CN108841826B - Application of arabidopsis long-chain non-coding RNA AtHAL6 in regulation and control of high-temperature stress tolerance of plants - Google Patents
Application of arabidopsis long-chain non-coding RNA AtHAL6 in regulation and control of high-temperature stress tolerance of plants Download PDFInfo
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Abstract
The invention discloses application of arabidopsis thaliana long-chain non-coding RNA AtHAL6 in regulation and control of high-temperature stress tolerance of plants/application in preparation of transgenic plants, wherein a nucleotide sequence of arabidopsis thaliana long-chain non-coding RNA AtHAL6 is shown as SEQ ID No. 1. The invention also provides a recombinant expression vector and a host cell containing the Arabidopsis thaliana long-chain non-coding RNA AtHAL 6. The invention clones long-chain non-coding RNA AtHAL6 from Arabidopsis thaliana, and verifies that the gene has the function of adjusting the high temperature resistance of plants in the seed germination stage, the early seedling development stage and the seedling stage. The invention provides theoretical basis and gene source for cultivating new species of crops.
Description
Technical Field
The invention relates to application of arabidopsis long-chain non-coding RNA AtHAL6 in regulation and control of high-temperature stress tolerance of plants, belonging to the technical field of molecular biology and biotechnology.
Background
Temperature is one of the most important environmental factors influencing the growth and development of plants, and slight change of temperature can greatly influence the behaviors of the plants such as growth and development. Global warming has resulted in climate changes such as extreme temperatures, causing significant losses to crop production (Hasanuzzaman et al, 2013; Lesk et al, 2016).
High temperature stress can destroy the photosynthetic system of plants, reduce the water content of plants, and affect cell division and growth (Hasanuzzaman et al, 2013). Plants have evolved complex and diverse systems to cope with high temperature stress due to sessile growth. There are genetic and epigenetic controls in plants in response to high temperature stress. Genetic regulation includes putative temperature receptors, heat shock transcription factors HSF and heat shock protein HSP response pathways, hormone regulatory networks and secondary metabolites (Bokszczanin et al, 2013; Qu et al, 2013). Epigenetic regulation includes DNA methylation, histone modification, chromatin remodeling, and regulation of small molecule RNAs, etc. Epigenetic modifications play an important role in maintaining genome stability, regulating plant growth and development, and responding to adversity stress (Liu and He, 2014).
The long-chain non-coding RNA refers to a type of RNA with the length of more than 200bp and without a protein coding function. Current work on lncRNA is mainly focused on animals, and lncRNA is found to have powerful and unique functions, including apparent regulation of chromatin silencing, transcriptional regulation, post-transcriptional regulation affecting splicing of spliceosomes, or competitive binding of miRNA to affect expression of target genes, etc. (Fatica and Bozzoni, 2014; TR et al, 2009). However, there is relatively little research in plants, and the current studies of biological functions and mechanisms of action of plant non-coding RNAs focus primarily on model plants, and less in crops other than rice.
Disclosure of Invention
Aiming at the prior art, the invention provides a new application of Arabidopsis thaliana long-chain non-coding RNA AtHAL6, namely an application of Arabidopsis thaliana long-chain non-coding RNA AtHAL6 in regulation and control of high temperature stress tolerance of plants. The invention also provides a recombinant expression vector capable of regulating and controlling the expression of the arabidopsis long-chain non-coding RNA AtHAL 6.
The invention is realized by the following technical scheme:
the arabidopsis long-chain non-coding RNA AtHAL6 has a nucleotide sequence shown in SEQ ID NO. 1.
The application of the arabidopsis long-chain non-coding RNA AtHAL6 in regulating and controlling the high-temperature stress tolerance of plants and the application in preparing transgenic plants. According to the invention, researches show that the arabidopsis long-chain non-coding RNA AtHAL6 can obviously improve the heat resistance of the transgenic arabidopsis in the seed germination stage, the early seedling development stage and the seedling strengthening stage. The invention provides theoretical basis and gene source for cultivating new species of crops.
A plant expression vector comprising the Arabidopsis thaliana long-chain non-coding RNA AtHAL 6. Preferably, the plasmid used by the plant expression vector is PBI 121.
A genetically engineered host cell comprising the plant expression vector described above, or having Arabidopsis thaliana long non-coding RNA AtHAL6 inserted into its genome.
The construction method of the genetically engineered host cell is a conventional technical means, and the plant expression vector is introduced into the host cell, so that the plant expression vector/Arabidopsis thaliana long-chain non-coding RNA AtHAL6 is effectively expressed in the host cell.
