Application of CmoBI1L gene in regulating and controlling salt tolerance of pumpkin

Abstract

The invention provides application of a CmoBI1L gene in regulation and control of salt tolerance of pumpkins, a nucleotide sequence of the CmoBI1L gene is shown as SEQ ID NO: 1, and an amino acid sequence coded by the CmoBI1L gene is shown as SEQ ID NO: 4. The salt tolerance of the pumpkin can be improved by overexpressing the CmoBI1L gene in a pumpkin root system by utilizing a root system transformation technology. The CmoBI1L gene provided by the invention can be used for regulating and controlling CmoHKT1 at the same time; 1 and CmoNHX4, and a new direction is provided for cultivation of salt-tolerant pumpkins.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology and specifically relates to CmoBI1L Application of genes in regulating salt tolerance in pumpkin. Background Art

[0002] Salt stress is an important cause of crop yield reduction. Soil salinization is particularly serious in facility cultivation environments with unreasonable fertilization and irrigation. Grafting is often used in production to improve the resistance of crops to biotic and abiotic stresses. Using salt-tolerant pumpkin as a rootstock for grafting can significantly improve the salt tolerance of salt-sensitive cucumbers (Peng et al. 2023). Maintaining good Na under salt stress + / K + Balance is the basis for plants to cope with salt stress. Plants have evolved many ion transporters to precisely regulate ion homeostasis in the body (Liu et al 2019). Among them, the high affinity potassium transporter HKT1 and Na + / H + The antiporter NHX is the Na + / K + The key to transport, HKT1 transporter is mainly expressed on the cell membrane, through the Na + Absorbed into the xylem parenchyma cells, thus preventing sodium ions from being transported to the aerial parts and causing salt damage (Kobayashi et al 2017). NHX transporters are mainly expressed on the tonoplast, which transports Na + Transported to the vacuole and compartmentalized to reduce Na in the cytoplasm + HKT1 and NHX have been found to be involved in plant salt stress responses in species such as Arabidopsis, rice, and maize (Leidi et al 2010, Moller et al 2009, Uchiyama et al 2023, Zhang et al 2023).

[0003] However, as a direct regulator of Na + / K + The regulatory mechanisms of HKT1 and NHX4, key genes for salt transport, remain poorly understood, and the co-regulatory factors are even more unknown. Therefore, researchers have been analyzing the regulatory mechanisms of HKT1 and NHX4 at the protein level in order to provide ideas for salt-tolerant germplasm innovation. In recent years, reports have found that AtPP2C49 interacts with AtHKT1;1 in Arabidopsis thaliana and negatively regulates the Na+ transport of AtHKT1;1. + Transport activity (Chu et al 2020), in pumpkin CmoCNIH1 promotes Cmo HKT1;1 accumulates on the plasma membrane, thereby promoting Na accumulation in pumpkin roots + , reducing Na + transport to the aboveground part (Wei et al 2023). Summary of the Invention

[0004] In view of this, in order to analyze the mechanism of pumpkin response to salt stress, the present invention provides a method that can be used simultaneously with CmoHKT 1;1 and CmoNHX 4 key genes for synergistic effects CmoBI1L .

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide CmoBI1L Application of genes in regulating salt tolerance of pumpkin, CmoBI1L The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] Furthermore, the CmoBI1L The amino acid sequence encoded by the gene is shown in SEQ ID NO:4.

[0007] The second object of the present invention is to provide a method for regulating the salt tolerance of pumpkin, wherein the salt tolerance of pumpkin is improved by overexpressing CmoBI1L Genetic realization.

[0008] In some specific embodiments, preferably, overexpression CmoBI1L The gene is expressed by transfecting bacteria containing an overexpression vector. The primer sequences for constructing the overexpression vector are as follows: Upstream primer: GATACTCGAGTAATCTAGATGATGGAAGCATTTTCATCTTTCTT; Downstream primer: CGAAAGCTCTGAGCTCCTAGTCCCTCCTCTTCTTCTTCT.

