CN117264970B - Application of populus euphratica PeHIT54 gene in improving salt tolerance of plants - Google Patents

Application of populus euphratica PeHIT54 gene in improving salt tolerance of plants Download PDF

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CN117264970B
CN117264970B CN202311469485.4A CN202311469485A CN117264970B CN 117264970 B CN117264970 B CN 117264970B CN 202311469485 A CN202311469485 A CN 202311469485A CN 117264970 B CN117264970 B CN 117264970B
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李建波
贾会霞
代新仁
赵金娜
杨艳飞
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Experimental Center Of Forestry In North China Chinese Academy Of Forestry
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    • 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

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Abstract

The invention discloses application of a populus euphratica PeHIT54 gene in improving salt tolerance of plants, wherein the sequence of the populus euphratica PeHIT54 gene is shown as SEQ ID No.1 or a degenerate sequence of the same protein encoded by the SEQ ID No.1, and the salt tolerance of the plants is improved by over-expressing the populus euphratica PeHIT54 gene in the plants. According to the invention, through comparing and analyzing the growth of the plant height, the ground diameter and the like of the PeHIT54 over-expressed transgenic poplar and the wild 84K poplar under the salt stress and the physiological index changes of photosynthetic rate, antioxidase activity and the like, the growth state of the PeHIT54 over-expressed transgenic plant under the salt stress is found to be obviously better than that of the wild 84K poplar, and the physiological index result also proves that the salt tolerance of the PeHIT54 over-expressed poplar is obviously better than that of the wild 84K poplar. The discovery reveals that the PeHIT54 gene can specifically improve the resistance of plants under salt stress, and provides important theoretical and practical significance for cultivating new salt-tolerant transgenic varieties in the field of tree molecular breeding.

Description

Application of populus euphratica PeHIT54 gene in improving salt tolerance of plants
Technical Field
The invention relates to the field of biotechnology, in particular to application of populus euphratica PeHIT54 gene in improving salt stress resistance of plants.
Background
Populus euphratica (Populus euphratica) is a arbor plant growing in saline-alkali areas, and has the outstanding advantages of saline-alkali resistance, drought resistance, environmental protection and the like. Has important theoretical and practical significance for improving saline-alkali areas, promoting ecological environment construction and recovery and improving economic benefits. The physiological and ecological characteristics and molecular mechanisms of populus euphratica are researched, and the mechanism of salt and alkali tolerance and drought tolerance of populus euphratica are explored, so that the method is a key for realizing improvement and utilization of a saline-alkali area, construction and recovery of an ecological environment and improvement of economic benefits.
Histidine trimer nucleoside binding protein 1 (Histidine triad nucleotide binding protein, hin 1) has cancer-inhibiting and disease-resistant functional properties in mammals and plants, respectively, but the function of hin 1 in plant salt tolerance is not yet clear. Currently, the study of the function of HINT is mainly focused on mammals, mainly comprising: (1) having nucleotide transferase and hydrolase activity; (2) Has gene transcription regulating effect, such as transcription factor MITF; (3) has obvious inhibition effect on tumor. In humans and mice, there are at least 3 pent gene members pent 1, pent 2 and pent 3.HINT1 and HINT2 are considered tumor suppressors. The human HINT1 gene mutation site is associated with diseases such as schizophrenia, nicotine dependency and autosomal recessive axonal neuropathy. Mice knocked out the HINT1 gene are far more susceptible to gastric, mastadenoma and ovarian tumors caused by carcinogens than normal mice. The HINT1 acts as a tumor inhibitor mainly by up-regulating the apoptosis control gene P53 and pro-apoptotic factor Bax, down-regulating the apoptosis inhibitor Bcl-2, and regulating the activity of cyclin-dependent kinase inhibitor P27. Mice knocked out the HINT2 gene develop fatty liver and affect the glycemic and thermoregulation system. In plants, the study of HINT is less, only functional studies in maize. The expression level of the corn Zm-HINT1 is induced by salicylic acid and jasmonic acid, and the over-expression of the gene can enhance the resistance of plants to fusarium graminearum.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides a new application of populus euphratica PeHIT54 gene in improving salt tolerance of plants.
The technical scheme of the invention is as follows: the application of the populus euphratica PeHIT54 gene in improving the salt tolerance of plants is provided, wherein the sequence of the populus euphratica PeHIT54 gene is shown as SEQ ID No. 1. Due to the degeneracy of the codons, the degenerate sequence encoding the same protein as SEQ ID No.1 (SEQ ID No. 2) also has the same effect.
