CN106191001A - The application in improving plant salt endurance of phospholipase PLD ζ 1 gene - Google Patents

Abstract

The invention discloses the application of the phospholipase PLDζ1 gene in improving the salt tolerance of plants. The PLDz1 gene is transferred into crops by means of molecular genetic transformation, and transformed plants overexpressing the PLDz1 gene are obtained. Overexpression of PLDz1 can significantly improve the salt tolerance of plants. Under salt stress, the malondialdehyde content and plasma membrane permeability of overexpressed PLDz1 plants were significantly lower than those of the control wild type, and their survival rate and plant height were significantly higher than those of the control wild type. However, the loss of PLDz1 gene expression significantly reduced the salt tolerance of the plants, and the biomass was significantly lower than that of the wild type, which further indicated that PLDz1 had a significant positive regulatory effect on salt tolerance. The invention provides a new approach and creates new germplasm materials for molecular breeding of crop salt tolerance.

Description

Application of Phospholipase PLDζ1 Gene in Improving Salt Tolerance of Plants

technical field

The invention belongs to the field of plant molecular breeding and biotechnology, and specifically relates to the application of phospholipase PLDζ1 gene in improving plant salt tolerance and increasing crop yield under salt stress.

Background technique

With the change of climate and environment and the continuous increase of population, the problem of global food security is worrying. In recent years, the frequent occurrence of drought and the continuous expansion of drought and salinized cultivated land have made the environmental stress of crop production more and more serious. Land salinization is one of the main problems faced by agricultural production. 1/5 of the cultivated land in the world is salinized (Yamaguc hi et al., Developing salt-tolerant crop plants: challenges and opportunities. Trends Plant Sci. 2005, 10:615-620.), and 1/3 of the farmland in my country is too salty As a result, crop yields have decreased, and the occurrence of drought and excessive application of chemical fertilizers have led to the continuous spread and expansion of salinized land; climate warming and sea level rise have caused more coastal cultivated land to be submerged and salinized. Therefore, drought and salt stress have become one of the main factors limiting agricultural production. With a large population in my country, how to efficiently and rationally utilize limited land and water resources and promote sustainable agricultural development is a top priority. In view of the current problems, two measures are mainly taken: one is to irrigate and improve the soil through engineering measures; the other is to cultivate new varieties of drought-resistant and salt-tolerant crops through biotechnology. The former is costly and difficult to carry out on a large scale, while the latter is an economical and effective measure. However, to solve this problem, conventional breeding alone has encountered a bottleneck and its potential is quite limited. We must explore the mechanism of drought and salt tolerance in plants, dig out key genes, break through the barriers that cannot be overcome by conventional hybridization through genetic engineering, and cultivate salt-tolerant, drought-resistant plants. New varieties of crops.

In the process of growth and development, plants often encounter drought and salt stress, and gradually establish a whole set of mechanisms including signal perception, conduction, physiology, biochemistry and morphological development to adapt to the environment during the long-term evolution process and minimize damage ( Vinocur et al., Recent advances in engineering plant tolerance to abiotic stress achievements and limitation. Curr Opin Biotechnol. 2005, 16:123-132). At present, humans have conducted a lot of research on the mechanism of plant salt and drought stress, and discovered a number of genes that are resistant to salt stress and drought and applied to genetic engineering to improve crops. According to the mechanism of their action, these genes can be classified into two categories: one category encodes effector molecules, mainly including: 1) osmoprotectants such as proline, betaine, etc. (Yoshiba et al., Regulation of levels of proline as an osmol yte in plants under water stress.Plant CellPhysiol.1997,38:1095-10102); 2) maintain intracellular ion balance protein such as Na ⁺ /H ⁺ antiporter (Apse et al., Salt tolerance conferred by overexpression of a vacuolar Na ⁺ /H ⁺ antiport in Arabidopsis.Science.1999,285:1256-1258); 3) antioxidant enzymes such as POD, SOD, CAT (Miller et al., Reactive oxygen signaling and abiotic stress.Physiol Plant.2008,133 (3): 481-9); 4) Functional proteins that protect cells such as LEA proteins, aquaporins, etc. :18073-18078). The other category is regulatory factors including: 1) transcription factors DREB, CBF, NAC, SKIP, etc. responsive gene expression, respectively, in Arabidopsis.PlantCell.1998,10:1391-1406; Denby et al., Engineering suffered and salinity tolerance implants: lessons from genome-wide expression profiling in Arabidopsis.Trends Biotechnol.2005,23:547-552; etc., A homolog of human ski-interacting proteinin rice positively regulates cell viability and stress tolerance.Proc NatlAcad Sci US A.2009,106:6410-6415); 2) SOS, calcium neurons and protein kinases that sense and transmit adversity signals etc. (Kiegerl et al., SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK. Plant Cell. 2000, 12: 2247-2258; Zhu, Salt and drought stress signal transduction in plants. Annu Rev Plant Biol. 2002, 53:247-273). The discovery and characterization of the above genes is encouraging. However, salt stress and drought are quantitative traits controlled by multiple genes (Denby et al., Eng ineering drought and salinity tolerance implants: lessons from genome-wide expression pro filing in Arabidopsis. Trends Biotechnol. 2005, 23:547-552; Sahi et al., Salt stress response in rice: genetics, molecular biology, and comparative genomics. Funct Integr Genomics. 2006, 6: 263-284), is regulated by multiple signaling or metabolic pathways, and each pathway has an interactive relationship, and its molecular mechanism is equivalent Breeding for complex, drought and salt tolerance is more challenging. Therefore, we need to analyze the signaling network and key nodes involved in plant response to adversity stress, and find out the pleiotropic factors with salt tolerance, drought resistance and high yield.

