CN116875629A - Coding gene for enhancing iron deficiency tolerance of plants and promoting iron accumulation of plants and application - Google Patents
Coding gene for enhancing iron deficiency tolerance of plants and promoting iron accumulation of plants and application Download PDFInfo
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Abstract
The invention relates to application of a coding gene for enhancing iron deficiency tolerance of plants and promoting iron accumulation of plants, belonging to the technical field of bioengineering. The coding gene sequence for enhancing the iron deficiency tolerance and iron accumulation of the plants is shown in a sequence table SEQ ID No: l is shown. The invention relates to a method for preparing a sequence table SEQ ID No: the coding gene for enhancing the tolerance of the plant to the iron deficiency stress is knocked out in the plant, so that the coding gene is not expressed in the plant, and the plant shows the iron deficiency tolerance character. The functional gene for enhancing the iron deficiency tolerance of plants and promoting the iron accumulation of the plants can provide new gene resources and technical support for the iron deficiency stress tolerance breeding of crops, and plant seeds with enhanced stress resistance and stress tolerance can be cultivated by using the functional gene.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a functional gene for enhancing iron deficiency tolerance of plants and promoting iron accumulation of plants, and simultaneously relates to a method for enhancing the tolerance of plants to iron deficiency environments by utilizing the gene, so that the response of the plants to the iron deficiency environments can be negatively regulated.
Background
The metallic element iron (Fe) accounts for 4.75% of the crust content, and is next to oxygen, silicon and aluminum, and the existing crust content is ranked fourth. Iron is an essential trace element essential in the growth and development process of animals and plants, and exists mainly in the animal or human body in the form of heme or transferrin in plasma, and the heme mainly acts to supply oxygen to the human body in the animal or human body, and when the human body ingests iron mainly from food, when the human body ingests iron-containing food, the human body firstly converts the ingested iron into ferrous form which is easily absorbed by the human body through a series of conversion in the stomach, and then the iron is absorbed by the small intestine. The free form of iron ions is toxic to body cells, so that free iron ions are not generally present in the body, which is also one of the characteristics of distinguishing iron elements from other metal elements. Investigation reports show that the variation of iron content in humans is affected by a number of factors such as age, weight, nutritional status, etc.
Iron element is involved in the formation of plant chloroplast organelles, is an essential element indispensable for plant photosynthesis, and can promote plant photosynthesis. The iron element, although high in the soil, exists mainly in the form of ferric iron which is not absorbed by the plants, which limits the absorption of effective iron by the plants due to its low solubility in alkaline soil. During long-term evolution, plants have developed an effective mechanism for uptake, transport and storage of iron in soil to maintain iron homeostasis. Since iron in plants is difficult to transfer from mature tissue parts to tender tissue parts, iron transport must be dependent on the xylem of the plant. The plants need to ensure an effective iron supply at various stages of their growth, so as to meet the iron demand during plant growth. Thus, iron deficiency is a common nutritional stress for plants. When the plants are stressed by the iron-deficiency environment, the normal growth and development of the plants are affected by affecting photosynthesis, mitochondrial respiration, nutrient transport and plant immunity of the plants, so that the yield and quality of crops are reduced. The phytophagous food not only provides a variety of necessary nutrients for human beings, but also is one of the main sources of iron intake for human beings.
At present, the human beings mainly improve the iron-deficiency environment of plants by means of foliar fertilization such as spraying ferrous sulfate, iron fulvate and the like. The traditional fertilization method has higher cost and poorer effect, so that the problem of iron deficiency of plants cannot be effectively solved. For serious soil iron deficiency, especially high pH and high carbonate content soil, the iron absorption, transportation and storage capacity of plants can be enhanced by exploring molecular mechanisms of iron absorption, transportation and storage of plants. The method is not only an environment-friendly way for solving the problem of iron deficiency of the soil, but also has important theoretical and economic significance for treating the problem of iron deficiency of the soil.