The plant expression vector and the genetically engineered host cell are applied to preparation of high-temperature-resistant transgenic plants (the high-temperature stress tolerance of which is superior to that of wild plants). The plant comprises Arabidopsis thaliana.
The invention clones a section of long-chain non-coding RNA AtHAL6 from Arabidopsis thaliana, connects the long-chain non-coding RNA AtHAL6 to an expression vector, and transforms Arabidopsis thaliana by using an Agrobacterium tumefaciens infection method, and data shows that the gene can obviously improve the heat resistance of transgenic Arabidopsis thaliana in the seed germination stage, the early seedling development stage and the seedling strengthening stage. The invention provides theoretical basis and gene source for cultivating new species of crops.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
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FIG. 1: and (3) germination conditions of the transgenic arabidopsis seeds and wild arabidopsis seeds on an MS culture medium treated at high temperature of 45 ℃ for 6h under normal conditions, wherein A: under normal conditions, the germination conditions of wild type and over-expression arabidopsis seeds on an MS culture medium; b: and (4) performing high-temperature treatment at 45 ℃ for 6h, and performing germination of wild type and overexpression arabidopsis seeds on an MS culture medium. WT represents wild-type arabidopsis seeds and OE represents transgenic arabidopsis seeds.
FIG. 2: carrying out seed germination rate statistics on transgenic arabidopsis seeds and wild type arabidopsis seeds under high-temperature treatment for 6 hours at 45 ℃, wherein the abscissa represents time, and the ordinate represents seed germination rate; WT represents wild type arabidopsis seeds, OE represents transgenic arabidopsis seeds; CK stands for control and HS stands for high temperature treatment at 45 ℃ for 6 h.
FIG. 3: survival conditions of transgenic arabidopsis seedlings and wild arabidopsis seedlings after normal condition and high-temperature treatment are shown, wherein A: under normal conditions, the survival conditions of wild type and overexpression arabidopsis seedlings after high-temperature treatment; b: survival conditions of the transgenic arabidopsis seedlings and the wild type arabidopsis seedlings after high-temperature treatment. WT stands for wild type Arabidopsis thaliana and OE for transgenic Arabidopsis thaliana.
FIG. 4: survival conditions of the transgenic arabidopsis seedlings and the wild type arabidopsis seedlings after high-temperature treatment; wherein the abscissa represents different materials and the ordinate represents survival rate; WT stands for wild type Arabidopsis thaliana and OE for transgenic Arabidopsis thaliana.
FIG. 5: the survival conditions of strong transgenic arabidopsis seedlings and wild arabidopsis seedlings after normal conditions and high-temperature treatment are as follows, wherein A: under normal conditions, the survival conditions of strong seedlings of wild type and over-expression arabidopsis thaliana after high-temperature treatment; b: the survival condition of the strong transgenic arabidopsis seedlings and the survival condition of the wild arabidopsis seedlings after high-temperature treatment. WT stands for wild type Arabidopsis thaliana and OE for transgenic Arabidopsis thaliana.
FIG. 6: the survival conditions of the strong transgenic arabidopsis seedlings and the wild arabidopsis seedlings after high-temperature treatment are shown, wherein the abscissa represents different materials, and the ordinate represents the survival rate; WT stands for wild type Arabidopsis thaliana and OE for transgenic Arabidopsis thaliana.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The basic operation methods of PCR, digestion, ligation, transformation, and other genetic engineering used in the examples can be performed according to the methods described in "third edition of molecular cloning Experimental Manual" (U.S.; J. Samum Brookfield, science publishers, published in 2002) and "principles and techniques of plant genetic engineering" (Wanggan, the works of the formula, the science publishers, 1998), unless otherwise specified.
Example 1: acquisition of Arabidopsis thaliana long-chain non-coding RNAATHAL6
(1) Extracting genomic DNA of an arabidopsis thaliana leaf;
(2) PCR amplification of atahal 6: using arabidopsis thaliana leaf DNA as a template, designing a primer according to the sequence of AtHAL6, carrying out PCR amplification, recovering and purifying PCR amplification products, and sequencing. The primers are as follows:
a forward primer: 5'-TCTAGAGGTGTCAACGTCTCCACTGG-3' (SEQ ID NO. 2).
Reverse primer: 5'-GAGCTCCGGGAATGAACAACGAGGCG-3' (SEQ ID NO. 3).
The PCR reaction system and amplification conditions are shown in Table 1.
TABLE 1
The reaction steps are as follows: pre-denaturation: 30s at 98 ℃; circulation conditions are as follows: circulating for 30 times at 98 ℃ for 10s, 55 ℃ for 10s and 72 ℃ for 30 s; and (3) post-extension: 10min at 72 ℃.
