CN118086330A - Salt stress-resistant cotton gene and application thereof - Google Patents
Salt stress-resistant cotton gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, and relates to application of cotton genes GHGH 3.6.6 and GHEXPA4 in plant salt stress tolerance. The invention obtains the gene silencing cotton plant with obviously reduced tolerance to salt stress through the VIGS technology and obviously improved tolerance to salt stress of the arabidopsis homozygous line over-expressing the gene through the transgenic technology, and is verified by the comparison of phenotype change, leaf wilting degree, in vitro leaf water loss rate, leaf relative water content and root length elongation after salt stress treatment and the like. The results show that the gene plays an important role in the plant salt stress response process, has important significance in improving the breeding and research of cotton salt tolerance, and can be applied to breeding stress-resistant cotton varieties.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to application of cotton genes GHGH 3.6.6 and GHEXPA4 in plant salt stress tolerance.
Background
Salt stress is a major environmental stress severely limiting crop production and threatening the sustainable development of agriculture. With the increasing global warming, salt stress is more and more frequent in some areas, further threatening crop production and grain safety. Salt stress can further increase intracellular Na + concentration, induce osmotic stress, ionic toxicity and oxidative stress, thereby disrupting many physiological and biochemical processes associated with cellular functions, leading to reduced photosynthesis, unbalanced nutrient absorption, reduced yield and quality loss. Salt stress can affect photosynthesis of plants, and excessive absorption of salt can cause plants to absorb insufficient nutrients and minerals, resulting in too low chlorophyll content in plants, further affecting photosynthesis. In addition, salt stress can influence plant respiration, influence membrane lipid and membrane protein of cells, hinder protein synthesis, cause osmotic stress and ion stress to plants, and excessive ion components in salt can cause inconsistent osmotic pressure inside and outside plant tissues, so that serious harm is caused to plants, and death of the plants can be caused when serious. Thus, identification of key genes and mechanisms of plant adaptation to salt stress is critical to improving plant adaptation and crop yield.
Auxins are involved in almost all aspects of plant development. Some studies indicate that biosynthesis of auxin is critical for salt resistance, while other studies provide evidence that increased salt resistance is associated with reduced levels of indole-3-acetic acid (IAA). IAA is the predominant form of auxin in plants, and auxin homeostasis is regulated by IAA coupling. The GRETCHEN HAGEN (GH 3) family of genes is involved in IAA binding to amino acids, forming reversible or irreversible IAA catabolites. IAA coupled with alanine, leucine and phenylalanine as transient storage compounds can be hydrolyzed to free IAA, whereas IAA coupled with aspartic acid or glutamic acid is metabolized by oxidation rather than hydrolyzed to free IAA. Research shows that GH3 family genes play an important role in plant development and stress response. GH3.3, GH3.5 and GH3.6 forward redundancy regulates indefinite numbers. The single mutants of gh3.3, gh3.5 or gh3.6 produced more adventitious roots than the wild type, whereas the triple mutants of gh3.3-1gh3.5-2gh3.6-1 showed significantly less adventitious roots. Overexpression OsGH-2 in rice reduces drought tolerance and abscisic acid (ABA) levels, while overexpression of GH3.5 in cotton enhances drought tolerance and salt tolerance. Therefore, GH3 family genes play an important role in stress such as salt resistance, drought resistance and the like. In recent years, single-cell sequencing technology can subdivide tissues into different cell groups, then quantitatively analyze the cells of the different cell groups, and combine with subsequent Marker genes to perform functional verification on the cell groups, so that the problem of heterogeneity of the cell groups is solved. In combination with the earlier published single cell sequencing article about Asian cotton root tip, we screen Marker gene GaGH3.6 (Ga 03G 2153) of root tip epidermal layer tissue, and perform VIGS experiment on the gene in Asian cotton, and the experimental result proves that the Marker gene has salt tolerance property. In order to further study the gene function of the epidermis Marker gene in upland cotton, the invention determines the homologous gene GHGH 3.6.6 (GH_A03G1858) of the epidermis Marker gene, and the invention expects to combine with the function verification of the subsequent transgenic cotton to obtain a new cotton variety with novel salt resistance, thereby making an important contribution to cultivating salt-resistant cotton.
Expansin is a cell wall loosening protein that can affect plant growth, development processes, and environmental stress responses. Studies have shown that changes in expression of tobacco α -expansin 4 (EXPA 4) affect tobacco susceptibility to tobacco mosaic virus infection through agrobacterium-mediated transient expression. Phenotype and tissue morphology differences of transgenic plants with altered gene expression indicate that EXPA4 is critical to the normal growth and development of tobacco. Studies of tobacco EXPA4 abiotic stress mutants have shown that RNAi mutants have increased sensitivity to salt and drought stress. Whereas tobacco over-expressed EXPA4 is more tolerant to salt and drought, manifesting as less cell damage, higher fresh weight, higher accumulation of soluble sugars and proline, and higher expression levels of multiple stress response genes. In addition, the over-expression lines are more sensitive to the viral pathogen TMV-GFP than the wild type or RNAi mutants. Compared to the wild type, after infection with TMV-GFP, the antioxidant system of the RNAi mutant, several defenses-related plant hormone induction and gene expression were down-regulated, while the antioxidant system of the RNAi mutant was induced and gene expression was up-regulated. In addition, EXPA4 overexpression also accelerates the disease development of pseudomonas syringae DC3000 on tobacco. Taken together, EXPA4 plays an important role in tobacco growth and response to abiotic stress. In order to further study the function of EXPA4 in cotton, RNA extraction and transcriptome sequencing are carried out on normal growth materials and salt stress materials of upland cotton (TM-1), transcriptome data are further analyzed, the analysis result shows that the expression level of a gene GHEXPA (GH_D09G 1670) in salt stress treatment materials is obviously higher than that in normal growth materials, and in order to further study the function of the gene in plants and whether the gene has the function of improving the salt tolerance of cotton in cotton, the function of the gene GHEXPA is further verified through a VIGS experiment and an over-expression Arabidopsis experiment.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to research cotton genes with salt stress resistance and application thereof.
The technical scheme of the invention is as follows:
a salt stress resistant gene GHGH 3.6.6 has a nucleotide sequence table shown in SEQ ID No. 1.
The gene GHGH 3.6.6 is applied to improving the salt tolerance of plants.
The application is to over-express a gene GHGH 3.6.6 in a plant, thereby improving the tolerance of the plant to salt stress.
A method for improving salt stress tolerance of plants comprises transforming gene GHGH 3.6.6 into plants by agrobacterium mediation by using plant over-expression vector, thereby obtaining salt tolerant transgenic plants.
The plant is cotton or Arabidopsis thaliana.
The cotton is upland cotton (TM-1).
A salt stress resistant gene GHEXPA4, the nucleotide sequence table of which is shown as SEQ ID No. 2.
The gene GHEXPA4 is applied to improving the salt tolerance of plants.
The application is to over-express gene GHEXPA-4 in plants, thereby improving the tolerance of the plants to salt stress.
A method for improving salt stress tolerance of plants comprises transforming gene GHEXPA4 into plants by agrobacterium mediation by using plant over-expression vector, thereby obtaining salt tolerant transgenic plants.
The plant is cotton or Arabidopsis thaliana.
The cotton is upland cotton (TM-1).