Biofilms are rich in signaling molecules, which are the starting point for sensing and transmitting external signals, as well as the site for material and energy exchange between cells and the outside world, and a barrier to prevent external infection and adversity damage. Phospholipase PLD catalyzes the production of the signaling molecule phosphatidic acid from membrane phospholipids. The invention relates to the positive regulation of a unique phospholipase PLDζ1 in plants in plant salt tolerance. Overexpression of its gene can significantly improve the salt tolerance of crops such as rice, and provide new genes and breeding for salt tolerance breeding of crops New germplasm material.

Contents of the invention

The purpose of the present invention is to provide the application of phospholipase PLDζ1 gene in enhancing crop salt tolerance and biological yield under salt stress conditions. The salt tolerance of plants can be significantly improved by cloning the full-length CDS of rice PLDζ1 and overexpressing PLDζ1 in rice. The degree of leaf tissue obstruction of PLDζ1 overexpressed plants under salt stress is significantly lower than that of the control wild type, and the cell viability of the plant is stronger than that of the wild type. The nucleotide sequence of the phospholipase PLDζ1 gene is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2. In addition, the lack of expression of PLDζ1 in rice resulted in significantly lower salt tolerance than that of the control wild type. Under salt stress, the plant growth, survival rate, plant height and biomass of the pldζ1 deletion mutant were significantly lower than that of the control wild type, further indicating that PLDζ1 Positive regulatory role in plant salt tolerance.

The present invention also found that PLDζ1 encodes an active phospholipase D, which catalyzes phosphatidylcholine to produce phosphatidic acid PA, and the activity of PLDζ1 has a significant impact on plant lipid metabolism, and its deletion mutation leads to a significant decrease in the phosphatidic acid content in the plant. Lipid content also changed. It shows that changing the expression level of PLDζ1 through molecular genetic manipulation can effectively regulate membrane lipid metabolism and signaling, and then affect plant response to salt stress signals. PLDζ1 can regulate the expression of genes related to sodium ion transport, regulate sodium ion transport and the homeostasis of sodium/potassium ions in plants, and then enhance the salt tolerance and plant growth of plants.

In addition, the present invention creates plant materials with different expression levels of PLDζ1, including PLDζ1 overexpression and deletion mutants. In particular, the overexpression plant material can be used for crop genetic improvement, and is a new germplasm material for cultivating salt-tolerant crops, and the obtained transgenic rice is suitable for planting in saline-alkali soil. PLDζ1 positively regulates the salt tolerance of plants, making it more feasible to improve the application of crop salt tolerance.

Positive effect of the present invention

1. The deletion of PLDζ1 in rice resulted in significantly lower salt tolerance than the control wild type, including significantly lower plant growth, survival rate, plant height and biomass than the wild type.

2. Overexpression of PLDζ1 can significantly improve plant growth and seed setting rate, especially under salt stress, the survival rate of overexpressed PLDζ1 plants is significantly higher than that of the control wild type, and overexpression of leaf malondialdehyde content and plasma membrane under salt stress The permeability was significantly lower than that of the control wild type, and its survival rate and plant height were significantly higher than that of the control wild type.

3. Under the condition of salt stress, PLDζ1 plays an important role in regulating the transport of sodium ions. The loss of PLDζ1 leads to significantly higher sodium ion content in plants than wild type, indicating that PLDζ1 enhances the salt tolerance of plants by regulating the balance of sodium and potassium ions .

4. PLDζ1 encodes active phospholipase D, which can catalyze phosphatidylcholine PC to generate signal molecule phosphatidic acid PA. Its activity does not depend on calcium ions, but requires PIP ₂ as an activation factor, and its activity is highest at pH 7. Its biochemical properties are similar to animal PLD. The deletion of PLDζ1 resulted in significantly lower phosphatidic acid PA content in plants than in the wild type, indicating that PLDζ1 has a regulatory effect on lipid metabolism in plants.

5. This study found that the deletion mutation of PLDζ1 led to the down-regulation of sodium ion transport-related genes such as NHX 1, SOS1 and SOS3, which hindered the efflux of sodium ions in the body and accumulated more sodium ions in the plant, and the deletion of PLDζ1 led to sodium ion transport. Significant up-regulation of ion homeostasis-related gene HKT1 enhanced plant uptake and transport of sodium ions from the environment. These results suggest that PLDζ1 is involved in the regulation of sodium ion uptake and transport.

6. The activity of PLDζ1 has a significant effect on the lipid components of the cell membrane, and its absence leads to significant changes in the lipid metabolism of the cell membrane under salt stress, and the overexpression of PLDζ1 can significantly enhance the salt tolerance of plants. This characteristic makes the application of the PLDζ1 gene more It works.

Description of drawings

Figure 1. Schematic diagram of PLDζ1 overexpression enhancing plant salt tolerance.

