVAFSFTGAKHLDATTTKTLCMDGYFIPLARLQLMNSPLV LQQSVRRAVPASWDPPALASFIENFGTHIITSVTIGGKDVIYVKQHHSSPLSAMEIKSYVHDIGNQRFSSTESLTSSSLLRYKDKSSDPSIFNSQGIYPQPTNAPFIAGNGKEDVTVIFRRRGGDDLEQSHTQWARTVRSSPDVIE MSFYPITLLLEGIKGKEHLERAISLYLEYKPQIEELRYFLEFQGPRVWAPVQTIFPGQQRKEPVCPHLQFSMMGQKLYVSQEQVSVGRKPVTGMRLNLEGSKSNRLCIYLQHLTSLPKILLPCWDTHVAIGAPKWQGPEEQDSRWF EPVKWKNFSHVSTAPIECPETFIGDDLSGVYIVTGAQLGVWDFGSRNVLYMKLLYSRLPGCTIRRSLWDHSPNDKSNKQVNCGSNNGDASSVTGENITGNKLSKFVDMTEMSKGPQDPPGHWLVTGGKLGVEKGRIVLRLKYSLLNY The amplified perforin CaMACPF6 The gene was inserted into an overexpression vector, and the overexpression vector was obtained through sequencing verification. pEGOEP35S-CaMACPF6-H-EGFP .

[0040] 2. Agrobacterium-mediated genetic transformation This invention utilizes Agrobacterium-mediated inflorescence transfection to transform wild-type Arabidopsis thaliana. The overexpression vector is then transferred into LB liquid medium containing rifampin and kanamycin by vigorous shaking. pEGOEP35S-CaMACPF6-H-EGFPAgrobacterium (GV1301, VidiBio) was used. When the bacterial suspension turned orange-yellow, the bacteria were harvested and resuspended in the infection solution (formulation shown in Table 1). The inflorescences of flowering wild-type Arabidopsis thaliana were immersed in the infection solution and gently rotated. The infected Arabidopsis thaliana were wrapped in black plastic bags and placed in a plant room for overnight cultivation, followed by normal cultivation (16 hours of light, 23℃ / 8 hours of darkness, 21℃) until seed harvest.

[0041] Table 1 Infection solution formulation

[0042] 3. Real-time quantitative PCR (qRT-PCR) detection The transformed plants were subjected to qRT-PCR detection to determine their... CaMACPF6 The relative expression levels of the gene were compared with those of the wild type. Testing confirmed that this invention successfully constructed an overexpression of perforin. CaMACPF6 The Arabidopsis thaliana genes were named respectively. Ca6OE-1, Ca6OE-2 ,That CaMACPF6 The relative expression level of genes, such as Figure 1 As shown. By Figure 1 It can be seen that, compared with the wild type, Ca6OE-1, Ca6OE-2 middle CaMACPF6 High gene expression indicates that it is CaMACPF6 Gene overexpression material. Subsequent use of overexpressed perforin. CaMACPF6 Arabidopsis thaliana genes Ca6OE-1, Ca6OE-2 Further experiments will be conducted.

[0043] Example 2: Cultivation of Arabidopsis thaliana Cultivation conditions: Wild-type (WT) Arabidopsis thaliana and negative control ( erfⅦ Arabidopsis thaliana and overexpression of perforin CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 Seeds were surface-sterilized with 20% disinfectant (20% NaClO) for 15 minutes, rinsed 5 times with sterile double-distilled water, and then resuspended in sterile water. The treated seeds were separated and evenly sown on solid 1 / 2 MS (Morishige and Skoog) medium (formulation as shown in Table 2, pH adjusted to 5.8, sterilized at 121℃ for 20 minutes). The sown seeds were vernalized at 4℃ in the dark for 3-4 days, and then transferred to the plant room and cultured under the conditions of 16 hours of light, 23℃ / 8 hours of darkness, and 21℃. The 10-day-old seedlings were transplanted into peat moss and placed in the plant room for further cultivation.