Further, by overexpressing the populus euphratica PeHIT54 gene in plants, the salt tolerance of the plants is improved.
Further, the plant is poplar.
Through carrying out whole genome association analysis on the salt tolerance of the seeds of natural populus euphratica, correlating to the PeHIT54 gene, screening the cDNA sequence of the HIT54 according to the sequence number in a populus euphratica database, designing a synthetic oligonucleotide primer according to the sequence, cloning to obtain the full-length cDNA sequence of the populus euphratica HIT54 gene by taking the cDNA after reverse transcription of the mRNA of the populus euphratica as a template, naming the full-length cDNA sequence as the PeHIT54, constructing the full-length cDNA sequence on an entry vector, constructing the full-length cDNA on a plant expression vector through recombination reaction, screening positive clones, and transforming the populus euphratica (GV 3101) into poplar by a leaf disc method. After the treatment, from the experimental results, the PeHIT54 gene derived from populus euphratica is driven by the CaMV 35S promoter, the transgenic plants cannot have bad agronomic characters, and the salt tolerance of the populus euphratica transformed with the PeHIT54 gene is improved.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, through comparing and analyzing growth indexes such as plant height, ground diameter and the like of the PeHIT54 over-expressed transgenic poplar and the wild 84K poplar under salt stress and physiological index changes such as photosynthetic rate, antioxidant enzyme activity and the like, the growth state of the PeHIT54 over-expressed transgenic plant under salt stress is found to be obviously better than that of the wild 84K poplar, and the physiological index result also proves that the salt tolerance of the PeHIT54 over-expressed poplar is obviously better than that of the wild 84K poplar. The discovery reveals that the PeHIT54 gene can specifically improve the resistance of plants under salt stress, and provides important theoretical and practical significance for cultivating new salt-tolerant transgenic varieties in the field of tree molecular breeding.
Drawings
FIG. 1 is a cloning electrophoresis pattern of the populus euphratica PeHIT54 gene provided in example 1 of the present invention;
FIG. 2 shows the real-time fluorescence quantitative PCR analysis of the expression level of PeHIT54 gene in Populus euphratica root under NaCl stress provided in example 2 of the present invention;
FIG. 3 is a map of plasmid pCAMBIA1302-PeHIT54 provided in example 3 of the present invention;
FIG. 4 is a PCR identification electrophoresis chart of transgenic poplar plants provided in example 4 of the present invention;
FIG. 5 is a graph showing quantitative data of transgenic poplar expression provided in example 4 of the present invention;
FIG. 6 is a schematic diagram showing comparison of salt treatment of transgenic poplar and wild type 84K poplar in soil as provided in example 5 of the present invention;
FIG. 7 is a schematic diagram of plant height and ground diameter of transgenic poplar and wild 84K poplar salt treated in soil provided in example 5 of the present invention;
FIG. 8 is a graph showing net photosynthetic rates of transgenic poplar and wild 84K poplar salt treatment in soil as provided in example 5 of the present invention;
FIG. 9 is a schematic representation of chlorophyll content of soil treated with transgenic poplar and wild type 84K poplar salt as provided in example 5 of the present invention;
FIG. 10 is a schematic representation of chlorophyll fluorescence after salt treatment of transgenic poplar and wild 84K poplar in soil as provided in example 5 of the present invention;
FIG. 11 is a schematic representation of DAB and NBT staining of leaves of transgenic poplar and wild 84K poplar salt treated in soil as provided in inventive example 5;
fig. 12 is a schematic diagram showing the content of leaf antioxidant enzyme SOD, POD, CAT after salt treatment of transgenic poplar and wild 84K poplar in soil provided in example 5 of the present invention.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
EXAMPLE 1 cloning of the PeHIT54 Gene of Populus euphratica
1.1 obtaining the Gene sequence of interest
According to sequence numbers in the Phytozome database populus tomentosa, cDNA sequences homologous to populus euphratica PeHIT54 genes are screened, primers are designed according to the sequences by using primer 5 software, and full-length amplification of the genes is carried out by PCR. The primers are shown in Table 1.
