CN107142266B - ZmRCI2-8 gene and application thereof in promoting plant germination and lateral root growth under abiotic stress condition - Google Patents

Abstract

The invention relates to a ZmRCI2-8 gene and application thereof in promoting plant germination and lateral root growth under the condition of abiotic stress. The ZmRCI2-8 gene has a nucleotide sequence shown as SEQ ID NO.1, and codes protein with an amino acid sequence shown as SEQ ID NO. 2. The invention further relates to application of the ZmRCI2-8 gene in improving abiotic stress resistance of plants, in particular to application in promoting the growth of lateral roots of plants and improving the germination rate of plant seeds. The ZmRCI2-8 gene of the invention participates in regulating and controlling nutrient absorption and photosynthesis or transpiration of leaves, can be used for improving the accumulation of plant carbohydrates, improving the nutritional quality and yield of seeds, promoting the germination of seeds and the growth of lateral roots under the condition of abiotic stress, participating in low-phosphorus regulation, and is expected to improve the phosphorus efficiency of plants and cultivate phosphorus-efficient crops.

Description

ZmRCI2-8 gene and application thereof in promoting plant germination and lateral root growth under abiotic stress condition

Technical Field

The invention belongs to the fields of gene engineering technology and physiological heredity, and particularly relates to a gene of a protein capable of responding to various abiotic stresses.

Background

Phosphorus is an important component of a variety of organic compounds, including nucleic acids, phospholipids, ATP, and the like. Phosphorus participates in physiological processes such as carbohydrate metabolism, nitrogen metabolism and fat metabolism in plants, regulates enzymatic reaction and cell signal transduction, and can regulate and control various important physiological and biological processes through protein modification. Meanwhile, the phosphorus can promote the root system to extend, reduce the transpiration strength of the leaves, improve the drought damage tolerance of the plants, or maintain and regulate the metabolism process in the crops, and improve the cold resistance of the plants. Phosphorus is a major element, but available phosphorus that plants can absorb from soil is very little, and at least one third of the available cultivated soil in the world is not absorbed by plants, and therefore phosphorus is considered to be a major limiting factor affecting plant growth and crop yield.

On the other hand, the more phosphate fertilizer that is applied, the better. If the phosphate fertilizer is excessively applied, the following problems are also caused: (1) excessive phosphorus nutrition promotes the crops to breathe vigorously, so that reproductive organs develop in advance, the crops mature prematurely, the grains are small, and the yield is low. (2) Inducing the zinc deficiency of the soil. The zinc in the soil reacts with excess phosphorus to produce zinc phosphate precipitates, or the excess phosphorus causes the soil to become alkaline, reducing the effectiveness of the zinc. (3) Leads the crops to lack silicon. According to researches, excessive phosphate fertilizer can cause silicon in soil to be fixed and not absorbed, so that silicon deficiency of crops is caused, and the silicon deficiency symptoms such as slender stems, poor lodging and disease resistance and the like are caused, and particularly, the influence on silicon-loving gramineous crops such as corn is larger. (4) The crops can be lack of molybdenum due to phosphorus, and the molybdenum deficiency symptom is shown. (5) Causing the accumulation of harmful elements in the soil. The phosphorus fertilizer contains harmful elements such as cadmium, lead, fluorine and the like, the application of the phosphate fertilizer can increase the cadmium and the like in soil, the annual growth amount of the cadmium and the like even reaches 0.15%, the cadmium has high effectiveness, and the cadmium is easy to be absorbed by crops and causes harm to people and livestock. (6) Causing deterioration of physicochemical properties of the soil, acidification (superphosphate) or alkalinity (calcium magnesium phosphate) of the soil and deterioration of physicochemical properties.