[0009] The third object of the present invention is to provide a recombinant vector, expression cassette or engineered bacteria containing the above sequence for use in regulating the salt tolerance of pumpkin or cultivating high salt tolerance crops.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This application found that through interaction protein screening CmoBI1L Can be used with CmoHKT 1;1 (gene number: CmoCh10G003830 , the specific sequence is shown in SEQ ID NO: 2) and can also interact with the vacuole membrane localized CmoNHX4 (Gene number: CmoCh10G011690 , the specific sequence is shown in SEQ ID NO: 3) interaction; for the first time, it was discovered that CmoHKT 1;1 and CmoNHX 4 regulatory factors. Further overexpressed in pumpkin roots through the melon rooting system CmoBI1L Then, we determined the pumpkin seedling phenotype, malondialdehyde content, root activity, relative conductivity and other related physiological indicators under salt stress. CmoBI1L Overexpression can improve the salt tolerance of pumpkin seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 For Example 1 CmoBI1L and CmoHKT 1;1 interaction verification result diagram.

[0012] Figure 2 For Example 1 CmoBI1L and CmoNHX 4. Interaction verification results diagram.

[0013] Figure 3 For Example 2 CmoBI1L The results of subcellular localization and tissue expression pattern of .

[0014] Figure 4 The wild type (WT) and overexpression CmoBI1L The results of pumpkin physiological index measurement are shown in Figure 2.

[0015] Figure 5 The wild type (WT) and overexpression CmoBI1L Sodium in pumpkin roots, leaves and stems + Content determination results diagram. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0017] Example 1 This embodiment provides CmoBI1L Respectively CmoHKT 1;1, CmoNHX The mutual verification process of 4 is as follows: CmoBI1LThe nucleotide sequence SEQ ID NO:1 is as follows: ATGGAAGCATTTTCATCTTTCTTTGATTCTCAAGCAGAATCCAGAAACCGTTGGAGCTATGATTCTCTCAAGAACTTCCGGCAGATTTCGCCGGTCGTTCAATCTCATCTCCAGATGGTTTACCTTACTCTCGGTTGTGCTTTAGTTGCATCAGCTGCTGGAGCATATCTGCATATCCTTTGGAACATTGGTGGTATTCTCACAACGCTTGCAGGTGTTGGAAGCATCGCATGGCTGATGGCCACTCCTCCTTATGAAGAGAAAAAGAGGATTTCTATTCTAATGGCGGCTGCTCTTCTCGAAGGAGCTTCAATTGGTCCTTTGATTGGTTTAGCTATTGAGATTGACTCGAGTGTTCTTGTCAGTGCCTTCGTGGGAACTGCGGTGGCCTTTGGTTGTTTCTCAGCTGCAGCCATGTTGGCAAGACGCAGAGAATTCCTCTATCTGGGTGGCCTACTTTCTTCTGGAATATCAATGTTACTCTGGTTGCATTTCGCCTCCTCCATTTTCGGTGGTTCTACTGCCGTTTTCAAATTTGAGTTGTACTTTGGGCTTTTGCTGTTTGTGGGCTACATGGTAGTTGATACTCAAGAGATAATTGAGAGGGCACATGTTGGTGATATGGACTATGTGAAGCATGCACTGACCCTGTTCACCGATTTCATCGGTGTTTTTGTCCGAATTCTCATTATAATGCTAAGGAACACTGCAGAGAAGAATGAGAAGAAGAAGAAGAAGAGGAGGGACTAG。

[0018] CmoHKT

[0019] CmoNHX

[0020] CmoBI1L Encoding nucleotide sequence SEQ ID NO:4 is as follows: MEAFSSFFDSQAESRNRWSYDSLKNFRQISPVVQSHLQMVYLTLGCALVASAAGAYLHILWNIGGILTTLAGVGSIAWLMATPPYEEKKRISILMAAALLEGASIGPLIGLAIEIDSSVLV SAFVGTAVAFGCFSAAAMLARRREFLYLGGLLSGISMLLWLHFASSIFGGSTAVFKFELYFGLLLFVGYMVVDTQEIIERAHVGDMDYVKHALTLFTFIGVFVRILIIMLRNTAEKNEKKKKKKRRD.