A. Phenotype of PLDζ1 overexpression plants under salt stress; B-C. Leaf malondialdehyde (MDA) content (B) and leaf quality of PLDζ1 overexpression plants at tillering stage and control wild type treated with 0, 75mM NaCl for 30 days Membrane permeability (C). Values represent mean ± SD (n=3), *P<0.05, **P<0.01.

Figure 2. Schematic diagram of the enhanced sensitivity of plants to salt stress caused by the deletion mutation of PLDζ1.

Phenotypes of the pldζ1 mutant and wild type at the tillering stage treated with 0, 50, and 150 mM NaCl for 21 days.

Figure 3. Schematic diagram of the loss of PLDζ1 leading to the decrease of plant height and biological yield of plants under salt stress, and the degree of cell membrane damage is more severe than that of the control wild type.

A. Determination of the plant height of the pldζ1 mutant and wild type at the tillering stage after being treated with 0, 50, 150 mM NaCl for 21 days. Values represent the mean ± standard deviation (n=6); B-D. The fresh weight of shoots (B), MDA content (C) and Measurement results of plasma membrane permeability (D). Values represent mean ± SD (n=3), *P<0.05, **P<0.01.

Fig. 4. Schematic diagram of the effect of PLDζ1 on Na ⁺ and K ⁺ content in plants under salt stress.

A, B. Sodium ion determination results of the pldζ1 mutant and wild type at the tillering stage after being treated with 0, 150 mM NaCl for 14 days; C, D. pldζ1 mutant and wild type at the tillering stage after being treated with 0, 150 mM NaCl for 14 days The results of the determination of potassium ions. Values represent mean ± SD (n=3), *P<0.05, **P<0.01.

Figure 5. Schematic diagram of PLDζ1 catalyzing phosphatidylcholine PC to generate phosphatidic acid PA.

PIP ₂ can significantly promote the catalytic activity of PLDζ1, and the activity of PLDζ1 is the highest at neutral pH 7.

Figure 6. Schematic diagram of the difference in expression of Na ⁺ transport-related genes in leaves of mutant pldζ1 and wild-type leaves under salt stress.

After treating the pldζ1 mutant and wild type at the tillering stage with 0 and 150 mM NaCl for 6 hours, the results of the expression of genes related to Na ⁺ transport were analyzed. Values represent mean ± SD (n=3), *P<0.05, **P<0.01.

detailed description

The accession number of the rice phospholipase PLDζ1 gene used in the examples of the present invention in the public nucleotide database GenBank (http://www.ncbi.nlm.nih.gov) is XM_015784967.

The technical solutions of the present invention, if not specified, are conventional solutions in the art; the reagents or materials, if not specified, are all derived from commercial channels.

Example 1:

PLDζ1 Gene Cloning and Overexpression Enhance Plant Salt Tolerance

1. Cloning of full-length cDNA of PLDζ1 gene and construction of plant overexpression vector:

Total RNA was extracted from the leaves of japonica rice Dongjin, and the first-strand cDNA was synthesized by reverse transcription RT-PCR using mRNA as a template, and then the cDNA was used as a template, using PLDζ1 sequence-specific primers PLDζ1FZ: 5'-GGGGTACCATGCAAGAATATCTGAACC-3' and PLDζ1RZ: 5'-CGGGATCCATGGAAAACTTGTGGAG-3' was amplified by PCR, and the amplified target fragment included the sequence shown in SEQ ID NO.1. The amplified target fragment was inserted into the restriction site of the plant overexpression vector pU1301D1 (modified by pCAMBIA1301, Hajdukiewicz et al., The small, versatile pPZPfamily of Agrobacterium binary vectors for plant transformation. PlantMol. Biol. 1994, 25: 989-994) Between Kpn I and BamH I, the pU1301D1:PLDζ1 recombinant plasmid was obtained. After restriction enzyme digestion and sequencing to verify the correct sequence of PLDζ1, the plasmid was transformed into Agrobacterium EHA105 strain.

2. Genetic transformation and establishment of PLDζ1 overexpressing rice plants:

Using Agrobacterium-mediated genetic transformation technology, the Agrobacterium containing the pU1301D1:PLDζ1 recombinant plasmid was used to infect the callus of Japonica rice Dongjin, and the specific steps were as follows:

1) induction of rice callus:

Shell the rice seeds, sterilize them with 75% ethanol for 1 min, then sterilize them with 0.05% mercuric chloride solution for 20 min, wash them with double distilled water for 5 times, sow them on the induction medium, and culture them in the dark at 26°C for about 4 weeks Obtain callus. The callus was transferred to the subculture medium for subculture to obtain embryogenic callus. The formula of callus induction medium includes N6 medium, 27.8mg/L FeSO ₄ 7H ₂ O, 37.3mg/L Na ₂ EDTA 2H ₂ O, 1mg/L niacin, 1mg/L VB6, 1mg/L VB1, 2mg/L glycine, 100mg/L inositol, 2.5mg/L 2,4-D, 0.3g/L proline, 0.6g/L hydrolyzed casein, 30g/L sucrose and 3g/L agarose, pH 5.9. The 2,4-D concentration in the subculture medium is 2mg/L, the proline concentration is 0.5g/L, and the other components are the same as those in the induction medium.

