CN113151296B - Tobacco heat shock protein related gene and application thereof - Google Patents
Tobacco heat shock protein related gene and application thereof Download PDFInfo
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- CN113151296B CN113151296B CN202110302810.2A CN202110302810A CN113151296B CN 113151296 B CN113151296 B CN 113151296B CN 202110302810 A CN202110302810 A CN 202110302810A CN 113151296 B CN113151296 B CN 113151296B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
The invention relates to a tobacco heat shock protein related gene and application thereof, wherein a nucleotide sequence is shown as SEQ ID NO. 1. The gene related to the tobacco heat shock protein is named as NtDnaJ/HSP40, the gene editing technology mediated by CR I SPR/Cas9 is utilized to knock out the NtDnaJ/HSP40 gene to obtain a gene editing plant with reduced expression quantity, fluorescent quantitative PCR is found, under normal conditions, NtDnaJ/HSP40 gene expression quantity analysis is carried out on different tissues in a gene editing plant control plant, and the result shows that the NtDnaJ/HSP40 gene expression quantity in three plant tissues of roots, stems and leaves is lower than that of the control plant in the editing plant, wherein the expression quantity in leaves is reduced most obviously. The method provides genetic materials and theoretical basis for the research of tobacco heat shock protein and the research of tobacco stress resistance.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a tobacco heat shock protein related gene and application thereof.
Background
After an organism is stressed, the synthesis of normal proteins in vivo is inhibited, and cells turn to synthesize a special class of proteins, which are named Heat Shock Proteins (HSPs). In addition to heat stress, other environmental stress factors such as drought, low temperature, heavy metal ions, etc. also induce the synthesis of HSPs in plants. HSPs are a class of proteins with very conserved amino acid sequences and functions. The HSPs heat shock protein has molecular chaperone function, mainly participates in transportation, folding, assembly and positioning of nascent peptide in organisms and renaturation and degradation of denatured protein, and plays an important role in cell life activities. Overexpression of HSPs heat shock proteins can increase the resistance of organisms to high temperature stress and peroxidative damage.
The tobacco is used as an important economic crop and a model crop in China, and has important significance in researching the molecular mechanism of the tobacco heat shock protein gene responding to abiotic adversity stress. In the aspects of cloning and function research of heat shock protein genes, researchers find that the tobacco heat shock protein HSP22 is highly related to the content of tobacco pigment substances, and the content of the pigment substances in tobacco is obviously reduced after the genes are silenced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a tobacco heat shock protein related gene and application thereof, and provides genetic material and theoretical basis for researching the stress resistance and gene function of tobacco.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a gene related to a tobacco heat shock protein has a nucleotide sequence shown in SEQ ID NO.1 and comprises 423bp bases, and the gene is derived from tobacco (Nicotiana tabacum) and is named as NtDnaJ/HSP 40.
Preferably, the amino acid sequence of the protein coded by the gene related to the tobacco heat shock protein is shown in SEQ ID NO.2 and comprises 140 amino acids.
The invention also provides application of the gene of the tobacco heat shock protein in the aspect of stress resistance of tobacco. After the gene is edited, the tobacco has reduced resistance to salt stress in the seed germination seedling stage. The real-time fluorescent quantitative PCR shows that under normal conditions, NtDnaJ/HSP40 gene expression level analysis is carried out on different tissues in a gene editing plant control plant, and the result shows that the NtDnaJ/HSP40 gene expression level in three plant tissues of roots, stems and leaves is lower than that of the control plant in the editing plant, wherein the expression level in leaves is reduced most obviously. The gene function research provides genetic materials and theoretical basis for the genetic improvement of tobacco.
The invention has the beneficial effects that:
according to the invention, a CRISPR/Cas9 editing vector for knocking out NtDnaJ/HSP40 genes is constructed through a CRISPR/Cas9 mediated gene editing technology, and a tobacco plant with the NtDnaJ/HSP40 genes edited is obtained through genetic transformation. According to the invention, salt stress treatment is carried out at different degrees in the seed germination seedling stage, and the growth (overground part and root length) of the NtDnaJ/HSP40 gene editing plant is obviously lower than that of a control (untransformed) plant under the condition of salt stress treatment.
