CN112430600B

CN112430600B - High and low temperature stress resistance related gene of non-heading Chinese cabbage and application thereof

Info

Publication number: CN112430600B
Application number: CN202011323325.5A
Authority: CN
Inventors: 袁凌云; 郑禹杉; 汪承刚; 朱世东; 黄兴学; 侯金锋; 唐小燕; 吴颖; 张泽根; 陈国户
Original assignee: Anhui Wanjiang Vegetable Industry Technology Research Institute Co ltd; Anhui Agricultural University AHAU
Current assignee: Anhui Wanjiang Vegetable Industry Technology Research Institute Co ltd; Anhui Agricultural University AHAU
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-05-31
Anticipated expiration: 2040-11-23
Also published as: CN112430600A

Abstract

The invention discloses a gene related to high and low temperature stress resistance of non-heading Chinese cabbage and application thereof, and relates to the technical field of genetic breeding, wherein the gene is BcrGLU 1, and the nucleotide sequence of a coding region of the gene is shown in SEQ ID NO. 1.

Description

High and low temperature stress resistance related gene of non-heading Chinese cabbage and application thereof

Technical Field

The invention relates to the technical field of genetic breeding, in particular to a gene related to tolerance of non-heading Chinese cabbage and application thereof.

Background

Non-heading Chinese cabbage (Brassica campestris L.ssp. chinensis var. rossularis Tsen) belongs to Brassicaceae Brassica plants. The taste is crisp and sweet, is rich in various nutrients and is highly favored by consumers. The non-heading Chinese cabbage is mainly planted in late autumn and is an important leaf vegetable source in winter and spring in many areas of China.

However, the temperature stress causes the reduction of the economic yield of the non-heading Chinese cabbage and the quality of the vegetable itself. The method is characterized in that the Chinese cabbage is planted in less than two seasons in spring and summer, in order to meet market demands and realize annual supply of the non-heading Chinese cabbage in various places, the problem that the non-heading Chinese cabbage is damaged in the production process due to improper environmental temperature is fundamentally solved, the cold resistance and the heat resistance of the non-heading Chinese cabbage are better improved, the contradiction that the supply of vegetables is insufficient in summer and autumn and low temperature areas in winter is further alleviated, the mechanism discussion on the heat resistance and the cold resistance of the non-heading Chinese cabbage is urgently needed, and a theoretical basis is provided for cultivation and breeding of the non-heading Chinese cabbage.

Disclosure of Invention

The invention aims to provide a gene related to high and low temperature stress resistance of non-heading Chinese cabbage, which can improve the high and low temperature resistance of the non-heading Chinese cabbage.

The invention also provides a protein coded by the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage.

The invention also provides a recombinant vector containing the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage and a construction method thereof.

The invention further provides a recombinant bacterium containing the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage and a construction method thereof.

Meanwhile, the invention also provides application of the gene, the recombinant vector and the recombinant bacterium in cultivating high and low temperature resistant non-heading Chinese cabbage varieties.

In order to achieve the purpose, the invention adopts the technical scheme that:

a gene related to high and low temperature stress resistance of non-heading Chinese cabbage is BcrGLU 1, and the nucleotide sequence of the coding region of the gene is shown as SEQ ID NO. 1.

The recombinant vector comprises the gene related to the cold resistance of the non-heading Chinese cabbage.

Further, the method for constructing the recombinant vector comprises the following steps: inserting the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage into the enzyme cutting site of the corresponding expression vector, and connecting the gene to the recombinant vector through ligase.

The recombinant strain comprises the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage.

The construction method of the recombinant bacterium comprises the step of introducing genes related to high and low temperature stress resistance of the non-heading Chinese cabbage into the thallus to obtain the recombinant bacterium.

The gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage, the recombinant vector and the recombinant bacterium are applied to cultivation of the high and low temperature stress resistance non-heading Chinese cabbage transgenic variety.

Further, the gene related to the high and low temperature stress resistance of the non-heading Chinese cabbage is introduced into a target plant to obtain a high and low temperature stress resistant transgenic plant.

Compared with the prior art, the invention has the following advantages:

the invention provides a gene related to high and low temperature resistance of non-heading Chinese cabbage, and provides possibility for cultivating new varieties of stress-resistant non-heading Chinese cabbage.