The invention discloses application of upland cotton genes GHGH 3.6.6 and GHEXPA4 in improving plant salt stress capability. According to the invention, the tolerance of GHGH 3.6.6 or GHEXPA4 gene-silenced cotton plants to salt stress is obviously reduced by a VIGS technology, the tolerance of an arabidopsis homozygous line over-expressing GHGH 3.6.6 or GHEXPA4 genes to salt stress is obviously improved by a transgenic technology, and the bidirectional verification is carried out on phenotype change, leaf wilting degree, in-vitro leaf water loss rate, leaf relative water content, germination rate statistics, root length elongation comparison and the like after salt stress treatment. The result shows that GHGH 3.6.6 and GHEXPA can play an important role in the plant salt stress response process, have important significance in improving the breeding and research of cotton salt tolerance, and can be applied to breeding stress-resistant cotton varieties.
Drawings
FIG. 1 shows phenotype (A) of albino Plants (PDS), wild Type (WT), empty load (TRV 2: 00) and gene silencing plants (TRV 2: GHGH 3.6) after VIGS injection and WT, TRV2 for cotton plants as well as cotton plants according to examples of the present invention: 00 and TRV2: GHGH 3.6A schematic diagram of gene expression level in 3.6 (B).
FIG. 2 is a graph showing the change in phenotype of plants subjected to a salt stress treatment before and after a VIGS injection provided in the examples of the present invention.
FIG. 3 is a schematic representation of DAB staining results after salt stress treatment of plants subjected to VIGS injection, provided in the examples of the present invention.
Fig. 4 is a graph showing statistical results of chlorophyll content (a), stem length (B), root length (C) and lateral root number (D) of VIGS-injected plants subjected to salt stress treatment according to an embodiment of the present invention.
FIG. 5 shows the acquisition of the transgenic GHGH 3.6.6 gene Arabidopsis thaliana and the detection of the expression level of the T2 generation GHGH 3.6.6 gene provided in the examples of the present invention. A: a GHGH 3.6.6 gene transgenic Arabidopsis positive screening process; b: PCR detection of T1 generation transgenic Arabidopsis thaliana conversion results, M is Marker 5000, OE-2, OE-23, OE-25, OE-26, OE-27 and OE-33 as transformation lines, WT as negative control; c: the expression level of GHGH 3.6.6 in T2-generation transgenic arabidopsis was analyzed by qRT-PCR.
FIG. 6 is a comparison of the root lengths of wild-type (WT) and transgenic GHGH 3.6.6 lines (OE-2 and OE-25) at different concentrations NaCl, mannitol and ABA provided in the examples of the invention. A. B: root length phenotype and root length elongation changes of wild type and transgenic GHGH3.6 gene lines at concentrations of 100 mM and 150 mM NaCl; C. d: root length phenotype and root length elongation changes of wild type and transgenic GHGH 3.6.6 gene lines at concentrations of 100 mM, 200mM, and 300 mM Mannitol; E. f: the wild type and the transgenic GHGH 3.6.6 gene lines had a change in root length phenotype and root length elongation at 0.5. Mu.M, 1. Mu.M and 2. Mu.M ABA concentration.
FIG. 7 shows GHGH 3.6.6 transgenic lines and wild-type phenotypes under salt stress treatment provided in the examples of the present invention. A: GHGH 3.6.6 transgenic lines and wild type phenotypes; b: comparison of the size and morphology of rosette leaves of GHGH 3.6.6 transgenic lines and wild type Arabidopsis in response to salt stress.
FIG. 8 is a diagram showing the GHGH 3.6.6 transgenic line and wild-type physiological index and biochemical trait determination under salt and drought stress treatment provided in the examples of the present invention. A: GHGH 3.6.6 DAB staining of rosette leaves of Arabidopsis transgenic lines; b: GHGH 3.6.6 trypan blue staining of arabidopsis transgenic rosette leaves; c: measuring chlorophyll content; d: measuring the water loss rate of the in-vitro leaves; e: blade relative moisture content was measured.
FIG. 9 is a graph showing the relative expression levels of GHEXPA genes analyzed by qRT-PCR according to the example of the present invention. A: expression levels of GHEXPA gene in different tissues of cotton under normal growth conditions; b: expression levels of GHEXPA4 in cotton seedlings under salt stress (200 mM NaCl).
Fig. 10 shows albino Plants (PDS), WT, TRV2 after VIGS injection of cotton plants provided in the examples of the present invention: 00. TRV2: GHEXPA4 phenotype (A), WT, empty (TRV 2: 00), gene expression profile in gene-silenced plants (B), overall phenotype change after salt stress treatment (C) and cotton true She Biaoxing change (D).
FIG. 11 is a schematic diagram showing DAB staining results after salt stress treatment of plants subjected to VIGS injection, provided in the examples of the present invention.
Fig. 12 is a graph showing statistical results of chlorophyll content (a), stem length (B), and root length (C) of VIGS-injected plants subjected to salt stress treatment according to an embodiment of the present invention.
FIG. 13 shows the acquisition of the transgenic GHEXPA.sup.4 Arabidopsis thaliana and the detection of the expression level of the T2 generation GHEXPA gene provided in the examples of the present invention. A: transgenic GHEXPA4 gene Arabidopsis positive screening process; b: PCR detection of T1 generation transgenic Arabidopsis thaliana conversion results, M is Marker 5000, OE-5, OE-7, OE-10, OE-17, OE-18, OE-20, OE-30, OE-45 and OE-47 as transformation lines, and WT is a negative control; c: the expression level of GHEXPA4 in T2-generation transgenic arabidopsis thaliana was analyzed by qRT-PCR.
FIG. 14 is a comparison of the root lengths of wild-type (WT) and transgenic GHEXPA4 lines (OE-18, OE-20 and OE-45) at different concentrations of NaCl and Mannitol provided in the examples of the present invention. A. B: root length phenotype and root length elongation changes of wild type and transgenic GHEXPA4 gene lines at concentrations of 100 mM, 150 mM, and 200 mM NaCl; C. d: root length phenotype and root length elongation changes of wild type and transgenic GHEXPA4 gene lines at concentrations of 100 mM, 200 mM, and 300mM Mannitol.
FIG. 15 is a diagram of GHEXPA a4 transgenic line and wild type phenotype under salt stress treatment provided in the examples of the present invention. A: GHEXPA4 transgenic lines and wild type phenotypes; b: comparison of the size and morphology of rosette leaves of GHEXPA4 transgenic lines and wild type Arabidopsis in response to salt stress.
FIG. 16 is a diagram showing the measurement of GHEXPA4 transgenic lines and wild-type physiological indexes under salt stress treatment provided in the examples of the present invention. A: measuring chlorophyll content; b: measuring the water loss rate of the in-vitro leaves; c: blade relative moisture content was measured.
FIG. 17 is DAB staining of GHEXPA.sup.4 Arabidopsis transgenic rosette leaves under salt stress treatment provided in examples of the present invention.
FIG. 18 is trypan blue staining of GHEXPA.sup.4 Arabidopsis transgenic rosette leaves under salt stress treatment provided in the examples of the present invention.
Wherein FIGS. 1-8 are related experiments of gene GHGH 3.6.6 and FIGS. 9-18 are related experiments of GHEXPA.