The gene NtDnaJ/HSP40 related to the tobacco heat shock protein is found by fluorescent quantitative PCR (polymerase chain reaction), under the normal condition, the expression quantity of the NtDnaJ/HSP40 gene is analyzed in different tissues in a control plant of a gene editing plant, and the result shows that the expression quantity of the NtDnaJ/HSP40 gene in three plant tissues of roots, stems and leaves is lower than that of the control plant in the editing plant, wherein the expression quantity in the leaves is reduced most obviously.
In conclusion, NtDnaJ/HSP40 genes are knocked out by using a CRISPR/Cas9 mediated gene editing technology to obtain a gene editing plant with reduced expression level under normal conditions, so that genetic materials and theoretical bases are provided for the research of tobacco heat shock proteins and the research of tobacco stress resistance.
Drawings
FIGS. 1A and 1B are graphs comparing the germination stages of control (untransformed) plants and gene-edited plants under different concentration treatment conditions (salt stress), wherein (FIG. 1A) is a schematic diagram of the distribution in the culture dish and (FIG. 1B) is a graph comparing salt stress;
FIG. 2 shows the relative expression levels of NtDnaJ/HSP40 in different tissues (roots, stems, leaves) of control (untransformed) plants and gene editing plants under normal conditions.
Detailed Description
The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.
In the embodiments of the present application, those who do not specify a specific technique or condition, and those who do follow the existing techniques or conditions in the field, and those who do not specify a manufacturer or a material used, are general products that can be obtained by purchasing.
The percentages are volume percentages and proportions are volume ratios, unless otherwise indicated.
The tobacco used in the following examples of the present application is Honghuadajinyuan, a commercial tobacco.
Example 1
This example is described briefly below with respect to the process of obtaining the tobacco heat shock protein-associated gene NtDnaJ/HSP 40.
Taking cultivated species tobacco safflower large gold element leaves as samples, extracting total RNA of the tobacco leaves by utilizing an RNA extraction kit, and carrying out reverse transcription to obtain cDNA for later use:
extracting total RNA of tobacco according to the instruction of the plant RNA extraction kit.
Mu.g of total RNA was extracted from leaves for reverse transcription in the following transcription system:
Total RNA 1μg
Oligo(dT)(10μM) 1.5μL
ddH 2 O up to 15μL
mixing the above system, placing in PCR, keeping temperature at 70 deg.C for 5min, removing, immediately placing on ice for 5min, and adding the following reagents into the system:
the system is put into a PCR instrument, is kept at 42 ℃ for 65min, 65 ℃ for 10min and 4 ℃, and then is stored in a refrigerator at the temperature of minus 20 ℃ for use.
By a homologous alignment method, referring to the sequence of an arabidopsis gene and the known partial gene sequence of tobacco, the sequence of an amplification primer is designed as follows:
F：5’-ATGGATTGTACGTTAAAG-3’，(SEQ ID No.3)
R：5’-TCAACCTAAGCATCTAAC-3’；(SEQ ID No.4)
and (3) performing PCR amplification by using the prepared cDNA as a template and the primers:
amplification system (50 μ L):
mixing, centrifuging and performing PCR amplification, wherein the PCR reaction conditions are as follows: 30 cycles of 95 ℃ for 10sec, 52 ℃ for 30sec, and 72 ℃ for 2.5 min; 10min at 72 ℃; hold at 12 ℃.
And purifying and sequencing the amplified product to obtain a gene NtDnaJ/HSP40 sequence related to the heat shock protein of the tobacco, wherein the base sequence of the gene NtDnaJ/HSP40 sequence is shown as SEQ ID No.1 and comprises 423bp bases in total. After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.2 and comprises 140 amino acids in total, and further comparative analysis shows that the protein contains a sequence with high homology and is highly conserved.