Drawings

FIG. 1 is a schematic diagram of the construction of 35S BcrGLU 1 GFP fusion protein vectors in wild-type and over-expressed transgenic lines of Arabidopsis plants and the results of detection of physiological changes associated with temperature stress;

FIG. 2 is a diagram showing the results of the measurement of oxidative damage and superoxide anion content and hydrogen peroxide content in WT and OE lines of Arabidopsis plants;

FIG. 3A graph showing the results of MDA content, electrolyte permeability and proline content tests in WT and OE lines of Arabidopsis plants.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Acquisition of target Gene

The preparation of DEPC water was carried out in a laboratory fume hood, while the instruments required for RNA extraction were left to soak in DEPC water for 24 h. And after soaking, placing the test instrument in an autoclave, sterilizing for 30min at 116 ℃, taking out the test instrument from the autoclave after sterilization, placing the test instrument in a constant-temperature drying box at 80 ℃, and drying for later use. Placing the leaf of the lindera aggregata required by the test in a mortar precooled by liquid nitrogen in advance, adding sufficient liquid nitrogen, then quickly grinding by using a grinding rod, adding a proper amount of liquid nitrogen during the grinding process when the liquid nitrogen is volatilized, and carrying out the RNA extraction test according to the instruction of the Takara RNA extraction kit after the grinding of the sample is finished. The RNA Extraction Kit from Takara (Takara MiniBEST Universal RNA Extraction Kit) was used, and the assay procedures are described in the Kit instructions. After the RNA extraction is finished, the concentration and the purity of the extracted RNA are detected by using a nucleic acid quantitative analyzer, and after the concentration and the purity are both detected to be qualified, reverse transcription reaction is carried out.

Reverse transcription reaction: for the reverse transcription, Takara's reverse transcription kit (PrimeScriptTM RT Master Mix (Perfect Real Time)) was used. The cDNA was synthesized by sequentially adding test reagents according to the system of Table 1.

TABLE 1

To obtain 35S: GFP-BcrGLU 1/Col-0 line, using specific primers as follows: LP SEQ ID NO 2RP SEQ ID NO 3, a co-coding sequence (CDS) encoding the full-length sequence of BcrGLU 1 was amplified from the above cDNA: SEQ ID NO 1.

Example 2

Construction of recombinant vectors

The fragment was introduced into the pDONR207 vector using BP enzyme (Invitrogen, Carlsbad, California). The inserted fragment was transferred to the destination vector pMDC43 using LR enzyme (Invitrogen, Carlsbad, California) for GFP fusion as shown in fig. 1A.

Example 2

Acquisition of transgenic plants

35S-BcrGLU 1: GFP infects wild Arabidopsis plants (strain GV3101) by Agrobacterium-mediated inflorescence. Over-expressed plants were selected on 1/2MS agar plates containing 50. mu.g/ml hygromycin B (Invitrogen, Carlsbad, California). Extracting RNA of the screened plant, obtaining cDNA thereof after reverse transcription, and using gene specific primers as follows:

BccrGLU1 LP：SEQ ID NO 4

BccrGLU1 RP：SEQ ID NO 5

qRT-PCR was performed using the above cDNA as a template to detect the expression level of the transgene. (as in FIG. 1B)

Example 3

Verification conclusion

WT and OE seedlings were planted on 1/2MS plates at 24 ℃ under 16h/8h (day/night) with a relative humidity of 75% and light intensity set at 200mmol m^-2s^-1. In order to determine the tolerance of transgenic Arabidopsis seedlings to temperature stress, the seedlings were subjected to temperature stress treatment. Two-week old seedlings were transferred to 4 ℃ for 48h of cold stress. The plants were transferred to 40 ℃ for high temperature stress for 48 h. Samples were then taken for analysis of transcript levels and physiobiochemical analysis. Both sets of temperature stress were performed in triplicate. Transgenic plants of Arabidopsis T3 generation were used in this study.

The analysis results are shown in fig. 1, 2 and 3, and the specific analysis in this example is as follows:

1) FIG. 1 shows the schematic diagram of the construction of 35S-BcrGLU 1 GFP fusion protein vector in FIG. 1(A), and the expression level of BcrGLU 1 in T3 transgenic plant in FIG. 1 (B). Wherein the gene expression level of the wild type WT is far lower than the expression level of over-expression transgenic lines (OE #1, OE #2 and OE #); FIG. 1(C) shows the enzyme activities of Fd-GOGAT in wild-type and overexpressed transgenic lines. FIG. 1(D) shows the content of reduced glutathione in the leaf. FIG. 1(E) shows the ratio of reduced glutathione to oxidized glutathione in leaf blades. And indicates that there was a significant difference in wild type tested P <0.01 and P <0.05 using Student's.