Plants need to take up phosphorus from the soil to sustain growth. Under the condition of phosphorus deficiency, the plant can reduce the consumption of phosphorus in the body through metabolic regulation such as photosynthesis, carbon metabolism and the like, thereby improving the utilization efficiency of phosphorus. In order to make the most use of the phosphorus source, the phosphorus in the plant body can be transferred from the aged tissue organ to the new tissue by using active substances such as phosphatase, nuclease and the like under the condition of phosphorus deficiency. The adaptive change process of plant response to low phosphorus stress is the result of space-time specific expression of a series of genes. After low phosphorus stress, the stress on rice, corn, the differential expression of a number of genes in plants such as white lupin and Arabidopsis thaliana, which are involved in a variety of physiological biochemical pathways in plants (Wasaki J et al 2006. transcriptional analysis of genes expressed with metabolic change using genes expressed by the expression of Phosphorus in rice leaves. Journal of Experimental Botany 57: 2049-calcium 2059; Mordue R et al 2007.Genome-wide reprogramming of metabolic regulation of networks of microorganisms in Arabidopsis thaliana. Cell expression 30: 85-Herpesent G.112: 76144. response to Phosphorus stress in plants and plants [ genes of genes and genes of plants ] 76144. response to phosphate stress in plants [ see. Puccin J.2006 ] and. transcriptional analysis of genes expressed with metabolic change using genes expressed with metabolic regulation of Phosphorus in rice leaves. Low-phosphorus-responsive Genes can be divided into two classes, one is an early fast-response gene, which is sensitive to phosphorus deficiency reactions and is expressed within hours of phosphorus deficiency treatment (Hammond JP et al 2003.Changes in gene expression in Arabidopsis genome expression and Development potential during phosphorus deficiency), the other is a transcription factor AtPHR1(Rubio V et al 2001.A controlled MYB transformation factor in transformed in phosphor modification in genome expression in Genes expression and in expression in Genes in genome expression and in expression in genome expression in Genes in genome expression and in genome expression in Genes in genome expression and in genome expression in Genes in which Genes are involved in phosphate signal transduction in microtubule plants and algae, and the other is a non-specific promoter region, which may also include Genes ATPHOSPB-conserved in-13, and non-specific promoter regions in plants in microtubule plants and algae (TAT V. 15. A-Biogene expression promoter region, also includes Genes-A-11. A. a gene expression promoter region of this invention can be used for the Genes of the genus of the Genes of, guo Ping Yi.2013. progress of research on functional genome of plant responding to low phosphorus stress.Biotechnology report 7: 1-6.); another class are late low-phosphorus stress responsive genes, which are mostly phosphorus deficiency-specific inducible genes, are subject to low-phosphorus stress for a long time, and are closely related to Plant morphological, physiological and metabolic responses (Hammond JP, Broadley MR, White PJ.2004.genetic responses to phosphorus deficiency. Annals of Botany (London)94: 323. beta.) such as acid phosphatase, phosphate transporter, etc. (Liu C et al 1998.Tomato phosphate transporter gene differential regulation in Plant tissues. Plant Physiology 116: 91-99.).

RCI2 (Rare-cold-absorbent) is a highly conserved small hydrophobic polypeptide that is ubiquitous in prokaryotes and eukaryotes. The RCI2/PMP3s family of genes are present in prokaryotic and eukaryotic genomes (Medina J et al 2007.Phylogenetic and functional analysis of Arabidopsis RCI2 genes. Phylogenetic and functional analysis of Arabidopsis RCI2 genes. Journal of Experimental Botany 58: 4333-4346.). The PMP3 family of proteins are named for the yeast Pmp3p protein, which contains 55 amino acid residues. The plant RCI2/PMP3s is similar in size (50-80 amino acid residues), sequence and structure to Pmp3p, including two highly hydrophobic regions and an intact membrane protein structure, but the mechanism of the cell ion homeostasis activity of RCI2/PMP3s is not clear (Navarre C and Goffeau A.2000.Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, high level contained small protein of yeast plasma membrane (a highly conserved small protein PMP3 deletion induced membrane hyperpolarization and salt sensitivity of yeast plasma membrane), EMBO Journal 25219: 2515-. Many Plant PMP3 genes were designated according to Arabidopsis ATRI2A/B as rare cryo-inducible genes (RCI2 or RCI2-like (RCI2-like)) (Capel J et al 1997.Two homo homologous low-temperature-induced hydrophilic proteins from Arabidopsis encode encoding highly hydrophobic proteins Plant physiologies 115: 569-.

The RCI2gene is involved in responding to a variety of abiotic stresses. According to the genetic characteristics and over-expression research of RCI genes, most RCI2genes are possibly involved in regulating stress tolerance, cell ion balance, stabilizing membrane structure and the like. However, the RCI2gene is sometimes up-regulated and sometimes down-regulated in expression when stressed. Salvia miltiorrhiza SmPMP3-1/-2 is up-regulated by both salt stress and ABA treatment during the seedling stage, SmPMP3-1 is up-regulated by low temperature and drought treatment, and SmPMP3-2 is down-regulated (Wang DH et al 2013.Molecular cloning and expression of two plasma membrane protein 3(SmPMP3) genes from Salvia milirathia (Molecular cloning and expression of two cytoplasmic membrane protein 3(SmPMP3) genes from Salvia miltiorrhiza Bunge.) Russian Journal of Plant Physiology 60: 155-. 164.). Corn has ten RCI2genes, the expression levels of which are all affected by drought stress, with ZmRCI2-8 being significantly down-regulated (Zhao Y et al 2014.Identification and characterization of the RCI2gene family in corn (Zea mays). Journal of Genetics 93: 655-666).