Example 2
By utilizing the tobacco heat shock protein related gene NtDnaJ/HSP40 obtained in the embodiment 1, the invention further constructs a CRISPR/Cas9 vector and obtains a gene editing plant by utilizing the leaf disc method for transformation.
Selecting a more specific 20nt nucleotide sequence (CTATGATAAGTCTGTACCAGAGG SEQ ID No.5) in the NtDnaJ/HSP40 gene as a guide sequence of the CRISPR/Cas9, connecting the sequence fragment with a CRISPR/Cas9 vector (provided by southwestern university) to obtain a transformed clone, carrying out PCR amplification detection, then sending a PCR positive clone to a sequencing company for sequencing confirmation, and finally obtaining the CRISPR/Cas9-NtDnaJ/HSP40 editing vector.
A genetic transformation test is carried out by using the CRISPR/Cas9-NtDnaJ/HSP40 editing vector plasmid constructed in the last step and taking a safflower macrogold as an example to knock out a tobacco heat shock protein related gene NtDnaJ/HSP40 in a plant body, and related experimental processes are briefly introduced as follows.
Dibbling tobacco seed in culture dish until 4 cotyledons (15-20d) grow, transferring into culture bottle (containing 80mL MS liquid culture medium) containing 2 strains, and irradiating at 25 + -1 deg.C with illumination intensity of 30-50 μmol/(m) 2 S) and the light time is 16h/d, and the culture is continued for 40d for standby.
The LBA4404 stored at-80 ℃ was taken out and electrically transformed into competent Agrobacterium cells, and frozen and thawed on ice. When the competence had just thawed, 2. mu.L of plasmid containing the edited NtDnaJ/HSP40 gene was added, gently mixed by flick, and placed on ice. And transferring the uniformly mixed liquid into a precooled electric rotor, placing the electric rotor into an electric rotor for conversion, adding 1mL of YEB liquid culture medium after the conversion is finished, mixing the YEB liquid culture medium with the conversion solution, and then placing the mixture in a shaking table at 28 ℃ and culturing the mixture for 1.5 to 2 hours at 200 rpm. The cells were centrifuged at 8,000rpm to discard the supernatant medium, and then suspended in 200. mu.L of YEB liquid medium, and plated on YEB solid medium containing 50mg/L rifampicin, 50mg/L streptomycin and 50mg/L kanamycin for 2-3d by inversion in the dark at 28 ℃.
Preparing a tobacco leaf disc into a square leaf disc with the side length of 1cm in an ultraclean workbench, and preparing agrobacterium colony containing CRISPR/Cas9-NtDnaJ/HSP40 editing vector by using MS liquid to form suspension bacterial liquid (OD) 600 0.6-0.8). And soaking and infecting the tobacco leaf discs for 10min by using the suspension agrobacterium liquid. Then, the leaf discs were placed on MS solid medium containing 2.0mg/L NAA +0.5 mg/L6-BA, incubated at 28 ℃ in the dark for 3 d. Then, carrying out subculture, and placing on an MS solid culture medium containing 2.0mg/LNAA, 0.5 mg/L6-BA, 250mg/L Cb and 50mg/L Kan, wherein the culture conditions are as follows: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m) 2 S), culturing at 25 deg.C for 8h/d in dark, culturing for 45-60d until differentiated bud forms, and changing differentiation culture medium every 7-10d for 5-6 times; culturing until a differentiated bud is formed; cutting off callus formed by existing differentiated bud, culturing on MS culture medium containing carbenicillin 500mg/L and kanamycin 50mg/L, culturing for 8-14d when the differentiated bud on callus grows to 2-4cm high and the culture condition is the same as that of differentiated culture; and (3) performing rooting culture on the regenerated plant, cutting a differentiated bud, inserting the cut differentiated bud into an MS culture medium containing 500mg/L carbenicillin and 50mg/L kanamycin to perform rooting culture, wherein the culture condition is consistent with the differentiation culture condition, culturing for 7-10 days to obtain a regenerated plant of the gene NtDnaJ/HSP40 mediated and transformed by the Agrobacterium LBA4404, then sampling leaves of the transformed plant, delivering a gene Hua Dagen to perform molecular detection, and determining to obtain an edited plant of the gene NtDnaJ/HSP 40.