TABLE 1PeHIT54 Gene cloning primers
The total RNA extracted from all samples was reverse transcribed into cDNA using the FastKing cDNA first strand synthesis kit. According to a high-fidelity enzyme PCR reaction system, gene cloning is carried out, wherein the reaction system is as follows: primeSTAR 10. Mu.l, forward primer 1. Mu.l; reverse primer 1. Mu.l; 2 μl of Populus euphratica cDNA; sterile ddH 2 O was made up to 20. Mu.l. The reaction procedure is: 95 ℃ for 5min; (95 ℃,10s;57 ℃,30s;72 ℃,50 s) 40 cycles; 72℃for 2min. The product recovery was performed with the novinaian gum recovery kit as shown in fig. 1.
1.2 Gene ligation T vector
Connecting the recovered target fragment with a T carrier, wherein the reaction system is as follows: t1 μl; 30ng of target gene; solution I5. Mu.l; sterile ddH 2 O is added to 10 mu l; the ligation was carried out overnight at 16 ℃. And (3) carrying out escherichia coli transformation on the connected vectors, picking monoclonal bacterial liquid, carrying out bacterial liquid PCR verification, and carrying out positive colony sequencing verification. After sequencing and inspection, the nucleotide sequence is shown as SEQ ID No.1, and the gene fragment is named as PeHIT54 and consists of 558bp bases.
Example 2 analysis of expression Properties of Populus euphratica PeHIT54 Gene (qRT-PCR)
2.1 primer design
Fluorescent quantitative PCR was performed based on the cloned full-length sequence of the gene, and Primer3 was used to design quantitative primers, intra-gene reference primers. The primers are shown in Table 2.
TABLE 2 intragenic reference primers
2.2 salt stress treatment is carried out on populus euphratica tissue culture seedlings
Transferring the normally grown poplar tissue culture seedling to 100mM NaCl culture medium for treatment, taking the root of the poplar tissue culture seedling to extract RNA, reversely transcribing the root into cDNA, and carrying out qRT-PCR reaction. The reaction system is as follows: SYBR qPCR Master Mix 10 μl; forward primer 0.4. Mu.l; reverse primer 0.4. Mu.l; 2 μl of Populus euphratica cDNA; sterile ddH 2 O was made up to 20. Mu.l. The reaction procedure is: 95 ℃ for 5min; (95 ℃,10s;60 ℃,30 s) 40 cycles; 95 ℃ for 15s;60 ℃ for 60s;95℃for 15s. The resulting data were analyzed as shown in fig. 2. The result shows that under the salt stress treatment, the PeHIT54 gene has the trend of increasing the expression range along with the increase of the treatment time, and the gene is proved to be induced by the salt stress.
EXAMPLE 3 construction of Populus euphratica PeHIT54 Gene expression vector
3.1 primer design
Constructing a plant over-expression vector, and designing a primer with a pCAMBIA1302 homology arm. The primers are shown in Table 3.
TABLE 3 primers with pCAMBIA1302 homology arms
3.2 construction of pCAMBIA1302-PeHIT54 overexpression vector
The PeHIT54 gene is amplified by taking F and R as primers, the product is recovered, the pCAMBIA1302 vector is subjected to double digestion and linearization, and the target gene is constructed to the vector by using the Novain homologous recombinase. The reaction system is as follows: 15ng of target gene; 105ng of carrier; 5 XCE II Buffer 2. Mu.l; 1 μl of Exnase II; ddH 2 O makes up 10. Mu.l. The reaction procedure is that the reaction is carried out for 30min at 37 ℃; cooling to 4 ℃ or immediately cooling on ice. And (3) carrying out escherichia coli transformation on the connected vectors, picking monoclonal bacterial liquid, carrying out bacterial liquid PCR verification, and carrying out positive colony sequencing verification. The pCAMBIA1302-PeHIT54 over-expression vector which is successfully constructed is shown in FIG. 3.