2) Agrobacterium infects the callus:

Use LB solid medium containing 50mg/L kanamycin and 20mg/L rifampicin to cultivate Agrobacterium, culture at 28°C for 2 days, pick a single colony for expansion culture, and culture at 28°C with shaking (200rpm) Until the OD ₆₀₀ of the bacterial solution is equal to 0.8. Place the embryogenic callus in the bacterial solution, incubate at 28°C for 30 minutes, blot the bacterial solution, put the callus in a petri dish with filter paper to dry, and then move to co-cultivation based on co-cultivation at 20°C 2 days. The formula of co-cultivation medium includes 1/2N6 medium, 27.8mg/L FeSO ₄ 7H ₂ O, 37.3mg/L Na ₂ EDTA 2H ₂ O, 1mg/L niacin, 1mg/L VB6, 1mg/L VB1, 2mg/L glycine, 100mg/L inositol, 2,4-D 3mg/L, 0.8g/L hydrolyzed casein, 20g/L sucrose and 7g/L agarose, pH5.6.

3) Obtaining of transformed plants:

The co-cultured callus was transferred out, rinsed 5 times with sterile water, and then soaked in sterile water containing 400 ppm carbenicillin for 30 min. Then suck it dry and place it on filter paper, transfer the dried callus to the selection medium (containing 50 mg/L hygromycin), culture in dark at 28°C for 20 days, and subculture once. Transfer the newly grown resistant callus to the differentiation medium, culture it under light at 28°C for 2 to 3 weeks, update the medium every 2 weeks, and transfer it to MS medium after the callus grows seedlings. Take root and cultivate, then harden and transplant. The screening medium formula includes N ₆ medium, 27.8mg/L FeSO ₄ ·7H ₂ O, 37.3mg/LNa ₂ EDTA·2H ₂ O, 1mg/L niacin, 1mg/L VB6, 1mg/L VB1, 2mg/L L glycine, 100mg/L inositol, 2.5mg/L 2,4-D, 0.6g/L hydrolyzed casein, 30g/L sucrose, 7g/L agarose, 1mg/L hygromycin and 200ppm carbenicillin, pH6.0. The bud differentiation medium is MS medium, 27.8mg/L FeSO ₄ 7H ₂ O, 37.3mg/L Na ₂ EDTA 2H ₂ O, 1mg/L niacin, 1mg/L VB6, 1mg/LVB1, 2mg/L L glycine, 100mg/L inositol, 2mg/L 6-BA, 2mg/L KT, 0.2mg/LNAA, 0.2mg/L IAA, 1g/L hydrolyzed casein, 30g/L sucrose and 3g/L agarose, pH 6.0.

4) PCR identification and Real-time PCR expression analysis of transformed plants:

Extract the leaf DNA of regenerated plants, use PLDζ1 specific sequence primers PLDζ1FZ: 5'-GGGGTACCATGCAAGAATATCTGAACC-3' and PLDζ1RZ: 5'-CGGGATCCATGGAAAACTTGTGGAG-3' to detect the leaf DNA by PCR, and use the DNA of WT (wild type japonica rice Dongjin) as negative As a control, it was detected whether the PLDζ1 gene was introduced into the regenerated plants. The results showed that 9 independent lines could amplify the target fragment with the same expected size, while the control wild-type WT did not amplify the target fragment, indicating that the full-length CDS sequence of PLDζ1 had been successfully introduced into rice plants. In addition, total RNA was extracted from leaves of transformed plants, cDNA was synthesized by reverse transcription, and the transformed plant lines obtained above were analyzed by real-time quantitative Real-time PCR. The results showed that the expression levels of most transformed strains were significantly higher than that of the control wild type. The rice transgenic plants overexpressing PLDζ1 were obtained for the following examples.

Example 2:

Overexpression of PLDζ1 enhances salt tolerance of plants

Two PLDζ1-OE independent strains OE-14 and OE-15 of the PLDζ1 overexpression rice transformed plants created in Example 1 were randomly selected for detailed analysis, and the non-transformed regenerated plants (wild type, WT) that had also undergone the tissue culture process were randomly selected. As a control, it was treated with different concentrations of NaCl solution (0, 50, 75mM) for one month at the tillering stage. The results found that under normal conditions, PLDζ1 overexpressed plants had no significant difference in plant height, leaf plasma membrane permeability, and malondialdehyde content between the corresponding wild-type plants. Significantly higher than the wild type, the leaf plasma membrane permeability and malondialdehyde content were significantly lower than the non-transformed wild type (Figure 1). After 75mM NaCl treatment for one month, the malondialdehyde content in leaves of overexpressed OE14 and OE15 plants was only 39% and 58% of the control wild type, respectively, and the plasma membrane permeability of leaves was only 38% and 73% of the wild type (Fig. 1 ). It shows that under salt stress, the damage degree of leaves of overexpression plants is significantly lower than that of control wild type. And under the stress of 150mM NaCl, the overexpression plants could harvest mature and plump seeds, while the control wild-type plants failed to obtain seeds. It indicated that the overexpression of rice PLDζ1 could enhance the salt tolerance of the plant and increase its seed yield. PLDζ1 positively regulates plant salt tolerance in response to salt stress.