[0021] 1.1 Vector construction Phanta Max Super-Fidelity DNA Polymerase (Novagen, P505) was used to amplify the cDNA of pumpkin. CmoBI1L and CmoHKT 1;1, CmoNHX 4 CDS, and then construct it into the destination vector through homologous recombination, and then proceed to subsequent steps such as firefly luciferase complementation imaging and immunoprecipitation. The specific primers (see Table 1), PCR system (see Table 2) and procedures (see Table 3) for each step are as follows: Table 1 Details of primer sequences for each step

[0022] Table 2 PCR reaction system details

[0023] Table 3 PCR reaction procedure details

[0024] 1.2 Yeast two-hybrid Will CmoHKT 1;1 was used as bait to screen for interacting proteins in the pumpkin membrane protein yeast library and sequence comparison revealed CmoBI1L Possibly and CmoHKT 1;1 interaction, and then the screening library results were verified by dot-to-dot yeast two-hybrid. In the SD / -LWHA four-deficient medium, the yeast of the negative control could not grow, while the experimental group (co-transfected CmoHKT 1;1-Cub / CmoBI1L -Nub) and positive control yeast can grow, confirming CmoBI1L and CmoHKT1;1 does interact ( Figure 1 a).

[0025] The same experimental steps as above also confirmed CmoBI1L and CmoNHX 4 There is indeed interaction ( Figure 2 a).

[0026] 1.3 Firefly luciferase complementation Will CmoHKT 1;1-nLUC and cLUC- CmoBI1L The plasmid was transformed into Agrobacterium EHA105 and injected into the lower epidermis of tobacco. After 2 days, fluorescein potassium salt was applied to the injection site and fluorescence was observed using a plant living imaging system. CmoHKT 1;1-nLUC / cLUC) compared to co-transfection CmoHKT 1;1-nLUC / cLUC- CmoBI1L The tobacco leaves showed obvious fluorescence ( Figure 1 b) Description CmoBI1L and CmoHKT 1;1 interactions in plants.

[0027] The same experimental steps as above also confirmed CmoBI1L and CmoNHX 4 Interactions in plants ( Figure 2 b).

[0028] 1.4 Co-immunoprecipitation Transient expression of HA- CmoHKT 1;1 and CmoBI1L -YFP fusion protein, the protein was extracted 2 days after injection and enriched with GFP magnetic beads, and then the protein in the sample was detected by Western Blot. The results are as follows Figure 1 As shown in c: In the input lane, all proteins can be detected, indicating that the proteins are expressed normally, while in the IP sample, the experimental group CmoBI1L -YFP can convert HA- CmoHKT 1;1 immunoprecipitated, while the negative control GFP could not, again indicating that CmoBI1L and CmoHKT 1;1 interactions exist in plants.

[0029] The same experimental steps as above also confirmed CmoBI1L and CmoNHX 4 Interactions in plants ( Figure 2 c).

[0030] Example 2 This embodiment targets CmoBI1L The expression pattern of α-amino acid ... 2.1 CmoBI1L Subcellular localization Will CmoBI1L The CDS was constructed into the 101YFP vector (Meng et al 2022) for expression CmoBI1L -YFP fusion protein, co-injected with the endoplasmic reticulum marker HDEL-mCherry (Nelson et al 2007), was observed in tobacco plants. CmoBI1L -YFP's yellow fluorescence overlaps with HDEL-mCherry's red fluorescence, indicating CmoBI1L Localized in the endoplasmic reticulum ( Figure 3 a).

[0031] 2.2 CmoBI1L Analysis of gene expression in tissues under salt stress qRT-PCR analysis CmoBI1L The expression changes in different tissues of pumpkin treated with salt for 24 hours showed: CmoBI1L The pumpkin roots, hypocotyls, and true leaves were significantly induced by salt stress, but the expression level in the cotyledons showed no significant change ( Figure 3 b). The CmoBI1L gene is mainly located in the endoplasmic reticulum and its expression is significantly induced and upregulated in multiple pumpkin tissues under salt stress.

[0032] Example 3 This embodiment provides CmoBI1L The salt stress phenotype and physiological indexes of overexpressed pumpkin were determined as follows: 3.1 CmoBI1L Overexpression pumpkin creation Phanta Max Super-Fidelity DNA Polymerase (Novagen, P505) was used to amplify the cDNA from pumpkin. CmoBI1L The CDS was sequenced and cloned (upstream primer: GATACTCGAGTAATCTAGATGATGGAAGCATTTTCATCTTTCTT, downstream primer: CGAAAGCTCTGAGCTCCTAGTCCCTCCTCTTCTTCTTCT), and then constructed into the pBSE403R vector by homologous recombination. The plasmid was then transformed into Agrobacterium rhizogenes K599 for infecting pumpkin.