Corn is one of the most important dual-purpose crops for food and feed in China, and phosphorus plays a critical role in the early growth and development of corn, so that a large amount of phosphate fertilizer is required to be supplemented in places where corn is planted, and a large amount of manpower and material resources are consumed.

In addition, soil salinization is a worldwide resource and ecological problem, and China has saline-alkali area of 3.6 multiplied by 10⁷hm²The land area of the secondary salinization soil is still continuously enlarged due to improper irrigation, large application of chemical fertilizers and the like, so that the arable area is reduced year by year, and researches indicate that the arable area is reduced by 30% in the next 25 years, so that the soil salinization seriously restricts the agriculture and forestry production and ecological environment construction. The salinized soil contains sulfate, chloride, carbonate and bicarbonate of sodium, potassium, calcium, magnesium and the like and has high content, so the salinized soil has serious influence on corn seeds, influences on the early germination rate and germination index of the corn seeds, hinders the germination of the corn seeds, reduces the antioxidant activity and antioxidant substance content of the corn seeds, influences on intracellular redox potential and K⁺/Na⁺The ion balance causes damage to corn cells, and further causes extremely low germination rate of corn seeds, even no germination.

Therefore, it is highly desirable to provide a method for breeding corn that is tolerant to both low phosphorus stress and high salt stress.

Disclosure of Invention

The technical problems to be solved by the invention are the problems of poor plant growth under low-phosphorus conditions and low germination rate under high-salt environment, and the problems caused by excessive application of phosphate fertilizer or soil deterioration, in particular, and the problem of soil salinization derivation.

In order to solve the above problems, the present invention provides in a first aspect the ZmRCI2-8 gene having a nucleotide sequence shown in SEQ ID NO. 1.

In a second aspect, the invention provides a ZmRCI2-8 protein, which has an amino acid sequence as shown in SEQ ID NO. 2.

In a third aspect, the present invention provides the use of the gene of the first aspect of the present invention for increasing the tolerance of a plant to abiotic stress.

The gene of the invention can respond to various abiotic stresses, is named ZmRCI2-8 and is derived from corn (Zea mays L.) of Zea. ZmRCI2-8 has 2 exons, 1 intron, and contains a 231bp open reading frame, and the sequence of the frame is shown in SEQ ID NO. 1. The protein encoded by the gene has 76 amino acids, contains a UPF0057 conserved domain, has two hydrophobic transmembrane domains, belongs to an RCI2 family, and has an amino acid sequence shown in SEQ ID No. 2.

The increase of the number of lateral roots is found by the research on the ZmRCI2-8 transgenic plants under the condition of low phosphorus, which indicates that the ZmRCI2-8 gene has the influence on the root system configuration. The germination rate of the transgenic line is obviously improved compared with that of the wild type under the condition of salt stress, which shows that the ZmRCI2-8 gene participates in a salt stress regulation mechanism and can improve the tolerance of the transgenic line to the salt stress. Therefore, the invention provides more effective gene resources for efficient absorption and utilization of phosphorus of crops, plays an important role in research of improving the nutrition high-efficiency performance of plants and corresponding abiotic stress in genetic engineering, and is expected to improve the phosphorus efficiency of plants and cultivate phosphorus-efficient crops. In addition, the gene of the invention also participates in the regulation of nutrient absorption and photosynthesis or transpiration of leaves, is used for improving the accumulation of plant carbohydrates and improving the nutritional quality and yield of seeds.

Drawings

FIG. 1 is a schematic diagram of the gene structure of ZmRCI2-8, in maize ZmRCI2-8 located on chromosome 9 (Chr.9: 105271795 and 105597302bp) and comprising 2 exons (indicated by black boxes in E1 and E2) and 1 intron (indicated by a broken line between the two exons).

FIG. 2 is a schematic diagram of the transmembrane structure of ZmRCI2-8, in which the full-length cDNA of ZmRCI2-8 is 1067bp, the ORF (open reading frame) is located at 373 bp of its 143-type nucleotide, the full-length is 231bp, and 76 amino acids are encoded. The transmembrane structure of the protein is predicted by TMHMM2.0, and the result shows that the protein contains 2 transmembrane domains. Solid line 1 represents the probability of transmembrane structure; solid line 2 represents the intramembrane probability; solid line 3 represents the extracellular probability.