Detailed Description
The following describes in further detail the specific embodiments of the present invention with reference to the drawings and examples. The methods used in the following examples are conventional methods unless otherwise specified; the reagents and materials used, unless otherwise indicated, are commercially available.
1. The application of cotton gene GHGH 3.6.6 in plant salt stress tolerance is provided.
Example 1
The effect of GHGH 3.6.6 genes on cotton in resisting salt stress is verified by using VIGS experiment
1.1 Plant VIGS vector TRV2: GHGH3.6 construction of 3.6
Upland cotton TM-1 is used as a VIGS injection material. After soaking the seeds for one day, transferring the seeds to wet filter paper and placing the wet filter paper into a greenhouse at 28 ℃ for germination, after the seeds germinate for six days (cotyledons are grown), transferring seedlings into a water culture box filled with Hoagland nutrient solution, and placing the water culture box into the greenhouse (at 28 ℃ in daytime and 25 ℃ at night, 16 h/8 h light/dark cycle) to enable the seedlings to grow normally. After about 5 days of growth (when the cotyledons of cotton seedlings were flattened), VIGS injections were performed on the seedlings. During injection, the back of cotyledons is scratched by the needle point of the injector, then the target gene, PDS and empty bacterial liquid (TRV 2: 00) are injected into the cotyledons and fully fill the whole cotyledons, and cotton seedlings are subjected to greenhouse dark culture for 24h after injection is completed; 24 After h, normal growth culture is carried out. The albino phenotype appeared after 12 days of cotton seedling growth injected with PDS, and the albino phenotype remained after 30 days, indicating successful gene silencing, while silencing effect was relatively stable (a in fig. 1). When the albino phenotype is stable, selecting cotton true leaves of WT, empty load and target genes (TRV 2: GHGH 3.6.6) to extract RNA, and reversely transcribing the RNA into cDNA to detect RT-qPCR. GHGH 3.6.6 at WT, TRV2:00 and TRV2 were tested using RT-qPCR: GHGH 3.6.6, the results show that: the GHGH 3.6.6 gene was expressed in WT and TRV2:00 without significant change, but in TRV2: the expression in GHGH 3.6.6 plants was significantly reduced and lower than WT and TRV2:00, indicating that GHGH 3.6.6 gene silencing was successful (B in fig. 1). After three true leaves grow on the plant, transferring the plant into a Hoagland nutrient solution containing 250 mM NaCl for salt stress treatment, and collecting the true leaves in two time periods of 0h and 48 h, freezing with liquid nitrogen and preserving at-80 ℃.
1.2 Phenotypic characterization of Gene-silenced cotton against salt stress
For WT, TRV2: 00. TRV2: GHGH 3.6.6 before salt stress treatment, WT, TRV2:00, TRV2: GHGH 3.6.6 seedlings were consistent in growth vigor. After 250 mM NaCl treatment 48 h, there was no significant difference in WT and TRV2:00 seedling phenotypes, whereas TRV2: GHGH 3.6.6 seedlings showed more pronounced leaf shedding and severe leaf yellowing and wilting (fig. 2), indicating a decrease in salt resistance of cotton plants after GHGH 3.6.6 gene silencing.
1.3 DAB GHGH 3.6.6) gene silenced plants, WT and empty (TRV 2:00 Dyeing)
Collecting 0 h and 48 h leaves of salt-treated WT, TRV2:00 and TRV2: GHGH 3.6.6 cotton plants, and verifying the damaged condition of the leaves after salt stress treatment by using a DAB dyeing test. Firstly, preparing DAB color development working solution: reagent a and reagent B were combined at 1: and (3) mixing uniformly in a volume ratio to obtain the DAB color development working solution. In this example, reagent A was used as 3 mL and reagent B was used as 57 mL. Then, cotton leaves subjected to salt stress treatment of 0 h and 48 h are placed into DAB chromogenic working solution to be chromogenic, and the time is controlled to be 10-16 h. After the color development is completed, absolute ethyl alcohol, 70% absolute ethyl alcohol and 50% absolute ethyl alcohol are used for gradient washing, leaf color is washed off, and different gradients are washed for 3 times, 1h times each time. DAB staining showed that under normal conditions, there was little ROS accumulation in WT, TRV2:00 and TRV2: GHGH 3.6.6 cotton leaves, and the brown appearance of the leaves was not apparent. After salt stress treatment, leaves of the cotton plants with TRV2: GHGH 3.6.6 exhibited brown regions, but leaves of the plants with WT and TRV2:00 were not significantly brown, indicating that under the same 250: 250 mM NaCl salt stress conditions, the TRV2: GHGH 3.6.6 plants were more severely damaged (FIG. 3), and salt resistance of the cotton plants was reduced after GHGH 3.6.6 gene silencing.
1.4 Physiological index change determination of GHGH 3.6.6 Gene-silenced Cotton plant under salt stress condition
The chlorophyll content (SPAD), cotton stem length, root length and lateral root number of the WT, TRV2:00 and TRV2: GHGH 3.6.6 cotton leaves were determined at salt treatments 0h and 48 h. Measurement of the relative chlorophyll content in cotton leaves using SPAD instrument we observed that WT, TRV2 at salt stress treatment 0 h: the chlorophyll contents of 00 and TRV2: GHGH 3.6.6 are basically consistent, and no obvious difference exists; however, after salt treatment 48h, the chlorophyll content of the TRV2: GHGH 3.6.6 plants was reduced more than that of the WT and TRV2:00 plants, indicating that the WT and TRV2:00 growth conditions were better under salt stress conditions than those of the genetically silenced plants (FIG. 4A). Furthermore, by comparison of stem length, root length and lateral root number, we found that after silencing GHGH 3.6.6 gene, the stem length and root length of the TRV2: GHGH3.6 plants were much shorter than that of WT and TRV2:00 plants, and that lateral root number was significantly reduced (B, C and D in FIG. 4), demonstrating that GHGH 3.6.6 might regulate cotton lateral root number growth and play a vital role in regulating stem and root growth. From the results we can see that the plants have reduced resistance to salt stress after GhGH3.6 silencing.
Example 2
Verifying the effect of GHGH 3.6.6 genes on salt stress resistance in Arabidopsis thaliana by using plant overexpression vectors
2.1 Construction of plant overexpression vector pCMBIA2300-35S: GHGH3.6
Gene cloning primers (F: ATTTGGAGAGGACAGGGTACCATGCCCGAAGCTCCCAAAAT; R: CTAGGTTAACCATGTGGTACCTTAGTTTTGAGTGCACCATTG) were designed based on the GHGH 3.6.6 gene CDS coding region sequence. The cDNA of upland cotton is used as a template, a target fragment of a gene GHGH 3.6.6 is obtained through PCR amplification, a plant over-expression vector pCMBIA2300 is cut by adopting restriction enzyme KpnI, and the target gene fragment and the vector fragment are recovered. And (3) connecting the target gene fragment and the carrier fragment, and transforming the connection product into the escherichia coli DH5 alpha competent cells. Obtaining a recombinant vector pCMBIA-35S containing a 35S promoter, wherein the recombinant vector pCMBIA-35S is GH3.6, extracting a plasmid containing a target gene after a sequencing result is correct, and transforming an agrobacterium GV3101 competent cell; after the agrobacterium grows out of single colony, colony PCR is carried out on the single colony, and the single colony with the PCR product consistent with the size of the target gene is obtained. Shaking the single colony, and preserving the thallus at-80 ℃ for later use. Up to this point, plant over-expression recombinant vector pCMBIA-35S GHGH3.6 was constructed successfully.