FIGS. 1A-B demonstrate that plants were grown using 35S-BcrGLU 1: GFP transformation was successful. Under normal growth conditions, no phenotypic differences were observed between wild type and overexpressed transgenic lines (OE), indicating that overexpression of BccrGLU1 did not cause phenotypic defects in transgenic arabidopsis seedlings. In the OE line, ferredoxin (Fd-GOGAT) activity was significantly higher than wild-type under normal conditions (fig. 1C). Under low and high temperature stress treatment, the reduced glutathione levels and the ratio of reduced glutathione to oxidized glutathione in the three over-expressed transgenic plants (OE #1, OE #2 and OE #7) were significantly higher than in the wild type (fig. 1D). The higher GSH/GSSG ratio in OE line is beneficial to antioxidant protection, and further improves the tolerance of plants under temperature stress (figure 1E).

2) With reference to FIG. 2, wherein FIG. 2 is the oxidation damage of WT and OE lines of Arabidopsis plantsThe lesions were stained by histochemical staining, 3-Diaminobenzidine (DAB), Nitro Blue Tetrazolium (NBT) (FIG. 2(A)), and hydrogen peroxide (H)₂O₂) Superoxide anion (O)^2-) And (4) measuring. As shown in fig. 2(B) for superoxide anion content. As shown in FIG. 2(C), hydrogen peroxide (H)₂O₂) And (4) content. And represents the respective P in the Student's assay<0.01 and P<At 0.05, there was a significant difference from the wild type plants.

FIGS. 2A-C demonstrate H under low temperature, high temperature stress₂O₂The content was significantly higher in Arabidopsis wild type plants than in over-expressed plants. Compared with wild plants, after low-temperature and high-temperature stress, Arabidopsis overexpresses O of transgenic plants₂ ^.-The production rate is also significantly reduced. These results were also confirmed by comparison with DAB and NBT staining results of wild type and overexpressing transgenic plant leaves. NBT, DAB staining after temperature stress indicated lower ROS levels in overexpressing transgenic plants compared to wild-type plants (fig. 2A, B). These results indicate that BccrGLU1 is involved in active oxygen scavenging to eliminate oxidative stress.

3) With reference to FIG. 3, wherein FIG. 3(A) is a schematic representation of the MDA content in WT and OE lines of Arabidopsis plant seedlings. FIG. 3(B) is the electrolyte permeability in WT and OE lines of Arabidopsis seedlings. FIG. 3(C) shows proline content in WT and OE lines of Arabidopsis seedlings. Indicates that there was a significant difference from wild type plants at P <0.01 and P <0.05, respectively, using Student's test.

Furthermore, we determined oxidative damage parameters under temperature stress to analyze redox homeostasis in OE lines. We determined the malondialdehyde content in Arabidopsis thaliana leaves. Under normal growth conditions, the OE system has no difference in MDA content and electrolyte permeability from WT. After low-temperature and high-temperature stress, the MDA content of the arabidopsis overexpression transgenic line is remarkably reduced compared with that of a wild type (figure 3A). When plants were subjected to low temperature, high temperature stress, the electrolyte permeability of the OE lines was lower than WT (fig. 3B). After low temperature stress, the stress is respectively reduced by 25.06 percent, 37.21 percent and 29.12 percent. Under normal growth conditions, the proline content in the three OE lines was higher than WT, whereas after low temperature, high temperature stress, the OE line proline content was significantly higher than WT (fig. 3C).

In conclusion, the BcrGLU 1 gene can maintain the redox homeostasis of plants under the condition of temperature stress, so that the plants are less damaged, and the tolerance of the plants under the condition of temperature stress is improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Anhui agricultural university, Anhui river vegetable industry and technology research institute of Anhui province, Limited liability company

<120> a gene related to high and low temperature stress resistance of non-heading Chinese cabbage and application thereof

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Claims

1.BccrGLU1The application of the gene in cultivating the transgenic variety of the non-heading Chinese cabbage with high temperature stress resistance or low temperature stress resistance is characterized in thatBccrGLU1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.