Example 3:

Isolation, Identification and Functional Identification of PLDζ1 Deletion Mutant

1. Isolation, identification and phenotype observation of mutant pldζ1:

In order to further verify and analyze the function of the PLDζ1 gene, the present invention isolated and obtained two independent mutant lines with T-DNA inserted into different sites of PLDζ1, named pldζ1-1 and pldζ1-2 respectively, wherein the T-DNA of pldζ1-1 was inserted into It is located in the 3'-UTR region of PLDζ1, while the T-DNA insertion of pldζ1-2 is located in the third intron of PLDζ1. The effect of T-DNA insertion on the expression of PLDζ1 was analyzed by semi-quantitative RT-PCR. The results showed that there was a small amount of expression of PLDζ1 in the pldζ1-1 homozygous mutant, but it was significantly lower than that of the wild type, while in the pldζ1-2 homozygous mutant PLDζ1mRNA could not be detected, which belonged to complete deletion mutant.

The analysis showed that PLDζ1 was induced by salt stress, which indicated that PLDζ1 might be involved in the process of salt stress response. In this study, the obtained mutant pldζ1-1 and pldζ1-2 materials were used to analyze the salt stress treatment. The mutants and corresponding wild-type seeds harvested at the same period were sown in soil (organic soil: vermiculite=1:1), and then the 3-4 leaf stage seedlings with the same growth potential were transplanted to potting soil (paddy field soil: organic soil =1:1). The salt stress treatment was started when the plants grew to the early stage of tillering, and the phenotype was observed after three weeks of treatment with different concentrations of NaCl solutions (0, 50, 150 mM). It was found that under normal conditions, the growth of the mutants pldζ1-1 and pldζ1-2 was not significantly different from the wild type, but under the salt stress treatment, the growth of the pldζ1-1 and pldζ1-2 mutant plants was significantly weaker than that of the corresponding wild type. type, its plant height was significantly lower than that of the wild type, and as the salt concentration increased, the mutant grew worse, and the difference in plant height from the wild type was greater (Fig. 2, A in Fig. 3). In order to analyze the effect of deletion mutation of PLDζ1 on the salt tolerance of plants, the plants in the tillering stage were treated with 200mM NaCl for 15 days, and then the fresh weight of the aboveground parts of the plants, the permeability of the plasma membrane of the leaves and the content of malondialdehyde were measured. The results showed that there was no significant difference between the pldζ1-1 and pldζ1-2 mutants under normal conditions and the wild type in terms of plant fresh weight, plasma membrane permeability and malondialdehyde content. However, under salt stress conditions, the mutant's plant height and shoot fresh weight were significantly lower than those of the wild type, and the plasma membrane permeability and malondialdehyde content of the mutant were significantly higher than that of the wild type, which was twice that of the wild type ( image 3).

2. Effects of rice PLDζ1 on Na ⁺ and K ⁺ contents in plants under salt stress:

In order to test whether rice PLDζ1 affects the uptake of Na ⁺ and K ⁺ in plants under salt stress, this study measured the Na ⁺ and K ⁺ contents in the pldζ1 mutant and the corresponding wild type under salt stress. 150mM NaCl solution was applied for two weeks at the tillering stage, and the roots, leaf sheaths, and leaf tissues of the pldζ1 mutant and wild-type plants were taken, and the Na ⁺ and K ⁺ contents of the samples were measured by atomic absorption spectrophotometer, respectively. It was found that under normal growth conditions, there was no significant difference in the Na ⁺ content of the root and leaf tissues between the pldζ1 mutant and the wild type (Fig. 4A, B). Under salt stress, the Na ⁺ contents in the root, sheath and leaf tissue of the two pldζ1 mutants were significantly higher than those of the corresponding wild type (Fig. 4A,B). The potassium ion K ⁺ content of the mutant leaf sheath was significantly lower than that of the corresponding wild type (Fig. 4C, D). It indicated that the deletion of PLDζ1 led to the enhancement of sodium ion accumulation in the plant, which affected the Na ⁺ /K ⁺ ion balance of leaf tissue, and the deletion mutation of PLDζ1 in rice caused plants to be sensitive to salt.

Example 4:

PLDζ1 prokaryotic protein expression, enzyme biochemical characteristics

Whether rice PLDζ1 encodes phospholipase D is still unclear, and its biochemical characteristics need further analysis. For this reason, the present invention constructs the prokaryotic expression vector pET28a (Novagen Company) of PLDζ1, and its restriction site is BamH I and SacI, and the primers used are PLDζ1FQ (5'-GGATCCATGCAAGAATATCTGAACC-3') and PLDζ1RQ (5'-GAGCTCATGGAAAACTTGTGGAGAC- 3'). The expressed PLDζ1 recombinant protein was used for enzyme activity analysis, and the protein induced by the pET28a empty vector strain was used as a negative control. Using phosphatidylcholine PC (ordinary PC: fluorescent PC = 8:2) as the substrate, the results showed that rice PLDζ1 could catalyze phosphatidylcholine to produce phosphate acid PA, while the protein containing empty carrier could not under the same conditions. A clear PA signal was detected. The present invention further analyzed the effect of phosphatidylinositol derivative PIP ₂ on the activity of PLDζ1, and found that the catalytic activity of rice PLDζ1 was dependent on PIP ₂ , and the activity of PLDζ1 was the highest at pH 7 (Figure 5). These results indicated that rice PLDζ1 gene encodes phospholipase D, which mainly catalyzes the production of PA from PC.