[0044] Table 2 Formulation of solid 1 / 2MS medium (1 L)

[0045] Example 3 Comparison of hypoxia stress tolerance between wild-type Arabidopsis and overexpressing Arabidopsis Wild-type (WT) Arabidopsis thaliana seeds and negative control seeds were used. erfⅦ Arabidopsis seeds and overexpression of perforin CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 The seeds were sown on 1 / 2 MS medium according to the method described in Example 2 and germinated in a 3% hypoxic environment. A control group was set up in a normal oxygen environment. The culture temperature and photoperiod were kept consistent for both groups (16 hours of light, 23°C / 8 hours of darkness, 21°C). After 15 days of culture, the germination of each Arabidopsis seed was observed and the germination rate was counted. At the same time, the root growth of each Arabidopsis was observed and the average root length was measured and counted.

[0046] Wild-type (WT) Arabidopsis thaliana, negative control ( erfⅦ Arabidopsis thaliana and overexpression of perforin CaMACPF6 Arabidopsis thaliana (genes) Ca6OE-1 , Ca6OE-2 The germination rates of seeds after hypoxia treatment were compared as follows: Figure 2 As shown; Figure 2 In the figure, 'a' represents a plate image of the germination of each Arabidopsis thaliana seed after 11 days of treatment with 3% hypoxia. Figure 2 In the figure, b represents the germination rate of Arabidopsis seeds after 11 days of treatment under 3% hypoxia. The horizontal axis represents the sample name, and the vertical axis represents the germination rate. Wild-type Arabidopsis, negative control Arabidopsis, and overexpressing perforin were included. CaMACPF6 The following is a comparison of root lengths of Arabidopsis thaliana seeds after hypoxia treatment. Figure 3 As shown; Figure 3 In the figure, 'a' represents the root length of each Arabidopsis thaliana after 11 days of treatment under 3% hypoxia. Figure 3 In Table 3, b represents the root length statistics of each Arabidopsis species after 11 days of treatment with 3% hypoxia. The horizontal axis represents the sample name, and the vertical axis represents the root length. Table 3 shows the germination rate and average root length statistics of each Arabidopsis species after 11 days of treatment with 3% hypoxia.

[0047] Table 3. Germination rate and root length of each strain after treatment with 3% hypoxia

[0048] Combination Figure 2 , Figure 3 As shown in Table 3, compared with the wild type, overexpression of perforin... CaMACPF6 The germination rate of Arabidopsis seeds from the gene-modified strain was significantly higher than that of the wild type, and its root length was also significantly longer. These results indicate that... CaMACPF6 Genes can enhance a plant's tolerance to hypoxia stress.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of perforin CaMACPF6 in improving plant tolerance to hypoxia stress, characterized in that, The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

2. The application of the gene encoding the perforin CaMACPF6 in improving plant tolerance to hypoxia stress, characterized by, The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

3. The application of a perforin CaMACPF6 expression promoter in improving plant tolerance to hypoxia stress, characterized in that... The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

4. The application of perforin CaMACPF6 in constructing flood-tolerant plant lines, characterized in that, The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

5. The application of the gene encoding the perforin CaMACPF6 in constructing flood-tolerant plant lines, characterized by, The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

6. The application of a perforin CaMACPF6 expression promoter in constructing flood-tolerant plant lines, characterized in that... The amino acid sequence of the perforin CaMACPF6 is shown in SEQ ID NO.

2.

7. The application according to any one of claims 1 to 6, characterized in that, The plant in question is either Arabidopsis thaliana or pepper.

8. The application according to claim 2 or 5, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. A method for constructing plant lines tolerant to hypoxia stress, characterized in that, The expression level of perforin CaMACPF6 in plants was increased by genetic modification or by using expression promoters; the amino acid sequence of said perforin CaMACPF6 is shown in SEQ ID NO.

2.

10. The method according to claim 9, characterized in that, The plant in question is either Arabidopsis thaliana or pepper.

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