2.2 Selection of transformed Arabidopsis thaliana and transgenic Arabidopsis thaliana
Sterilizing wild Arabidopsis seeds with 0.1% mercuric chloride for 5 min times, washing with sterilized water for 3-5 times, dibbling the seeds onto 1/2MS solid medium, and placing the medium into a refrigerator at 4deg.C for vernalization for 2 days to break seed dormancy. After 2 days, the medium was placed in an Arabidopsis greenhouse (temperature 22 ℃,16 h light/8 h darkness). When the arabidopsis seedlings in the culture medium develop to 3-4 leaves, the seedlings are transferred into a nutrition pot filled with nutrition soil for growth. Cutting off the open flowers when the arabidopsis flowers, leaving the unopened buds for transformation of the arabidopsis by a dip-in method. The heavy suspension used for the infestation contains Silweet-77. Mu.L/L, MS 2.15 g/L, sucrose 50 g/L, AS 200mmol/mL, pH 5.7-5.8. To increase the transformation efficiency, the infection was once a week and four times in total. After each infestation, 24 h were cultivated in the dark, after which normal growth was carried out in the greenhouse and the seeds harvested after maturation were T0 generation. The T0 generation seeds are planted on a 1/2MS solid culture medium containing 50 mg/L Kan + in a dibbling mode, seedlings which can normally grow on the culture medium are positive seedlings, and the positive seedlings are transplanted to culture and harvest the T1 generation seeds.
Sowing the T1 generation seeds on a 1/2MS selection medium containing 50 mg/mL Kan +, continuing positive screening, counting the proportion of positive Miao Yufei positive seedlings (about 3:1) in the selection medium, leaving the positive Miao Bao according with the proportion, transplanting the positive seedlings into nutrient soil for seed harvesting, wherein the positive seedlings are transgenic single copy lines. Seeds of the identified positive lines were harvested as T2 generation seeds (a in fig. 5). Meanwhile, T1 generation Arabidopsis leaves were collected, DNA was extracted, and the target gene was amplified, and expression of gene GHGH 3.6.6 was screened to obtain strains (OE-2, OE-25, OE-26, OE-27 and OE-33) containing the target gene (FIG. 5B). The T2 generation seeds are inoculated again to germinate on a solid selection medium containing 50 mg/mL Kan +, and the surviving Arabidopsis thaliana is selected for transplanting and seed collection. Meanwhile, taking T2 generation leaves, carrying out DNA extraction and qRT-PCR analysis, and detecting the quality and the expression quantity of a positive strain, as shown in a graph C in fig. 5, wherein the expression quantity of two transgenic lines, namely OE-2 and OE-25, is relatively high, and is determined to be a high expression GHGH 3.6.6 gene homozygous line, and after the T2 generation grows mature, harvesting T3 generation seeds, and carrying out the next experiment.
2.3 Overexpression GHGH 3.6.6 root Length determination of Arabidopsis germination phase in response to different stress treatments
(1) Preparing a solid culture medium: 100 and 150 mM NaCl were added to 1/2MS medium to simulate salt stress environment, and salt stress treatment was performed on Arabidopsis thaliana. 100, 200 and 300mM Mannitol are added into 1/2MS culture medium to simulate drought environment, and drought treatment is carried out on the arabidopsis germination period. The Arabidopsis seeds were subjected to ABA treatment by adding 0.5. Mu.M, 1. Mu.M, 2. Mu.M ABA to 1/2MS medium.
(2) Root length determination of arabidopsis: the transgenic lines (OE-2, OE-25) and Wild Type (WT) were sown in 1/2MS solid media treated with different stresses, after 8 days of growth in an Arabidopsis greenhouse, the initial root length was determined, and then transferred to 1/2MS solid media treated with different stresses, respectively, and grown vertically for 8 days, the root length was measured again, and the elongation of the main root was calculated. Experiments in both the different treatment groups and the control group were performed in at least three biological replicates.
The results of root length measurement of Arabidopsis thaliana in germination stage are shown in FIG. 6. The results show that: during seed germination, wild Type (WT) and transgenic lines (OE-2, OE-25) showed no significant difference in the growth of new roots; root elongation length of transgenic lines (OE-2, OE-25) was longer compared to wild-type (WT) under salt stress and drought stress treatments. Furthermore, under ABA stress, the root elongation length of transgenic lines (OE-2, OE-25) was longer than that of the wild type, indicating that overexpressed GHGH 3.6.6 arabidopsis was more active and tolerant to salt, drought and ABA treatments than the wild type.
2.4 Overexpression GHGH 3.6.6 Arabidopsis response to salt stress tolerance
Wild Type (WT) and transgenic lines (OE-2, OE-25) grown on 1/2MS solid medium Arabidopsis thaliana was placed in an Arabidopsis thaliana greenhouse for 8 days (Arabidopsis thaliana seedlings were grown to 4-5 cotyledons) and then transferred to nutrient soil for soil culture growth. After two weeks of earth culture growth, wild-type and transgenic arabidopsis thaliana was subjected to salt stress treatment of 200 mM NaCl, stress treatment for 7 days, phenotypes were observed, and chlorophyll content of a control group (non-salt stress treated group) and a salt stress treated group were measured with a SPAD instrument. The phenotype of GHGH 3.6.6 transgenic lines (OE-4, OE-25) and wild-type (WT) and the size and morphology of rosette leaves in response to salt stress are compared as shown in A, B of FIG. 7.
The results show that: the wild type and GHGH 3.6.6 transgenic lines (OE-2, OE-25) had better phenotypes and no significant differences in growth status before salt stress treatment; after salt stress treatment, arabidopsis plants suffered varying degrees of damage. After salt stress treatment is carried out on wild plants, the rosette leaves of the arabidopsis are withered in a large area, and most of the plants are withered and dying; leaves of GHGH 3.6.6 transgenic lines showed wilting, but the plants were more active overall and did not die (A, B in fig. 7). Thus, salt stress has less effect on transgenic lines than wild type.
2.5 DAB and trypan blue staining of salt stress treated pre and post overexpressing GHGH 3.6.6 Arabidopsis and WT rosette leaves
(1) DAB staining: DAB staining A DAB staining kit (Jiancheng Bioengineering Institute, nanjing, china) was used for the staining method, and the staining method was referred to the DAB staining experiment of the leaves of the GH_A03G1858 (GHGH 3.6) gene-silenced cotton plants. Three leaves per group were repeated three times.
(2) Trypan blue staining: the trypan blue dye solution was 0.4% trypan blue working solution. Wild Type (WT) and transgenic line (OE-2, OE-25) Arabidopsis leaves before and after salt stress treatment are taken and placed into a 50 mL centrifuge tube, trypan blue working solution is added into the centrifuge tube to completely cover the Arabidopsis leaves, the centrifuge tube is placed into a boiling water bath to be boiled for 2 min, after cooling, the leaves are taken out and decolored by chloral hydrate (2.5 g/mL), the chloral hydrate solution is replaced every 24 h until the background color of the Arabidopsis leaves is eliminated, and finally the Arabidopsis leaves are washed by sterile water. Three leaves per group were repeated three times.