Example 2: construction of recombinant expression vectors
Mu.l of AtHAL6 obtained in example 1 above was ligated with pMD19-T Vector according to the instructions of pMD19-T Vector system manufactured by Takara. Coli DH 5. alpha. strain was then transformed and cultured overnight on LB plates coated with 5-bromo-4-chloro-3-indole-. beta. -D-galactoside and X-gal with ampicillin (100. mu.g/ml). Selecting white colonies, culturing overnight in an LB liquid culture medium and carrying out colony PCR identification; meanwhile, plasmid DNA is extracted by an alkaline method for sequence determination, and a recombinant pMD19-T vector is prepared.
The recombinant pMD19-T vector was digested with restriction enzyme Xba I and restriction enzyme Sac I, and then ligated with vector PBI121 digested with restriction enzyme Xba I and restriction enzyme Sac I. The ligation product is transformed into DH5 alpha cells, then the cells are cultured on LB solid plates containing kanamycin (50 mu g/ml), and the colonies are subjected to PCR identification and enzyme digestion analysis of plasmid DNA; the recombinant expression vector PBI121-AtHAL6 was prepared.
Example 3: acquisition of transgenic Arabidopsis
The recombinant expression vector PBI121-AtHAL6 was transformed into competent cells of Agrobacterium GV3101, and a single clone of Agrobacterium transformed with the recombinant expression vector PBI121-AtHAL6 was selected and inoculated into LB liquid medium containing 50mg/L kanamycin and cultured with shaking at 28 ℃ for two days. The fermentation broth was centrifuged at 3000rpm/min for 5 minutes and the resulting Agrobacterium pellet was suspended in an infection solution containing 5% sucrose and 0.03% Silwet L-77.
Columbia ecotype wild type Arabidopsis thaliana (Col-0) (purchased from American Arabidopsis thaliana biological resource center, ABRC, http:// www.biosci.ohio-state. edu/pcmb/facts/ABRC/abrchome. htm) was transformed by inflorescence dip dyeing, and seeds (T) bearing the current generation of transgenic Arabidopsis plants were harvested (T-0)1Passage), germinated seeds were screened in MS medium containing 50mg/Lkan (kanamycin). T to be germinated1Transplanting the seedling to culture soil, and harvesting the seeds (T)2Generations) and then through the same screening process to obtain homozygous T3Transgenic Arabidopsis seed transformed with PBI121-AtHAL 6. Finally, will T3The transgenic arabidopsis seeds are directly sown in culture soil to grow T3Transgenic arabidopsis plants grow to bloom for about two weeks under long-day conditions.
For T3The transgenic plant identification is carried out on transgenic arabidopsis thaliana, and comprises the following steps: primary screening of resistance culture medium, PCR amplification to further screen positive plant,Identifying positive plants by RTPCR expression quantity; finally, selecting a strain with high expression quantity from the positive plants obtained by identification, harvesting seeds of the strain, preparing transgenic arabidopsis seeds, and carrying out anti-adversity phenotype identification. Two strains are finally obtained and named as OE2 and OE4 respectively.
Example 4: transgenic Arabidopsis thaliana heat-resistant phenotype identification
The experiment was set up with the following two controls:
wild type control: wild type Arabidopsis thaliana (Col-0) not transformed with any plasmid;
empty vector control: according to the obtaining of T3The method for transferring transgenic Arabidopsis thaliana with recombinant expression vector PBI121-AtHAL6 comprises transferring empty vector PBI121 into wild Arabidopsis thaliana, and subculturing to obtain T3Transgenic Arabidopsis thaliana transformed with PBI121 was used to prepare empty vector control seeds.
(1) Seed germination experiment under high temperature condition
Taking wild type arabidopsis seeds, empty vector control seeds and transgenic arabidopsis seeds, performing a seed germination experiment on an MS culture medium, and performing high-temperature treatment at 45 ℃ for different time under the experimental conditions that the photoperiod is 16 hours of illumination and 8 hours of darkness; light intensity of 300-2(ii) S; the temperature under the illumination condition is 22-24 ℃, and the relative humidity is 70-90%; the temperature under dark condition is 18-20 deg.C, and relative humidity is greater than 90%. Normal conditions were used as controls.
The seed germination rate is counted every 12 hours, the experiment is repeated for 3 times, the measurement results are shown in figure 1 and figure 2 (WT represents wild type arabidopsis seeds, OE represents transgenic arabidopsis seeds, and empty vector control seeds and wild type arabidopsis seeds have the same phenotype, so that empty vector control is not shown in the figure), and the germination rate of the transgenic arabidopsis seeds after high-temperature treatment at 45 ℃ in a culture medium is obviously higher than that of the wild type arabidopsis seeds and the empty vector control seeds.