The model plant Arabidopsis thaliana is widely used in research fields of plant genetics, crop biology, molecular biology and the like. The arabidopsis thaliana flower bud picking device has the characteristics of simple structure, high propagation coefficient, strong vitality, self-pollination, easiness in transformation and the like, so that the arabidopsis thaliana serving as a research object can reach an expected research target faster and better. The whole genome sequencing of the arabidopsis is completely finished, so that a new way for crop stress-resistant genetic improvement is provided by researching the molecular mechanism of the plant responding to the iron deficiency environment through the arabidopsis. Searching and finding new functional genes with independent intellectual property based on the Arabidopsis sequencing database (www.arabidopsis.org) is a hotspot in the field of International botanical research, where Arabidopsis has about 1.3 hundred million base pairs and 2.9 ten thousand genes. At present, the function research of most genes is not clear, the research of the gene functions by using mutant screening technology is an effective experimental method, and the CRISPR/Case9 mediated gene editing technical scheme can be used at presentThe method can also adopt methods such as T-DNA insertion, RNAi interference, transposon insertion, EMS mutagenesis and the like to knock out functional genes to be explored, and cultivate transformed plant tissues into mutant plants. In recent years, studies on Arabidopsis mutant plants have found some functional genes involved in regulating the iron deficiency stress response of plants, such as:FIT、bHLH38、bHLH39、bHLH100、bHLH101、bHLH34、bHLH104、bHLH105、 bHLH115、PYE、MYB10、MYB72、MYC1、WRKY12etc. In the face of the problem of iron deficiency of plants on a global scale, it is important to find genes which enhance the iron deficiency tolerance of plants and promote the accumulation of iron in plants and to elucidate the functions of the genes.
Disclosure of Invention
The invention aims to provide a coding gene for enhancing the iron deficiency tolerance of plants and promoting the iron accumulation of plants, and a second aim of the invention is to provide the application of the coding gene as a negative regulatory factor in regulating the iron deficiency tolerance and accumulation of plants.
As shown in a sequence table SEQ ID No: the application of the coding gene shown in the formula I in enhancing the iron deficiency tolerance of plants and promoting the iron accumulation of the plants is characterized in that: sequence listing SEQ ID No: l the coding gene for enhancing the iron deficiency tolerance of the plant and promoting the iron accumulation of the plant is knocked out in the plant, namely the coding gene is not expressed in the plant, and the plant shows the property of enhancing the iron deficiency tolerance, and is arabidopsis thaliana.
Sequence listing SEQ ID No: the coding gene for enhancing the iron deficiency tolerance and accumulation of the plants shown in the step I realizes the knocking out of the WRKY13 gene of the negative regulatory factor in the arabidopsis through a T-DNA insertion method.
The beneficial technical effects of the invention are as follows:
1. according to the published genome sequence of the Arabidopsis database, WRKY13 is a member of the Arabidopsis WRKY transcription factor family, and the applicant finds that the plant shows tolerance to iron deficiency stress under the treatment of iron deficiency stress after the WRKY13 gene mutation, which indicates that the WRKY13 gene responds to the regulation of the iron deficiency stress of the plant. Therefore, the gene function is researched, and further research results show that the content of Fe in a WRKY13 mutant plant body is higher than that of a wild plant, which shows that the knock-out gene WRKY13 can increase the absorption of Fe by arabidopsis thaliana, so that the content of Fe in the arabidopsis thaliana is increased, and the property of tolerance to iron deficiency stress is further shown.
2. After the WRKY13 gene is mutated, the iron deficiency tolerance and iron accumulation of plants can be enhanced, new gene resources and technical support are provided for stress-tolerant genetic improvement of crops, and plant seeds with enhanced stress resistance and stress tolerance can be cultivated by using the WRKY13 gene.
Drawings
FIG. 1 is a chart of phenotyping of WRKY13 mutant plants with iron addition and iron deficiency.
FIG. 2 is a graph showing chlorophyll content detection of WRKY13 mutant plants with iron addition and iron deficiency.
FIG. 3 is a graph showing root length analysis of WRKY13 mutant plants with iron addition and iron deficiency.
FIG. 4 is a graph showing analysis of iron content of WRKY13 mutant plants with iron addition and iron deficiency.
FIG. 5 is a chart of phenotyping of WRKY13 overexpressing plants with iron addition and iron deficiency.
FIG. 6 is a graph showing chlorophyll content detection of WRKY13 over-expressed plants with iron addition and iron deficiency.
FIG. 7 is a graph of root length analysis of WRKY13 overexpressing plants with iron addition and iron deficiency.
FIG. 8 is a graph of analysis of iron content in WRKY13 over-expressed plants with iron addition and iron deficiency.
Detailed Description
The invention is further described by way of examples with reference to the accompanying drawings.