DAB and trypan blue staining results are shown in FIG. 8, A, B. DAB staining results showed that in the control group, the accumulation of ROS was almost absent in the leaves of wild-type Arabidopsis and GHGH 3.6.6 transgenic lines, and the leaves showed almost no brown; after salt stress treatment, both wild type arabidopsis and transgenic leaves appeared brown, but the brown region on transgenic leaves was significantly less than that of wild type arabidopsis leaves, and the degree of staining was shallower, and GHGH 3.6.6 transgenic arabidopsis was less damaged under the same salt stress treatment (a in fig. 8). Trypan blue staining results showed that in the control group, the wild type arabidopsis and GHGH 3.6.6 transgenic leaves hardly appeared blue-stained areas; after salt stress treatment, wild type Arabidopsis leaves were stained dark blue, while transgenic leaves were stained light blue, and the more severely the cells were damaged, the darker the color of the leaves stained by trypan blue. Thus, the GHGH 3.6.6 transgenic line received less cellular damage under salt stress conditions than wild type Arabidopsis (B in FIG. 8).
2.6 Determination of chlorophyll content, in vitro leaf loss Rate and leaf relative Water content
Transgenic lines (OE-2, OE-25) and Wild Type (WT) were grown for 4 weeks under normal conditions, and after salt stress treatment for 7 days, chlorophyll content was measured on rosette leaves of control and treatment groups using a SPAD meter; the chlorophyll content is shown in figure 8C. Leaf chlorophyll content of wild type arabidopsis thaliana and GHGH 3.6.6 transgenic lines were not different under non-salt stress treatment; after salt stress, the chlorophyll content of transgenic arabidopsis leaves is reduced less than that of wild type arabidopsis leaves, which indicates that the growth condition of transgenic arabidopsis is better than that of wild type arabidopsis under salt stress treatment.
The leaves of Arabidopsis were harvested for physiological measurements, including leaf relative water Content (RELATIVE LEAF WATER Content, RLWC) and in vitro leaf Loss (Excised LEAF WATER Loss, ELWL). All experiments were performed in at least three biological replicates.
Loss rate of in vitro leaf: 12 leaves of Arabidopsis thaliana of substantially uniform size were taken, fresh Weight (FW) of the sample was weighed, then the leaves were placed in an Arabidopsis thaliana greenhouse for 24 hours, withered Weight (WW) of the sample was weighed, then the leaves were placed in an oven at 50℃for drying for 24 hours, and Dry Weight (DW) of the sample was weighed. The calculation formula is as follows: excised LEAF WATER Loss (ELWL) = (FW-WW)/DW.
Blade relative water content: fresh Weight (FW) of the sample was immediately measured from 12 leaves of arabidopsis thaliana of substantially uniform size, leaf 24h was immersed in distilled water in an arabidopsis thaliana greenhouse, the leaf surface was dried by suction with paper, and the sample Saturated Weight (SW) was measured. The leaves were then placed in a 50 ℃ oven to dry 48 h and the sample Dry Weight (DW) was weighed. The calculation formula is as follows: RELATIVE LEAF WATER Content (RLWC) = [ (FW-DW)/(SW-DW) ]. Times.100%
The results of the ELWC and ELWL measurements are shown in figure D, E of figure 8. The results show that: under normal conditions, there were no significant differences in the physiological index determinations of the transgenic lines (OE-2, OE-25) and the wild-type (WT), but the GHGH 3.6.6 transgenic line exhibited positive effects compared to the wild-type when salt stress was applied to each line.
The water loss rate of the in vitro leaf of GHGH 3.6.6 transgenic line after salt stress treatment was significantly higher than that of wild type arabidopsis thaliana, indicating that after overexpression of GHGH 3.6.6 gene, the water holding capacity of the plant was increased (D in fig. 8). Leaf relative water content of GHGH 3.6.6 transgenic lines after salt stress treatment was significantly higher than that of wild type arabidopsis (E in fig. 8). From the results we can see that the salt resistance of GHGH 3.6.6 transgenic lines is significantly increased compared to the wild type.
The invention obtains GHGH 3.6.6 gene silencing cotton strain through VIGS experiment, and by comparing tolerance of wild cotton (WT), empty-load plant (TRV 2: 00) and gene silencing strain (TRV 2: GHGH 3.6.6) to salt stress, the result shows that phenotype and physiological index of gene silencing strain (TRV 2: GHGH 3.6) under salt stress treatment are worse than those of wild type and empty-load plant; the invention obtains the transgenic arabidopsis thaliana pure line of the over-expression GHGH 3.6.6 by a transgenic technology, and the result shows that the over-expression GHGH 3.6.6 arabidopsis thaliana has stronger activity and tolerance compared with a wild type when coping with salt, drought stress and ABA treatment in a seed germination period by comparing the tolerance of the wild type arabidopsis thaliana and the transgenic arabidopsis thaliana to salt stress; after salt stress treatment, arabidopsis plants suffered varying degrees of damage. After salt stress treatment is carried out on wild plants, the rosette leaves of the arabidopsis are withered in a large area, and most of the plants are withered and dying; the leaf of GHGH 3.6.6 transgenic line shows wilting, but the whole activity of the plant is better, no death phenomenon exists, and compared with the wild type, the influence of salt stress on the transgenic line is smaller; in addition, the water loss rate of the in vitro leaf blade of GHGH 3.6.6 transgenic line is obviously higher than that of wild arabidopsis after salt stress treatment, which shows that after the GHGH 3.6.6 gene is overexpressed, the water holding capacity of the plant is improved. After salt stress treatment, the leaf relative water content of GHGH 3.6.6 transgenic line is obviously higher than that of wild arabidopsis; from the results, the salt resistance of GHGH 3.6.6 transgenic line is obviously improved compared with the wild type, which shows that the gene plays a remarkable role in plant salt stress tolerance.
2. Application of cotton gene GHEXPA4 in plant salt stress tolerance.
Example 1
RNA extraction of cotton tissue (root, stem, flower and leaf) and GHEXPA gene qRT-PCR analysis
Under normal growth conditions (day 28 ℃, night 25 ℃,16 h/8 h light/dark cycle), when upland cotton (TM-1) grows to a trefoil period, taking a proper amount of samples of roots, stems, leaves and flowers of upland cotton seedlings, wrapping with tinfoil paper, quick-freezing in liquid nitrogen, grinding into powder with liquid nitrogen, and extracting sample RNA using a EASYspin Plus plant RNA rapid extraction kit of beijing ideley biology company. In addition, after salt stress treatment is carried out on cotton leaves in a three-leaf period of upland cotton in a one-heart period, tender leaves of cotton are taken at 0,1, 3, 6, 12 and 24h, and sample RNA is extracted. The concentration of each RNA sample was determined using a spectrophotometer (NanoDrop 2000), the mass of each RNA sample was determined using agarose gel electrophoresis, and RNA of acceptable quality and appropriate concentration was retained for subsequent reverse transcription experiments.