(2) High-temperature phenotype identification of transgenic arabidopsis at seedling stage
Placing the seedling with the same growth and just flattened cotyledon normally germinated on MS plate in high temperature culture room, and culturing under the conditions of 16 hr light period and 8 hr light periodIt is dark; light intensity of 300-2(ii) S; the temperature under the illumination condition is 45 ℃, and the relative humidity is 70-90%; the temperature under dark conditions was 45 ℃ and the relative humidity was greater than 90%. After 2 hours of culture, the cells were recovered for one week, photographed and counted for survival, and the experiment was repeated 3 times, with normal conditions as a control. The determination results are shown in fig. 3 and 4 (WT represents wild type arabidopsis thaliana, OE represents transgenic arabidopsis thaliana, and empty vector control and wild type control have the same phenotype, so the empty vector control is not shown in the figure), and the survival rate of the transgenic arabidopsis thaliana seedlings after high-temperature treatment at 45 ℃ is obviously higher than the germination rate of wild type arabidopsis thaliana seeds and empty vector control seeds.
(3) High-temperature phenotype identification of transgenic arabidopsis at seedling stage
And (3) carrying out high-temperature treatment on the transgenic arabidopsis line which normally grows in the matrix and grows consistently and the wild arabidopsis seedling together. As shown in FIGS. 5 and 6 (WT for wild type Arabidopsis, OE for transgenic Arabidopsis; empty vector control is not shown in the figure because the phenotype of the empty vector control is identical to that of the wild type Arabidopsis), the wild type and the overexpression lines grow without difference under normal growth conditions. After high-temperature treatment, the seedlings of wild arabidopsis and transgenic arabidopsis show a phenotype of reduced drought resistance: wild type Arabidopsis lines have a high general wilting speed and a low survival rate. In contrast, transgenic arabidopsis thaliana wilts slowly and has a high survival rate. Compared with wild type, the heat resistance of the transgenic arabidopsis line is obviously improved.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
Sequence listing
<110> Shandong university of agriculture
<120> application of Arabidopsis thaliana long-chain non-coding RNA AtHAL6 in regulation and control of high temperature stress tolerance of plants
<141> 2018-06-28
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gtgttgagcg ataaacttta aagatgtttt ccgtgagtgc tgtcatattc gtcaaagatg 180
gtggaagcat aaggtggggg agaaagttcg agaaagtttg agaaagttga aggaacatgt 240
gtttttgtcc gagcttccca agctctatgc tgttggagaa ctctgcaagg atcttgccac 300
cttcagggaa aagcttcaaa agagagtaac aaagaggaaa atctgaaaca aagaagaatc 360
aagaagatga tatcctgatg cccggcaagc aaaagcttct ccaatgttgg aaaccttgat 420
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Claims (6)
1. The application of the arabidopsis long-chain non-coding RNA AtHAL6 in regulating and controlling high-temperature stress tolerance of plants and/or in preparing heat-resistant transgenic plants, wherein the nucleotide sequence of the arabidopsis long-chain non-coding RNA AtHAL6 is shown as SEQ ID No. 1.
2. Use according to claim 1, characterized in that: when the gene is specifically applied, a plant expression vector containing the arabidopsis long-chain non-coding RNA AtHAL6 is introduced into plant cells or seeds, so that the arabidopsis long-chain non-coding RNA AtHAL6 is effectively expressed, and a transgenic plant with the heat resistance higher than that of a wild plant is obtained.
3. The application of the plant expression vector in preparing heat-resistant transgenic plants; the plant expression vector contains Arabidopsis thaliana long-chain non-coding RNA AtHAL6, and the nucleotide sequence of Arabidopsis thaliana long-chain non-coding RNA AtHAL6 is shown in SEQ ID No. 1.
4. Use according to claim 3, characterized in that: the heat resistance of the transgenic plant is stronger than that of a wild plant.
5. The application of genetically engineered host cells in the preparation of heat-resistant transgenic plants; the genetically engineered host cell has an arabidopsis long non-coding RNA AtHAL6 inserted into its genome; the nucleotide sequence of the arabidopsis long-chain non-coding RNA AtHAL6 is shown in SEQ ID NO. 1.
6. Use according to claim 5, characterized in that: the heat resistance of the transgenic plant is stronger than that of a wild plant.
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| CN114807137A (en) * | 2021-07-07 | 2022-07-29 | 忻州师范学院 | Potato high-temperature response lncRNA and application thereof |
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