Example 3
The plants determined to be NtDnaJ/HSP40 gene knockout plants by molecular detection in example 2 are used for harvesting to obtain gene editing materials. Then, carrying out stress resistance treatment on the seeds, selecting NtDnaJ/HSP40 gene edited tobacco seeds and control (untransformed) seeds, selecting plump seeds without obvious defects for surface disinfection, and then respectively dibbling the seeds in an MS culture medium for culture; the culture conditions were: the culture temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m) 2 "s"), was horizontally placed under the condition of 16h/d for cultivation and observed.
Preparation of salt stress (100mmol/L, 150mmol/L, 200mmol/L NaCl) treatment medium and MS medium.
When the tobacco seeds grow for about 3-4 days, selecting seeds (10-20 seeds) at the same period when the seeds are exposed to white, and carefully transferring the seeds to a salt stress (100mmol/L, 150mmol/L and 200mmol/L NaCl) treatment culture medium and an MS culture medium in the step (2); the culture conditions were: the culture temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m) 2 "s"), cultured for 12-25 days under the condition of 16h/d of illumination time, vertically placed for culture, and observed. And (4) placing the MS culture medium to expose white so as to prevent the inconsistent germination time due to different seed vigor. The seedlings are vertically placed and cultured, and the seedlings are vertically downward, so that the root length can be measured conveniently.
Observing and recording the root length, the state of the root, the leaf size, the leaf color and the like, and preliminarily determining the stress resistance of the tobacco seeds in the germination period.
Seeds of both (control) and gene editing material were treated in the same dish (results are shown in FIG. 1).
Example 4
In order to detect the expression condition of the tobacco heat shock protein NtDnaJ/HSP40 gene under different stress treatments, the expression quantity of the gene in leaves is detected by a real-time fluorescent quantitative PCR method. SYBR Premix Ex Taq TM II from TaKaRa was used as a real-time fluorescent quantitative PCR reagent. The specific detection method comprises the following steps:
selecting tobacco plants in the bud stage, and sampling root systems, stems and leaves of NtDnaJ/HSP40 gene editing plants and control (untransformed) plants under normal conditions; and then the label is placed in liquid nitrogen for storage, so that subsequent RNA extraction and real-time fluorescence quantitative PCR detection test can be conveniently carried out.
The NtDnaJ/HSP40 qPCR amplification primers were as follows:
qPCR-Nt NtDnaJ/HSP40-F1：TTTTCAACAGGAGCACAGGC(SEQ ID NO.6)
qPCR-Nt NtDnaJ/HSP40-R1：CACCATTTATTTGCATTGCCCG(SEQ ID NO.7)
the primers for amplifying the internal reference gene (18S) are as follows:
18S-F：CCTACGCTCTGTATACATTAGC(SEQ ID NO.8)
18S-R：GTGTTGAGTCAAATTAAGCCGC(SEQ ID NO.9)
three technical replicates were performed for each sample, the reaction system was as follows:
amplification conditions: 30s at 95 ℃; at 95 ℃ for 10s and at 60 ℃ for 10s, for 40 cycles.
Dissolution curve: 95 ℃ for 10s, 60 ℃ for 60s, 95-0.29 ℃/s.
The results show that the tobacco heat shock protein related gene NtDnaJ/HSP40 is expressed in different tissues (roots, stems, leaves) under normal conditions (the results are shown in FIG. 2).
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (2)
1. Tobacco heat shock protein related geneNtDnaJ/HSP40The gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. Tobacco heat shock protein related geneNtDnaJ/HSP40The use of (A), characterized by gene editingNtDnaJ/ HSP40The obtained plants have reduced resistance to salt stress;
the gene editing is a CRISPR/Cas9 mediated gene editing technology, and is used for knocking outNtDnaJ/HSP40The CRISPR/Cas9 editing vector is obtained after genetic transformationNtDnaJ/HSP40Edited tobacco plants occur.
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