[0033] The test material was the salt-tolerant pumpkin material "Fengle Jinjia". The seeds were germinated and sown in 50-hole trays and cultured in an artificial climate chamber. When the cotyledons flattened, they were cut obliquely about 2 cm below the cotyledons to prepare explants. 100 μL of the PCR-positive bacterial solution was added to 50 mL of LB liquid medium containing 50 mg / L Strep and 50 mg / L Kana. The culture was shaken at 28°C and 200 rpm for 14-18 hours until the OD 600 = 0.8-1.0. Centrifuge at 6000 rpm for 8 min, then resuspend in MS liquid medium containing 200 mM AS and 0.1% sucrose to OD 600 = 0.8-1.0. Then, 1 mL of the resuspended bacterial solution was pipetted into a 1.5 mL centrifuge tube for infection. The hypocotyl cut of the pumpkin explant was immersed in the bacterial solution in the 1.5 mL centrifuge tube and allowed to stand for 30 minutes to complete infection. The cut was then inserted into a 500 mL incubator containing sterilized vermiculite moistened with MS liquid medium (containing 0.1% sucrose and 200 mM AS) and incubated in the dark at 23°C for 4 days (co-cultivation). After co-cultivation, the cut hypocotyl cut of the explant was inserted into a seedling tray containing sterilized vermiculite moistened with 1 / 2 Hoagland's nutrient solution. The tray was covered to maintain moisture. Non-fluorescent roots were removed weekly. During the second removal, fluorescent roots began to gradually emerge. Seedlings with fluorescent roots larger than 2 cm were transferred to 1 / 2 Hoagland's nutrient solution for hydroponics. During this period, non-fluorescent roots were removed weekly. Transgenic plants with fully fluorescent roots were obtained in approximately two weeks.

[0034] 3.2 CmoBI1L Overexpression enhances salt tolerance in pumpkin When pumpkin had three leaves and one heart, it was treated with 100mM NaCl for 9 days. The results showed that the root system overexpressed CmoBI1L The degree of salt damage to pumpkins overexpressing α-glucan was less than that of wild type. The yellowing and wilting of leaves of wild type plants were more serious, while CmoBI1L The pumpkins grew well and the leaves were less affected by salt damage ( Figure 4 a).

[0035] 3.3 CmoBI1L Determination of physiological indicators of overexpressed pumpkin under salt stress The wild type pumpkin and transgenic pumpkin were sampled after salt treatment for 9 days. CmoBI1L The relative expression of genes, malondialdehyde content, relative conductivity, and root activity. The results showed that compared with the control, the overexpression CmoBI1L The tolerance of transgenic pumpkin to salt stress can be improved by increasing the root activity and leaf chlorophyll (SPAD) content of pumpkin seedlings under salt stress, and reducing the relative electrical conductivity (REC) of roots and leaves and the malondialdehyde (MDA) content of leaves ( Figure 4, where c is the chlorophyll (SPAD) content, d is the root activity, e is the malondialdehyde (MDA) content, f is the root relative electrical conductivity (REC), and g is the leaf relative electrical conductivity (REC). CmoBI1L Gene positively regulates pumpkin salt tolerance, overexpression CmoBI1L It can reduce the damage caused by salt stress to pumpkin seedlings.

[0036] 3.4 CmoBI1L Effect of overexpression on Na + Effect of content Overexpression CmoBI1L Na in the material + Further analysis of the changes in the content of CmoBI1L Increased Na in the root system + content, and significantly reduced the Na + content( Figure 5 ), thereby improving the salt tolerance of pumpkin.

[0037] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0038] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. CmoBI1L The application of the gene in regulating the salt tolerance of pumpkin is characterized by: described CmoBI1L The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that described CmoBI1L The amino acid sequence encoded by the gene is shown in SEQ ID NO:

4.

3. A method for regulating the salt tolerance of pumpkin, characterized in that: The method improves the salt tolerance of pumpkin by overexpression CmoBI1L Genetic realization.

4. The method according to claim 3, characterized in that Overexpression CmoBI1L The gene is expressed by transfecting bacteria containing an overexpression vector. The primer sequences for constructing the overexpression vector are as follows: Upstream primer: GATACTCGAGTAATCTAGATGATGGAAGCATTTTCATCTTTCTT; Downstream primer: CGAAAGCTCTGAGCTCCTAGTCCCTCCTCTTCTTCTTCT.

5. Use of a recombinant vector, expression cassette or engineered bacteria containing the sequence of claim 1 in regulating the salt tolerance of pumpkin or cultivating high salt tolerance crops.

Images