Example 5:

Effects of PLDζ1 on the expression of genes related to lipid metabolism and sodium ion transport in rice

1. Effect of PLDζ1 on rice lipid metabolism:

For analyzing the influence of PLDζ1 on lipid metabolism, the present invention utilizes the pldζ1 mutant and corresponding wild-type material planted in organic soil (nutrient soil: vermiculite=1:1) to be in the tillering stage, extracts normal growth and salt stress (50mM NaCl ) to treat leaf lipids for four days, and determine the lipid content in plants by high-throughput mass spectrometry. The results showed that the contents of phosphatidic acid (PA), digalactosyldiacylglycerol (DGDG), phosphatidylcholine (PC) and phosphatidylglycerol (PG) in the pldζ1 mutant under normal conditions were significantly lower than those of the wild type, while The contents of monogalactosyldiacylglycerol (MGDG) and phosphatidylserine (PS) were significantly higher than those of wild type. Under the condition of salt stress, the content and mole percentage of monogalactosolipid diacylglycerol (MGDG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), phosphatidylserine (PS) in mutant pldζ1 and digalactose diacylglycerol (DGDG) contents were significantly higher than wild type, while phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylcholine (PC) contents were significantly lower than those of wild type. This indicates that PLDζ1 is involved in the regulation of lipid metabolism under normal and salt stress conditions.

2. The effect of PLDζ1 on the expression of genes related to sodium ion transport:

In this study, real-time quantitative Real-time PCR was used to analyze the expression of sodium ion transport-related genes. The pldζ1-1 mutant and wild type grown in hydroponic fluid and at the tillering stage were treated with 150mM NaCl stress, and then the total RNA of rice leaves treated for 0 and 6h was extracted, and the expression of related genes was quantitatively analyzed. Under normal conditions, the expression levels of the HKT1 gene that transports Na ^{+ ions into the cell, the NHX1 gene that transports Na + ions} into the vacuole, and the related SOS3 gene in the SOS signal transduction pathway were not significantly different between the mutant and the wild type However, the expression of HKT1 was inhibited under salt stress, and the expression in the pldζ1-1 mutant was significantly higher than that of the wild type (Figure 6), which indicated that PLDζ1 and its product PA could inhibit the expression of HKT1, thereby preventing extracellular Na ⁺ entry; while the expression level of NHX1 under salt stress is opposite to that of HKT1 gene, which is significantly induced by salt stress, and the expression level of NHX1 in the mutant is significantly lower than that of the wild type, indicating that PLDζ1 can promote the expression of NHX1 and enhance Na ⁺ enters the vacuole; while the expression of SOS1 and SOS3 genes in the wild type is significantly higher than that in the mutant under salt treatment conditions, so PLDζ1 can promote their expression, thereby enhancing the excretion of Na ⁺ into the body. These results indicated that PLDζ1 may participate in the regulation of the expression of genes related to sodium ion transport, and then regulate the protection of plants from the damage of excessive sodium ions.

SEQUENCE LISTING

<110> Huazhong Agricultural University

<120> Application of Phospholipase PLDζ1 Gene in Improving Salt Tolerance of Plants

<130> Application of Phospholipase PLDζ1 Gene in Improving Salt Tolerance of Plants

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 2805

<212> DNA

<213> Rice

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ccagggaaag actactataa ccccagggaa tctgagccaa attcctggga ggacacaatg 1080