The extracted qualified RNA was reverse transcribed into cDNA using HISCRIPT III SuperMix for qPCR (+ GDNA WIPER) reverse transcription kit (Vazyme Biotech, nanjing, china), and qRT-PCR analysis was performed on the ABI7500fast platform using SYBR dye method with cotton Ghactin (F: ATCCTCCGTCTTGACCTTG; R: TGTCCGTCAGGCAACTCAT) as an internal reference gene and qRT-PCR analysis specific primer (F: TGCATGTGGGTACGGGAATC; R: GACACCATCTGGGGTCGTTT) of the target gene GHEXPA. The relative expression level of the target gene was calculated using the 2 -ΔΔCt algorithm, and at least 3 biological replicates were performed for each cDNA sample.
A: expression levels of GHEXPA gene in different tissues of cotton under normal growth conditions; b: expression levels of GHEXPA4 in cotton seedlings under salt stress (200 mM NaCl).
The expression levels of GHEXPA gene in different tissues of cotton under normal growth conditions are shown in FIG. 9A: the expression level of the gene is highest in flowers, and secondly, in stems and roots, and lowest in leaves. It was demonstrated that the gene was expressed mainly in flowers and stems. The expression level of GHEXPA gene in cotton seedlings under salt stress (200 mmol/L NaCl) is shown in FIG. 9B: the gene starts to up-regulate expression after salt stress treatment 1h, the expression quantity gradually rises along with the prolonging of the salt stress treatment time, peaks at the time of salt stress treatment 6h, and then the expression quantity drops, but the expression quantity of 12 h and 24h is still higher than 0 h. The GHEXPA gene is capable of highly up-regulating expression under salt stress treatment, and is a gene responding to salt stress.
Example 2
Use of VIGS experiments to verify GHEXPA gene's effect in cotton against salt stress
2.1 Plant VIGS vector TRV2: construction of GHEXPA4
Upland cotton (TM-1) is used as a VIGS injection material. After soaking the seeds for one day, transferring the seeds to wet filter paper and placing the wet filter paper into a greenhouse at 28 ℃ for germination, after the seeds germinate for five days (cotyledons are grown), transferring seedlings into a water culture box filled with Hoagland nutrient solution, and placing the water culture box into the greenhouse (at 28 ℃ in daytime and 25 ℃ at night, 16 h/8 h light/dark cycle) to enable the seedlings to grow normally. After about 5 days of growth (when the cotyledons of cotton seedlings were flattened), VIGS injections were performed on the seedlings. During injection, the back of cotyledons is scratched by the needle point of the injector, then the target gene, PDS and empty bacterial liquid (TRV 2: 00) are injected into the cotyledons and fully fill the whole cotyledons, and cotton seedlings are subjected to greenhouse dark culture for 24 h after injection is completed; 24 After h, normal growth culture is carried out. Albino phenotype appeared after 12 days of cotton seedling growth injected with PDS, and albino phenotype remained after 30 days indicating successful gene silencing, while silencing effect was relatively stable (a in fig. 10). When the albino phenotype is stable, selecting the cotton true leaves of WT, empty load and target gene (TRV 2: GHEXPA 4) to extract RNA, and reversely transcribing the RNA into cDNA to detect RT-qPCR. GH_D09G1670 (GHEXPA 4) was tested at WT, TRV2:00 and TRV2 by RT-qPCR: GHEXPA4, and the results show that: the GHEXPA4 gene was expressed in WT and TRV2:00 without significant change, but in TRV2: expression in GHEXPA4 plants was significantly reduced and below WT and TRV2:00, indicating successful GHEXPA gene silencing (B in fig. 10). After three true leaves grow on the plant, transferring the plant into Hoagland nutrient solution containing 200 mM NaCl for salt stress treatment, and collecting the true leaves in two time periods of 0h and 72 h, freezing with liquid nitrogen and preserving at-80 ℃.
2.2 Phenotypic characterization of Gene-silenced cotton against salt stress
For WT, TRV2: 00. TRV2: GHEXPA4 is subjected to salt stress treatment, and under the control condition, WT, TRV2:00, TRV2: GHEXPA4 seedlings were consistent in growth vigor. After 200 mM NaCl treatment 72 h, there was no significant difference in WT and TRV2:00 seedling phenotypes, whereas TRV2: GHEXPA4 seedlings showed smaller leaves and more severe yellowing and wilting of the leaves (C and D in FIG. 10), indicating a significant decrease in salt resistance of cotton plants after GHEXPA gene silencing compared to wild type.
2.3 Physiological index change determination of GHEXPA gene silencing cotton plant under salt stress condition
The chlorophyll content (SPAD), cotton stem length and root length of the cotton leaves of WT, TRV2:00 and TRV2: GHEXPA4 were determined at salt treatments 0h and 72 h. Measurement of the relative chlorophyll content in cotton leaves using SPAD instrument we observed that WT, TRV2 at salt stress treatment 0 h: the chlorophyll content of 00 and TRV2: GHEXPA4 are basically consistent, and no obvious difference exists; however, after salt treatment 72 h, the chlorophyll content of the three plants was reduced compared to treatment 0h, and the chlorophyll content of the TRV2: GHEXPA4 plants was reduced more than that of the WT and TRV2:00 plants, indicating that the growth conditions of WT and TRV2:00 under salt stress conditions were better than that of the gene silencing plants (A in FIG. 12). Furthermore, by comparing stem length to root length, we found that after silencing GHEXPA4 gene, the stem length and root length of the TRV2: GHEXPA4 plant were much shorter than that of the WT and TRV2:00 plants (B and C in FIG. 12), suggesting that GHEXPA4 may play a critical role in regulating stem and root growth. From the results we can see that GHEXPA plants have reduced resistance to salt stress after silencing.
2.4 DAB staining of GHEXPA gene-silenced plants, WT and empty (TRV 2:00) before and after salt stress treatment
Leaves of salt-treated WT, TRV2:00 and TRV2: GHEXPA4 cotton plants 0 h and 72 h are collected, and the damage condition of the leaves after salt stress treatment is verified by using a DAB dyeing test. Firstly, preparing DAB color development working solution: reagent a and reagent B were combined at 1: and (3) mixing uniformly in a volume ratio to obtain the DAB color development working solution. In this example, reagent A was used as reagent 4 mL and reagent B was used as reagent 76 mL. Then, cotton leaves subjected to salt stress treatment 0 h and 72 h are placed into DAB chromogenic working solution to be chromogenic, and the time is controlled between 12 and 16 and h. After the color development is completed, absolute ethyl alcohol and 70% absolute ethyl alcohol are respectively used for gradient washing, leaf color is washed off, and different gradients are respectively washed 3 times, each time is 3 h. DAB staining showed that under normal conditions, there was little accumulation of ROS in the WT, TRV2:00 and TRV2: GHEXPA4 cotton leaves, and the brown appearance of the leaves was not apparent. After salt stress treatment, leaves of the cotton plants with TRV2: GHEXPA4 appeared brown areas, but leaves of the cotton plants with WT and TRV2:00 were not obvious brown, which indicates that under the same 200 mM NaCl salt stress condition, the TRV2: GHEXPA4 plants were more severely damaged (FIG. 11), and salt resistance of the cotton plants was reduced after GHEXPA gene silencing.