Example 4 genetic transformation of the Populus euphratica PeHIT54 gene
The constructed pCAMBIA1302-PeHIT54 over-expression vector is transferred into agrobacterium GV3101 by a heat shock method. The PeHIT54 gene is transferred into poplar through agrobacterium mediation, and the transformation steps are as follows: hybrid poplar clone 84K tissue culture seedlings for genetic transformation were irradiated at a culture temperature of 23-25℃for 16/8h (day/night). Agrobacterium containing pCAMBIA1302-PeHIT54 expression vector infects 84K leaf disks at od600=0.6-0.8. The infected leaf discs were co-cultured for 3 days in the dark at 22.+ -. 2 ℃ on adventitious bud induction medium MS minimal medium supplemented with 0.5 mg/L6-BA and 0.05mg/L NAA. The cocultured leaf discs were transferred to a SIM containing 3mg/L hygromycin B and 200mg/LTimentin, and resistant adventitious buds were induced and selected under conditions of a culture temperature of 23-25℃and an illumination of 16/8h (day/night). After about 30 days of induction culture, the resistant adventitious shoots were transferred to rooting medium containing 3mg/L hygromycin B and 200mg/L Timentin (RIM, 1/2. Times. MS minimal medium supplemented with 0.05mg/L IBA and 0.02mg/L NAA) until adventitious roots were induced. gDNA is extracted by using a CTAB method to extract the DNA of the rooted plant leaves, and PCR verification is carried out. The results are shown in FIG. 4, and 20 positive PeHIT54 transgenic poplar plants are obtained. And extracting RNA from the 20 positive seedlings obtained by screening, and verifying the transgene expression quantity by qRT-PCR. As a result, as shown in FIG. 5, the transgenic poplar lines (OE-1, OE-7) with appropriate expression levels of PeHIT54 were selected for subsequent salt-resistant phenotypic assay.
EXAMPLE 5 salt resistance analysis of Populus euphratica PeHIT54 transgenic poplar
84K transgenic tissue culture seedlings growing for 30 days under the same conditions are transplanted into soil, 84K poplar and PeHIT54 transgenic poplar with similar sizes and growth conditions are selected for salt stress treatment for 30 days after 2 months, 100ml of 100mM NaCl solution is irrigated every 2 days, and 100ml of control plants are irrigated every 2 days. During the treatment, the 84K poplar and PeHIT54 transgenic poplar were monitored for plant height, net photosynthetic rate, enzyme activity, etc.
5.1PeHIT54 salt stress phenotyping
During stress treatment, poplars treated for 20d and 30d were photographed. The results are shown in FIG. 6. The phenotype differences of 84K poplar and PeHIT54 transgenic poplar were not significant under normal conditions. After 30 days of 100mM NaCl treatment, most 84K poplar leaves turned yellow, even partially necrotic. In contrast, peHIT54 transgenic poplar maintained normal growth and leaf color.
5.2 plant height and ground diameter analysis
During stress treatment, the plant height and ground diameter of the plants were measured with tape and vernier calipers for 84K poplar and PeHIT54 transgenic poplar treated for 0d, 20d and 30 d. The plant height and the ground diameter have the precision of 0.1cm and 0.001cm respectively. The results are shown in FIG. 7A, and under normal conditions, the plant heights of the plants have no obvious difference; under the condition of salt stress, the plant height of 84K poplar is reduced by 18.58 percent, and the plant height of PeHIT54 transgenic poplar is reduced by 11.64 to 11.88 percent;
as shown in fig. 7B, there is no obvious difference in ground diameter under normal conditions; under the salt stress treatment, the ground diameter of 84K poplar is reduced by 21.00%, and the transgenic poplar is reduced by 3.74-6.47%.
5.3 net photosynthetic Rate analysis
The net photosynthetic rate adopts LI-6400 type photosynthetic apparatus, and the light intensity is set to be 1000 mu mol.m -2 ·s -1 Control leaf chamber CO 2 The concentration (Ca) was 400.+ -.10. Mu. Mol.mol -1 The atmospheric temperature was 28.+ -. 1 ℃. And selecting 84K poplar and PeHIT54 transgenic poplar with similar 3-6 morphologies and basically consistent growth vigor, and measuring parameters under normal and salt treatment. The results are shown in FIG. 8, where the net photosynthetic rate of 84K poplar and PeHIT54 transgenic poplar is decreasing, and the rate of decrease of 84K poplar is significantly higher than that of transgenic poplar, and after 2 days, the photosynthetic rate of 84K poplar is lower than that of transgenic poplar.
5.4 chlorophyll fluorescence analysis
Chlorophyll content was measured using an ultraviolet spectrophotometer and data recorded. The leaves are soaked with 95% ethanol, 0.1 g of the leaves are cut off respectively for grinding, and after proper grinding, the volume is fixed to 25ml by using 95% ethanol, and the dilution multiple is 1. Then, colorimetry was performed at wavelengths 665nm, 649nm and 470nm, and the concentrations of chlorophyll a (Ca), chlorophyll b (Cb) and carotenoid (C-class) were calculated by absorbance values, respectively, and the total chlorophyll concentration was calculated according to the following formula.
C a ＝13.95A665-6.88A649
C b ＝24.96A649-7.32A665
Total = C a +C b
Class c= (1000 a470-2.05 xc a -114.8×C b )/245
Chlorophyll content (mg/g) = (concentration x extract volume x dilution factor)/fresh sample weight.