aaagatgagc tggaccgaac gaaatatcct cgcatgcctt ggcatgatgt tcagtgtgct 1140

ctctacggtc caccttgtcg ggatgttgca aggcattttg ttcagcgctg gaattatgca 1200

aagaggaaca aagctcccaa tgagcaagga attcccttgc tgatgcctca tcaccacatg 1260

gtaattccac attacaaggg cataggccaa gaaattaata gtgaggctga tggtaaacaa 1320

aatcatgaca aggattgtga tgttaaaaaa ccagtctctg tggactcacg ggagtcttgt 1380

caggacattc cattgctttt acctcaagag cttgaaccac cggcattgcc caatggagat 1440

ttaagagtga acgattaga cgccaaccac tctgatcacc tacacaaaac aagcttcaat 1500

cagcctttgc tcaaccggaa ggcaaagttg gattcttccc gacaggattt gcccatgaga 1560

ggtttcgtag acaatataag ttcccttgag tcatcatcta ttaggcattt tgattcttcc 1620

aaagaagaaa agtatcacat ggataagaac tggtgggaga tgcaagaaag aggagatcaa 1680

gttgcttcag tacttgatat agggcaagtt ggtccaaggg caacttgtca ttgtcaggta 1740

attagaagtg ttggtcaatg gtcagctgga accactcaaa tcgaaggaag catccacaat 1800

gcctattttt ctcttattga aaaggcagaa cattttgtat atatcgagaa tcaatttttc 1860

atatcaggcc tttcaggaga tgaaacgata aaaaatcgtg tattggaagc attatacagg 1920

cgcatacttc gggcagagag agaaaaaaag cgcttcaaag ccatcataat aatacctctg 1980

ttacctggtt ttcagggagg cattgatgat ggcggagctg catctgtgag agcaattatg 2040

cattggcaat accggactat ctgtagaggc cctaattcta tacttcagaa tctatatgat 2100

gttattggtc caaaagcaca tgattatatc tcattttatg gtcttagagc acatggtaga 2160

ctatgcgaag gaggtccctt ggtcactaat cagatttatg tccacagtaa gttgatgata 2220

attgatgacc gcatcacatt gattggatca gctaacataa atgatagaag cttgcttgga 2280

tcaagagatt ctgagattgc cgtggtcatt gaagataaag aagttgttag ttccaaaatg 2340

aatggaaaac cttgggaagc tgggaagttt tctctaagcc tacgtctctc tctctgggca 2400

gagcaccttg gccttcatcg aggagaggtc agccatatta tggaccctat cgatgactca 2460

actttcaaaa atatctggat ggccactgct aagacaaata ccatgattta ccaagatgtc 2520

ttctcatgtg tacctaatga tcttatccat tcaagggccc aatttcggca gagctttgct 2580

cactgcaggg ataaaatcgg tcacaataca atcgatttgg gggttgccca agagaagctg 2640

gaaacctacc aggacggcga tctcaagggt acggacccta tagaaagatt gcagatgatc 2700

aaaggtcacc ttgtttcttt cccgttggat ttcatgtccc aagaggactt gagaccatat 2760

ttcagtgaaa gtgaatatta tacgtctcca caagttttcc attag 2805

<210> 2

<211> 934

<212> PRT

<213> Rice

<400> 2

Met Gln Glu Tyr Leu Asn His Phe Leu Gly Asn Leu Asp Ile Val Asn

1 5 10 15

Ser Pro Glu Val Cys Lys Phe Leu Glu Val Ser Cys Leu Ser Phe Leu

20 25 30

Pro Glu Tyr Gly Pro Lys Leu Lys Glu Asp Tyr Val Ser Val Gly His

35 40 45

Leu Pro Lys Ile Gln Lys Asp His Lys Glu Asn Cys Cys Ser Cys Gly

50 55 60

Leu Phe Ser Cys Cys Lys Ser Ser Trp Gln Lys Val Trp Val Val Leu

65 70 75 80

Lys Pro Gly Phe Leu Ala Leu Leu Lys Asp Pro Phe Asp Pro Lys Leu

85 90 95

Leu Asp Val Leu Ile Phe Asp Ala Leu Pro His Met Asp Ile Ser Gly

100 105 110

Glu Gly Gln Ile Ser Leu Ala Lys Glu Ile Lys Glu Arg Asn Pro Leu

115 120 125

His Phe Gly Leu Gln Val Ser Ser Gly Gly Gln Thr Leu Lys Leu Arg

130 135 140

Thr Arg Ser Ser Ser Lys Val Lys Asp Trp Val Ser Ala Ile Asn Ala

145 150 155 160

Ala Arg Gln Thr Pro Glu Gly Trp Cys Tyr Pro His Arg Phe Gly Ser

165 170 175

Phe Ala Pro Pro Arg Gly Leu Met Pro Asp Gly Ser Met Val Gln Trp

180 185 190

Phe Ile Asp Gly Glu Ala Ala Phe Gln Ala Ile Ala Ser Ser Ser Ile Glu

195 200 205

Gln Ala Lys Ser Glu Ile Phe Ile Thr Gly Trp Trp Leu Cys Pro Glu

210 215 220

Leu Phe Leu Arg Arg Pro Phe Gln His His Gly Ser Ser Arg Leu Asp

225 230 235 240

Ala Leu Leu Glu Ala Arg Ala Lys Gln Gly Val Gln Ile Tyr Ile Leu

245 250 255

Leu Tyr Lys Glu Val Ala Leu Ala Leu Lys Ile Asn Ser Leu Tyr Ser

260 265 270

Lys Gln Lys Leu Leu Asn Ile His Glu Asn Val Lys Val Leu Arg Tyr

275 280 285

Pro Asp His Phe Ser Ser Gly Val Tyr Leu Trp Ser His His Glu Lys

290 295 300

Ile Val Ile Val Asp Asn Gln Val Cys Tyr Leu Gly Gly Leu Asp Leu

305 310 315 320

Cys Phe Gly Arg Tyr Asp Asn Ser Ala His Lys Leu Ser Asp Val Pro

325 330 335

Pro Val Ile Trp Pro Gly Lys Asp Tyr Tyr Asn Pro Arg Glu Ser Glu

340 345 350

Pro Asn Ser Trp Glu Asp Thr Met Lys Asp Glu Leu Asp Arg Thr Lys

355 360 365

Tyr Pro Arg Met Pro Trp His Asp Val Gln Cys Ala Leu Tyr Gly Pro

370 375 380

Pro Cys Arg Asp Val Ala Arg His Phe Val Gln Arg Trp Asn Tyr Ala

385 390 395 400

Lys Arg Asn Lys Ala Pro Asn Glu Gln Gly Ile Pro Leu Leu Met Pro

405 410 415

His His His Met Val Ile Pro His Tyr Lys Gly Ile Gly Gln Glu Ile