Example 3
Verifying the effect of GHEXPA gene on salt stress resistance in Arabidopsis thaliana by using plant overexpression vector
3.1 Construction of plant overexpression vector pCMBIA2300-35S GHEXPA4
Gene cloning primers (F: ATTTGGAGAGGACAGGGTACCATGTCCGCTGTTGCTCC; R: CTAGGTTAACCATGTGGTACCTCAAACGCGGAAATTCTTTCC) were designed based on the GHEXPA gene CDS coding region sequence. The cDNA of upland cotton is used as a template, a target fragment of a gene GHEXPA4 is obtained through PCR amplification, a plant over-expression vector pCMBIA2300 is cut by adopting restriction enzyme KpnI, and the target gene fragment and the vector fragment are recovered. And (3) connecting the target gene fragment and the carrier fragment, and transforming the connection product into the escherichia coli DH5 alpha competent cells. Obtaining a recombinant vector pCMBIA which contains a 35S promoter 2300-35S GHEXPA, extracting a plasmid containing a target gene after a sequencing result is correct, and transforming an agrobacterium GV4404 competent cell; after the agrobacterium grows out of single colony, colony PCR is carried out on the single colony, and the single colony with the PCR product consistent with the size of the target gene is obtained. Shaking the single colony, and preserving the thallus at-80 ℃ for later use. Up to this point, plant over-expression recombinant vector pCMBIA-35S GHEXPA4 was constructed successfully.
3.2 Selection of transformed Arabidopsis thaliana and transgenic Arabidopsis thaliana
Sterilizing wild Arabidopsis seeds with 0.1% mercuric chloride for 5min times, washing with sterilized water for 3-5 times, dibbling the seeds onto 1/2MS solid medium, and placing the medium into a refrigerator at 4deg.C for vernalization for 2 days to break seed dormancy. After 2 days, the medium was placed in an Arabidopsis greenhouse (temperature 22 ℃,16 h light/8 h darkness). When the arabidopsis seedlings in the culture medium develop to 3-4 leaves, the seedlings are transferred into a nutrition pot filled with nutrition soil for growth. Cutting off the open flowers when the arabidopsis flowers, leaving the unopened buds for transformation of the arabidopsis by a dip-in method. The heavy suspension used for the infestation contains Silweet-77. Mu.L/L, MS 2.15 g/L, sucrose 50 g/L, AS 200mmol/mL, pH 5.7-5.8. To increase the transformation efficiency, the infection was once a week and four times in total. After each infestation, 24 h were cultivated in the dark, after which normal growth was carried out in the greenhouse and the seeds harvested after maturation were T0 generation. The T0 generation seeds are planted on a 1/2MS solid culture medium containing 50 mg/mL Kan +, the seedlings which can normally grow on the culture medium are positive seedlings, and the positive seedlings are transplanted to culture and harvest the T1 generation seeds.
Sowing the T1 generation seeds on a 1/2MS selection medium containing 50 mg/mL Kan +, continuing positive screening, counting the proportion of positive Miao Yufei positive seedlings (about 3:1) in the selection medium, leaving the positive Miao Bao according with the proportion, transplanting the positive seedlings into nutrient soil for seed harvesting, wherein the positive seedlings are transgenic single copy lines. Seeds of the identified positive lines were harvested as T2 generation seeds (a in fig. 13). Meanwhile, T1 generation Arabidopsis leaves were collected, DNA was extracted, and the target gene was amplified, and the expression of gene GHEXPA4 was screened to obtain the strains (OE-5, OE-7, OE-10, OE-17, OE-18, OE-20, OE-30, OE-45, and OE-47) containing the target gene (FIG. 13B). The T2 generation seeds are inoculated again to germinate on a solid selection medium containing 50 mg/mL Kan +, and the surviving Arabidopsis thaliana is selected for transplanting and seed collection. Meanwhile, taking T2 generation leaves, carrying out DNA extraction and qRT-PCR analysis, and detecting the quality and the expression quantity of a positive strain, as shown in a graph C in fig. 13, wherein the expression quantity of three transgenic lines, namely OE-18, OE-20 and OE-45, is relatively high, the three transgenic lines are determined to be a high-expression GHEXPA gene homozygous line, and after the T2 generation grows mature, harvesting T3 generation seeds, and carrying out the next experiment.
3.3 Overexpression GHEXPA root Length determination of Arabidopsis thaliana germination phase response to different stress treatments
(1) Preparing a solid culture medium: 100, 150 and 200 mM NaCl were added to 1/2MS medium to simulate salt stress environment, and salt stress treatment was performed on Arabidopsis thaliana. 100, 200 and 300mM Mannitol are added into 1/2MS culture medium to simulate drought environment, and drought treatment is carried out on the arabidopsis germination period.
(2) Root length determination of arabidopsis: transgenic lines (OE-18, OE-20 and OE-45) and Wild Type (WT) were sown in 1/2MS solid media treated with different stresses, after 8 days of growth in an Arabidopsis greenhouse, the initial root length was determined, and then transferred to 1/2MS solid media treated with different stresses, respectively, and grown vertically for 8 days, root length was measured again, and the elongation of main roots was calculated. Experiments in both the different treatment groups and the control group were performed in at least three biological replicates.
The results of root length measurement of Arabidopsis thaliana in germination stage are shown in FIG. 14. The results show that: during seed germination, wild Type (WT) showed no significant difference in root growth from the new-born transgenic lines (OE-18, OE-20, and OE-45); root elongation length of transgenic lines (OE-18, OE-20 and OE-45) was longer compared to wild-type (WT) under salt stress and drought stress treatments. This demonstrates that overexpressed GHEXPA, arabidopsis thaliana has greater activity and tolerance compared to wild-type when dealing with salt and drought stress treatments.
3.4 Overexpression GHEXPA Arabidopsis thaliana response to salt stress tolerance
Wild Type (WT) and transgenic lines (OE-18, OE-20 and OE-45) sown on 1/2MS solid medium Arabidopsis thaliana was placed in an Arabidopsis thaliana greenhouse for 8 days (Arabidopsis thaliana seedlings were grown to 4-5 cotyledons) and then transferred to nutrient soil for soil culture growth. After two weeks of earth culture growth, wild-type and transgenic arabidopsis thaliana was subjected to salt stress treatment of 200 mM NaCl, stress treatment was performed for 8 days, phenotypes were observed, and chlorophyll content of a control group (non-salt stress treated group) and a salt stress treated group was measured with a SPAD instrument. The phenotype of GHEXPA4 transgenic lines (OE-18, OE-20, and OE-45) and wild-type (WT) and the size and morphology of rosette leaves in response to salt stress are compared as shown in A, B of FIG. 15.
The results show that: the wild type and GHEXPA4 transgenic lines (OE-18, OE-20 and OE-45) had better phenotypes and no significant differences in growth status before salt stress treatment; after salt stress treatment, arabidopsis plants suffered varying degrees of damage. After salt stress treatment is carried out on the wild plants, the leaves of the arabidopsis rosettes wither in a large area, only three or four leaves are green, and other leaves are all dry; leaves of GHEXPA4 transgenic lines showed wilting, but only one or two leaves of the whole plant were dried up, and no death occurred (A, B in FIG. 15). Thus, salt stress has less effect on transgenic lines than wild type.