Sequence listing SEQ ID No:1 and the coding gene for enhancing the iron deficiency tolerance of plants and promoting the accumulation of plant iron is a mutant which is obtained from screening an American Arabidopsis germplasm resource pool and responds to the iron deficiency stress of plants.
Example 1
Screening and functional analysis of iron deficiency resistant arabidopsis plants
1. Acquisition of WRKY13 mutant
Mutant seeds obtained from the U.S. arabidopsis germplasm resource center were cultivated on an iron-deficient medium. From which plants resistant to iron deficiency manifestations are obtainedThe strain is identified and found to be the mutantWRKY13Mutants with deleted gene function, designated aswrky13- 1(seed number: salk_ 064346C). To more reliably analyze the function of the gene mutant, we obtained another from the U.S. Arabidopsis germplasm resource centerWRKY13Mutant plants were designated aswrky13-2(seed number: salk_ 075362C).
2.WRKY13Iron deficiency comparison of mutant plants with wild type plants
Referring to FIG. 1, for Wild Type (WT) andWRKY13iron deficiency stress phenotype analysis is carried out on the gene function deficiency mutant material, namely wild type and wild typeWRKY13The gene function deletion mutant is simultaneously sown on a solid culture medium with iron and with iron deficiency, and is placed in a constant temperature greenhouse (the photoperiod is 16 hours of illumination and 8 hours of darkness) at 22 ℃ for vertical culture. After two weeks observations found that growth on the sideroblasts mediumWRKY13The mutant has no obvious difference compared with the wild type plant and grows on the iron-deficiency solid culture mediumWRKY13The mutant root length is obviously longer than that of wild plants, andWRKY13the leaves of the mutant are obviously greener than those of the wild plant.
3.WRKY13Chlorophyll content detection of mutant plants under iron addition and iron deficiency
Referring to FIG. 2, wild type andwrky13-1，wrky13-2the material is directly inoculated on a culture medium with iron and with iron deficiency for vertical culture, and the material is vertically cultured for two weeks under normal illumination. Detecting wild type and iron deficiency under iron adding and iron deficiency conditions respectivelywrky13-1，wrky13-2Chlorophyll content in plants, which was found to grow on iron-supplemented mediaWRKY13The mutant plants and the wild plants have no significant difference in chlorophyll content; growing under the condition of iron deficiency stress cultureWRKY13Chlorophyll content in the mutant plants (see fig. 2) was significantly higher than in the wild type.
4.WRKY13Root length analysis of mutant plants under iron addition and iron deficiency
Referring to FIG. 3, wild type andwrky13-1，wrky13-2the material is directly inoculated on a culture medium with iron and with iron deficiency, and is subjected to normal lightCulturing was performed vertically under the irradiation conditions for two weeks. Under the conditions of iron adding and iron deficiency respectivelyWRKY13Root length of mutant plants and wild type plants. As a result, it was found that, under the condition of adding iron,WRKY13the fresh weight of the mutant plant has no obvious difference with the wild type; and in the condition of iron deficiency, in the case of iron deficiency,WRKY13the root length of the mutant plants was significantly longer than that of the wild type.
5.WRKY13Analysis of iron content of mutant plants under iron addition and iron deficiency
See a in fig. 4 and B in fig. 4, wild type andwrky13-1，wrky13-2the material is directly inoculated on a culture medium with iron and with iron deficiency, and is vertically cultured for two weeks under normal illumination. Respectively detect under different conditionsWRKY13Iron content in leaves and roots of mutant plants and wild type plants. As a result, it was found that, under normal conditions,WRKY13the iron content in the leaves and roots of the mutant plants is not obviously different from that of the wild type; and in the condition of iron deficiency, in the case of iron deficiency,WRKY13the iron content in the leaves and roots of the mutant plants is significantly higher than that of the wild type.