420 425 430

Asn Ser Glu Ala Asp Gly Lys Gln Asn His Asp Lys Asp Cys Asp Val

435 440 445

Lys Lys Pro Val Ser Val Asp Ser Arg Glu Ser Cys Gln Asp Ile Pro

450 455 460

Leu Leu Leu Pro Gln Glu Leu Glu Pro Pro Ala Leu Pro Asn Gly Asp

465 470 475 480

Leu Arg Val Asn Asp Leu Asp Ala Asn His Ser Asp His Leu His Lys

485 490 495

Thr Ser Phe Asn Gln Pro Leu Leu Asn Arg Lys Ala Lys Leu Asp Ser

500 505 510

Ser Arg Gln Asp Leu Pro Met Arg Gly Phe Val Asp Asn Ile Ser Ser

515 520 525

Leu Glu Ser Ser Ser Ile Arg His Phe Asp Ser Ser Ser Lys Glu Glu Lys

530 535 540

Tyr His Met Asp Lys Asn Trp Trp Glu Met Gln Glu Arg Gly Asp Gln

545 550 555 560

Val Ala Ser Val Leu Asp Ile Gly Gln Val Gly Pro Arg Ala Thr Cys

565 570 575

His Cys Gln Val Ile Arg Ser Val Gly Gln Trp Ser Ala Gly Thr Thr

580 585 590

Gln Ile Glu Gly Ser Ile His Asn Ala Tyr Phe Ser Leu Ile Glu Lys

595 600 605

Ala Glu His Phe Val Tyr Ile Glu Asn Gln Phe Phe Ile Ser Gly Leu

610 615 620

Ser Gly Asp Glu Thr Ile Lys Asn Arg Val Leu Glu Ala Leu Tyr Arg

625 630 635 640

Arg Ile Leu Arg Ala Glu Arg Glu Lys Lys Arg Phe Lys Ala Ile Ile

645 650 655

Ile Ile Pro Leu Leu Pro Gly Phe Gln Gly Gly Ile Asp Asp Gly Gly

660 665 670

Ala Ala Ser Val Arg Ala Ile Met His Trp Gln Tyr Arg Thr Ile Cys

675 680 685

Arg Gly Pro Asn Ser Ile Leu Gln Asn Leu Tyr Asp Val Ile Gly Pro

690 695 700

Lys Ala His Asp Tyr Ile Ser Phe Tyr Gly Leu Arg Ala His Gly Arg

705 710 715 720

Leu Cys Glu Gly Gly Pro Leu Val Thr Asn Gln Ile Tyr Val His Ser

725 730 735

Lys Leu Met Ile Ile Asp Asp Arg Ile Thr Leu Ile Gly Ser Ala Asn

740 745 750

Ile Asn Asp Arg Ser Leu Leu Gly Ser Arg Asp Ser Glu Ile Ala Val

755 760 765

Val Ile Glu Asp Lys Glu Val Val Ser Ser Lys Met Asn Gly Lys Pro

770 775 780

Trp Glu Ala Gly Lys Phe Ser Leu Ser Leu Arg Leu Ser Leu Trp Ala

785 790 795 800

Glu His Leu Gly Leu His Arg Gly Glu Val Ser His Ile Met Asp Pro

805 810 815

Ile Asp Asp Ser Thr Phe Lys Asn Ile Trp Met Ala Thr Ala Lys Thr

820 825 830

Asn Thr Met Ile Tyr Gln Asp Val Phe Ser Cys Val Pro Asn Asp Leu

835 840 845

Ile His Ser Arg Ala Gln Phe Arg Gln Ser Phe Ala His Cys Arg Asp

850 855 860

Lys Ile Gly His Asn Thr Ile Asp Leu Gly Val Ala Gln Glu Lys Leu

865 870 875 880

Glu Thr Tyr Gln Asp Gly Asp Leu Lys Gly Thr Asp Pro Ile Glu Arg

885 890 895

Leu Gln Met Ile Lys Gly His Leu Val Ser Phe Pro Leu Asp Phe Met

900 905 910

Ser Gln Glu Asp Leu Arg Pro Tyr Phe Ser Glu Ser Glu Tyr Tyr Thr

915 920 925

Ser Pro Gln Val Phe His

930

Claims (8)

1. plant phosphorus lipasePLDz1Gene application in improving plant salt endurance.

2. the molecular genetic manipulation method promoting salt tolerance of crop, it is characterised in that overexpressionPLDz1 It is remarkably reinforced crop Such as the biological yield under the salt tolerance of rice plant and salt stress, gene delection is expressed then makes plant salt tolerance substantially reduce, Biological yield is significantly lower than wild type.

3. changePLDz1Expression can significantly modify the salt tolerance of plant, its Main Function is by sodium in regulation and control plant body The transhipment of ion and homeostasis；

OverexpressionPLDz1Plant survival rate and growth under salt stress can be made to be substantially better than comparison wild type, improve the salt tolerant of plant Property, andPLDz1Deletion mutation causes under salt stress significantly increasing and reducing salt tolerance of sodium ions content in plant body.

4. cloned by PLDz1 gene and enzyme activity assay showsPLDz1The albumen of coding belongs to Choline phosphatase z.

5. the PLDz1 gene as described in claims 1 and 2, its CDS sequence is by description nucleotide and gal4 amino acid Nucleotide sequence composition in sequence 1.

6.CDS sequence similarity is more than or equal to 50% of CDS sequence described in claims 5.

7. the PLDz1 gene as described in claims 1 and 2, the aminoacid sequence of its coded protein is by nucleoside in description Aminoacid sequence composition in acid and protein amino acid sequence 2.

8. protein amino acid sequence similarity is more than or equal to 50% of the aminoacid sequence described in claims 7.