As shown in FIG. 9, chlorophyll in 84K poplar leaves under salt stress showed a significant decrease in the content significantly lower than in PeHIT54 transgenic poplar.
Chlorophyll fluorescence was measured using Yaxin-1161G, first, plants were dark adapted for 30min, 84K poplar was measured for the same leaf position as PeHIT54 transgene Yang Xuanqu, and the following indicators were measured:
maximum photochemical efficiency of PS ii: fv/Fm
Actual photochemical efficiency of PS ii: Φpsii= (Fm '-Fs)/Fm'
Photochemical quenching coefficient of PS ii: qp= (Fm ' -Fs)/(Fm ' -Fo ')
PS ii non-photochemical quenching coefficient: qn=fm/Fm'
The results are shown in FIG. 10, and under normal conditions, the indexes are not different between 84K poplar and PeHIT54 transgenic poplar; after salt stress treatment, chlorophyll fluorescence parameters of 84K poplar suddenly drop, fv/Fm, ΦpsII, qP and qN are respectively reduced by 8.79%, 49.08%, 52.3333% and 16.4349%, and the ratio is obviously higher than that of a transgenic strain. The method shows that under the condition of salt stress, the photoinhit 54 transgenic poplar has better photoinhibition, optical system reaction center, optical energy utilization rate and optical energy conversion energy thermodynamic capability than those of non-transgenic strains, and has stronger photoprotection capability.
5.5NBT and DAB analysis
1.97g of Tris-HCl is fixed to 250mL, the pH value is adjusted to 5.5, 0.25g of DAB powder is added and uniformly shaken, and 1mg/mL DAB staining solution is prepared. After 91.5mL of the solution A and 85mL of the solution B of the PBS buffer were mixed, the volume was set to 400mL, and then 0.2g of NBT powder was added and shaken well to prepare 0.5mg/mL of NBT dye. Adding acetic acid, glycerol and absolute ethanol in the volume ratio of 1:1:3 into a conical flask, mixing and shaking uniformly to prepare decolorization liquid.
Taking a clean 50mL test tube, placing the blade of the sample to be tested into the test tube, then adding a proper amount of staining solution to enable the staining solution to be completely immersed, starting vacuumizing, enabling the blade to be immersed into the bottom of the test tube, wrapping the test tube with tinfoil, placing the test tube in a constant temperature incubator for staining for 6-8 hours, and setting the temperature to be 37 ℃. After dyeing, the leaves are clamped by forceps and put into decolored leaves, the leaves are boiled in a water bath at 100 ℃ for 5min until chlorophyll is completely decolored, the leaves are washed by absolute ethyl alcohol after being cooled to room temperature, and the water indicated by the leaves is sucked by filter paper, so that the coloring condition of active oxygen in the leaves is observed. As shown in FIG. 11, DAB and NBT had a darker coloring effect at 84K poplar under salt stress treatment, while PeHIT54 transgenic plants had a lighter coloring effect, and were not normally differentiated. The active oxygen content in the PeHIT54 transgenic poplar is lower than that of 84K poplar under the stress of salt.
5.6 analysis of antioxidant enzyme Activity
SOD, POD, CAT all adopt Soxhaust kit to detect under ultraviolet spectrophotometer. The results are shown in fig. 12, in normal conditions, POD and SOD activities of PeHIT54 transgenic poplar were not significantly different from 84K poplar (B in fig. 12 a and 12), but CAT activity was slightly higher than 84K poplar (C in fig. 12). However, after salt stress, the POD, SOD and CAT activities of transgenic plants were significantly higher than those of 84K poplar (A-C in FIG. 12).
In conclusion, through the measurement of growth and physiological indexes, the salt resistance of the PeHIT54 transgenic poplar is obviously enhanced, and the salt resistance of the poplar is finally improved by regulating and controlling the activity of antioxidant enzyme so as to maintain the stability of active oxygen under salt stress.

Claims (2)

1. Populus euphratica (Linn.) SprengPeHIT54Use of a gene for improving salt tolerance of poplar, said populus euphraticaPeHIT54The gene sequence is shown as SEQ ID No.1 or degenerate sequence which codes the same protein as SEQ ID No. 1.
2. Use according to claim 1, characterized in that the populus euphratica is overexpressed in poplarPeHIT54The gene can raise the salt tolerance of poplar.
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