3.5 DAB overexpressing GH_D09G1670 (GHEXPA) Arabidopsis and WT rosette leaves before and after salt stress treatment and staining
(1) DAB staining: DAB staining was performed using DAB staining kit (Jiancheng Bioengineering Institute, nanjing, china), and the staining method was referred to the GH_D09G1670 (GHEXPA 4) gene-silenced cotton plant leaf DAB staining assay. Three leaves per group were repeated three times.
(2) Trypan blue staining: the trypan blue dye solution was 0.4% trypan blue working solution. Placing the leaves into a 50mL centrifuge tube, adding trypan blue working solution into the centrifuge tube to completely cover the arabidopsis leaves, placing the centrifuge tube into a boiling water bath to boil for 2 min, taking out the leaves after cooling, decoloring with chloral hydrate (2.5 g/mL), replacing the chloral hydrate solution every 24h until the background color of the arabidopsis leaves is eliminated, and finally cleaning with sterile water. Three leaves per group were repeated three times. The results of staining of Wild Type (WT) and transgenic lines (OE-18, OE-20 and OE-45) Arabidopsis DAB and trypan blue before and after salt stress treatment are shown in FIGS. 17 and 18. DAB staining results showed that in the control group, the wild type Arabidopsis and GHEXPA4 transgenic lines showed little accumulation of ROS in the leaves and little brown leaves; after salt stress treatment, both wild type arabidopsis and transgenic leaves appeared brown, but the brown region on transgenic leaves was significantly less than that of wild type arabidopsis leaves, and the degree of staining was shallower, and GHEXPA4 transgenic arabidopsis was less damaged under the same salt stress treatment (fig. 17). Trypan blue staining results showed that in the control group, the wild type arabidopsis and GHEXPA4 transgenic leaves hardly appeared blue-stained areas; after salt stress treatment, wild type Arabidopsis leaves were stained dark blue, while transgenic leaves were stained light blue, and the more severely the cells were damaged, the darker the color of the leaves stained by trypan blue. The GHEXPA4 transgenic line was therefore less subject to cell damage under salt stress conditions than wild-type arabidopsis (fig. 18).
3.6 Determination of chlorophyll content, in vitro leaf loss Rate and leaf relative Water content
Transgenic lines (OE-18, OE-20 and OE-45) and Wild Type (WT) were grown for 4 weeks under normal conditions, and after salt stress treatment for 8 days, chlorophyll content was measured on rosette leaves of control and treatment groups using a SPAD meter; the chlorophyll content is shown in fig. 16 a. Leaf chlorophyll content of wild type arabidopsis thaliana and GHEXPA4 transgenic lines were not different under non-salt stress treatment; after salt stress, the chlorophyll content of the transgenic arabidopsis leaves is obviously higher than that of the wild arabidopsis leaves, which indicates that the growth condition of the transgenic arabidopsis is better than that of the wild arabidopsis under the salt stress treatment.
The leaves of Arabidopsis were harvested for physiological measurements, including leaf relative water Content (RELATIVE LEAF WATER Content, RLWC) and in vitro leaf Loss (Excised LEAF WATER Loss, ELWL). All experiments were performed in at least three biological replicates.
Loss rate of in vitro leaf: 12 leaves of Arabidopsis thaliana of substantially uniform size were taken, fresh Weight (FW) of the sample was weighed, then the leaves were placed in an Arabidopsis thaliana greenhouse for 24 hours, withered Weight (WW) of the sample was weighed, then the leaves were placed in an oven at 50℃for drying for 24 hours, and Dry Weight (DW) of the sample was weighed. The calculation formula is as follows: excised LEAF WATER Loss (ELWL) = (FW-WW)/DW.
Blade relative water content: fresh Weight (FW) of the sample was immediately measured from 12 leaves of arabidopsis thaliana of substantially uniform size, leaf 24h was immersed in distilled water in an arabidopsis thaliana greenhouse, the leaf surface was dried by suction with paper, and the sample Saturated Weight (SW) was measured. The leaves were then placed in a 50 ℃ oven to dry 48 h and the sample Dry Weight (DW) was weighed. The calculation formula is as follows: RELATIVE LEAF WATER Content (RLWC) = [ (FW-DW)/(SW-DW) ]. Times.100%
The results of the ELWL and RLWC measurements are shown in figure B, C of figure 16. The results show that: under normal conditions, there were no significant differences in the physiological index determinations of the transgenic lines (OE-18, OE-20 and OE-45) and the wild-type (WT), but the GHEXPA4 transgenic line exhibited positive effects compared to the wild-type when salt stress was applied to each line.
The water loss rate of the in vitro leaf of GHEXPA4 transgenic line after salt stress treatment was significantly higher than that of wild type arabidopsis thaliana, indicating that the water holding capacity of the plant was increased after overexpression of GHEXPA gene (B in fig. 16). Leaf relative water content of GHEXPA4 transgenic lines after salt stress treatment was significantly higher than that of wild type arabidopsis (C in fig. 16). From the results we can see that the salt resistance of the GHEXPA4 transgenic line is significantly increased compared to the wild type.
The invention obtains GHEXPA4 gene silencing cotton line through VIGS experiment, and the phenotype and physiological index of the gene silencing line (TRV 2: GHEXPA) under salt stress treatment are worse than those of wild type and empty-load plants by comparing the tolerance of the wild type cotton (WT), the empty-load plant (TRV 2: 00) and the gene silencing line (TRV 2: GHEXPA 4) to salt stress; the invention obtains the transgenic arabidopsis thaliana pure line of the over-expression GHEXPA by a transgenic technology, and the result shows that the over-expression GHEXPA arabidopsis thaliana has stronger activity and tolerance compared with a wild type when coping with salt and drought stress treatment in a seed germination period by comparing the tolerance of the wild type arabidopsis thaliana and the transgenic arabidopsis thaliana to salt stress; after salt stress treatment, arabidopsis plants suffered varying degrees of damage. After salt stress treatment is carried out on wild plants, the rosette leaves of the arabidopsis are withered in a large area, and most of the plants are withered and dying; leaf of GHEXPA4 transgenic line has wilting, but the whole activity of plant is better, no death phenomenon occurs, and compared with wild type, the influence of salt stress on transgenic line is smaller; in addition, the water loss rate of the in vitro leaf blade of GHEXPA4 transgenic line is obviously higher than that of wild arabidopsis after salt stress treatment, which shows that after the GHEXPA gene is overexpressed, the water holding capacity of the plant is increased. After salt stress treatment, the leaf relative water content of GHEXPA4 transgenic line is obviously higher than that of wild arabidopsis; from the results, the salt resistance of GHEXPA-4 transgenic line is obviously improved compared with the wild type, which shows that the gene plays a remarkable role in plant salt stress tolerance.
Claims (6)
1. A salt stress resistant gene GHGH 3.6.6 has a nucleotide sequence table shown in SEQ ID No. 1.
2. Use of the gene GHGH, 3.6 according to claim 1 to increase salt tolerance in plants.
3. The use according to claim 2 for over-expressing a gene GHGH 3.6.6 in a plant, thereby increasing the tolerance of the plant to salt stress.
4. A method for improving salt stress tolerance of a plant, wherein the gene GHGH 3.6.6 of claim 1 is transformed into the plant by agrobacterium mediation by using a plant over-expression vector, so as to obtain a salt tolerant transgenic plant.
5. The plant of claim 4 is cotton or Arabidopsis.
6. The cotton of claim 5 is upland cotton (TM-1).
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