Example 2
Cultivation of Arabidopsis plants sensitive to iron deficiency stress phenotype
1.WRKY13Gene over-expression transgenic linesWRKY13-OE1、OE2Is obtained by (a)
To further verify the function of the gene in the regulation of iron deficiency stress of plants, we constructedWRKY13Gene over-expression vector35S:WRKY13). First, target fragment amplification is performed. The wild arabidopsis is normally cultured on an MS culture medium for two weeks, total RNA of plants is extracted, cDNA is synthesized by reverse transcription, and PCR is carried out by taking the synthesized cDNA as a template to amplify a sufficient amount of target products. And then taking the PCR product as a template to carry out secondary amplification, wherein the purpose is to introduce enzyme cutting sites. And (3) carrying out enzyme digestion and recovery on the PCR product and a vector pCAMBIA 1301. The recovered and purified target DNA fragment and the vector were then ligated overnight with T4-DNA ligase. Transferring the connecting solution into DH5 alpha, detecting and screening out positive clones, and sequencing. After the sequencing result is determined to be correct, the agrobacterium GV3101 is transformed by using an electric shock transformation method. The agrobacterium GV3101 after electric shock transformation is activated and coated on LB medium plate containing double antibody (Kan, gen). Single colonies were randomly picked and positive colonies were detected by PCR. Amplifying and culturing agrobacterium in LB culture solution containing double antibody (kan, gen) and transforming Arabidopsis wild type plant by soaking method to obtainWRKY13A transgenic line over-expressing a gene.
2.WRKY13Iron deficiency comparison of overexpressed plants with wild-type plants
Referring to FIG. 5, wild type andWRKY13-OE1andOE2simultaneously, the seeds are sowed in a culture dish with the diameter of 90 mm, the culture medium is solid culture medium with added iron and deficient iron, and the culture medium is placed in a constant-temperature illumination incubator with the temperature of 22 ℃ (the photoperiod is 16 hours illumination and 8 hours darkness) for vertical culture. After two weeks, it can be observed that: transgenic plantsWRKY13-OE1AndOE2phenotype of vertical culture directly inoculated onto iron-added or iron-deficient medium (see FIG. 5) with wild-type material, wherein the iron-added medium is a control. Wild type and grown on iron-added mediaWRKY13-OE1、OE2No significant difference; when wild type andWRKY13-OE1andOE2when the material is directly inoculated on a culture medium with iron deficiency stress for culture,WRKY13-OE1andOE2exhibiting a phenotype that is susceptible to iron deficiency stress compared to the wild type.
Referring to FIG. 5, wild type andWRKY13-OE1andOE2simultaneously, the seeds are sowed in a culture dish with the diameter of 90 mm, the culture medium is solid culture medium with added iron and deficient iron, and the culture medium is placed in a constant-temperature illumination incubator with the temperature of 22 ℃ (the photoperiod is 16 hours illumination and 8 hours darkness) for vertical culture. After two weeks, it can be observed that: wild type and wild typeWRKY13-OE1AndOE2the materials are directly inoculated on a culture medium with iron and a culture medium with iron deficiency for vertical culture phenotype, and the phenotype is shown in figure 5; wherein the iron-supplemented medium is used as a control, and wild type grown on the iron-supplemented medium are used as the controlWRKY13-OE1AndOE2plants were not significantly different in chlorophyll (see fig. 6), root length (see fig. 7), iron content in leaves and roots (see a in fig. 8 and B in fig. 8), and the like. While the wild type andWRKY13-OE1andOE2when the material is directly inoculated on an iron-deficiency culture medium for vertical culture，WRKY13-OE1AndOE2plants exhibited significant iron deficiency sensitivity traits. Under the stress of iron deficiency, the iron-free alloy is prepared,WRKY13-OE1andOE2the chlorophyll content (see fig. 6), root length (see fig. 7), and iron content (see a in fig. 8 and B in fig. 8) were significantly lower than those of the wild type. The above-mentioned results show that,WRKY13-OE1andOE2the wild type shows a property more sensitive to iron deficiency stress.
Claims (2)
1. As shown in a sequence table SEQ ID No: the application of the coding gene shown in the formula I in enhancing the iron deficiency tolerance of plants and promoting the iron accumulation of the plants is characterized in that: sequence listing SEQ ID No: l the coding gene for enhancing the iron deficiency tolerance of the plant and promoting the iron accumulation of the plant is knocked out in the plant, namely the coding gene is not expressed in the plant, and the plant shows the property of enhancing the iron deficiency tolerance, and is arabidopsis thaliana.
2. The use according to claim 1, characterized in that: sequence listing SEQ ID No: the coding gene for enhancing the iron deficiency tolerance and accumulation of the plants shown in the step I realizes the knocking out of the WRKY13 gene of the negative regulatory factor in the arabidopsis through a T-DNA insertion method.
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