FIG. 3 is a maize ZmRCI2-8 promoter cis-acting element analysis, using PLACE (http:// www.dna.affrc.go.jp/PLACE/signalscan. html) database to analyze ZmRCI2-8 promoter cis-acting element, finding that ZmRCI2-8 promoter contains a series of plant adversity stress response elements including stress induction response element ABRELATED 1, phosphorus starvation response element P1BS, drought, low temperature and ABA response element MYC, gibberellin and cytokinin response element. Wherein the phosphorus starvation response element P1BS is present in most phosphorus starvation response genes, and two P1BS cis-elements are also present in the OsRCI2-8 promoter sequence induced by phosphorus deficiency, thereby presuming that ZmRCI2-8 is a phosphorus deficiency induced response gene.

FIG. 4 shows the evolutionary relationship between the maize ZmRCI2-8 protein and Arabidopsis AtRCI2(A-H) and rice RCI 2.

FIG. 5 shows the tissue expression characteristics of ZmRCI2-8 in the tasseling stage of maize inbred line B73 under normal nutritional conditions. Under the condition of sufficient field nutrients, vegetative organs (roots, joints, internodes and leaves) and reproductive organs (female ears and male ears) are harvested in the tasseling stage of a maize inbred line B73, and the expression levels of ZmRCI2-8 in the roots are taken as reference, so that the expression levels of ZmRCI2-8 in each tissue are sequentially leaves, joints, roots, internodes, male ears and female ears from high to low. The expression level of ZmRCI2-8 in leaves was 4.5 times higher than that of roots, and the level in nodes was 1.9 times higher than that of roots. The expression level of ZmRCI2-8 in the female ear and the male ear is only 10% of that in the root. The ZmRCI2-8 has higher expression in leaves and roots in the maize tasseling period. Wherein, the roots and the leaves have important function on the absorption or metabolism of nutrients and water, and the high and low expression level of ZmRCI2-8 influences the transpiration strength of the leaves and the absorption function of the roots.

FIG. 6 is the subcellular localization of the GFP-ZmRCI2-8 fusion protein, EGFP no-load fluorescence signal and ZmRCI2-8-EGFP fluorescence signal, respectively. As can be seen from the figure, ZmRCI2-8 is expressed in the plasma membrane, consistent with the results predicted by SignalP4.1Server and WoLF PSORT software.

FIG. 7 is the expression profile in ZmRCI2-8 roots (after 10 days of nitrogen, phosphorus, or potassium deficiency), and it can be seen that ZmRCI2-8 expression is strongly induced by low phosphorus only, significantly upregulated in both roots and aerial parts ten days after low nitrogen, low phosphorus, and low potassium treatment of maize B73. The relative expression level of ZmRCI2-8 in roots under low-phosphorus condition is up-regulated by 32.6 times compared with normal nutrition condition, and the expression level of ZmRCI2-8 has no significant difference under low nitrogen or low potassium.

FIG. 8 shows the expression characteristics of ZmRCI2-8 under the condition of aboveground nitrogen, phosphorus and potassium nutrient stress for 10d (after nitrogen, phosphorus or potassium deficiency for 10 d), and it can be seen that the relative expression level of ZmRCI2-8 in the aboveground part of the maize B73 inbred line is up-regulated by 31.2 times compared with normal nutrition, and the expression level of ZmRCI2-8 under the condition of low nitrogen or low potassium is not significantly different from that of normal nutrition. The results indicate that the ZmRCI2-8 gene is expressed in both maize roots and the above ground, and its expression is up-regulated by low phosphorus specific induction.

FIG. 9 is a phenotype analysis of ZmRCI2-8 overexpression lines (soil culture 12d Col-0 and the overall growth of overexpression lines L10, L12 and L16, scale bar of the figure is 1cm), transgenic lines are screened to T3 generation to obtain homozygous lines, T3 generation transgenic homozygous lines are soil cultured and the phenotype is observed. The Col-0 was used as a control to observe the phenotype of Arabidopsis transgenic lines at different growth stages. Through early-stage soil culture observation, the number of rosette leaves, the size of leaves, the leaf color, the leaf shape and the bolting time of the L10, the L12 and the L16 are not obviously different compared with Col-0.

FIG. 10 is a phenotype analysis of ZmRCI2-8 overexpression lines (the overall growth vigor of soil culture 27d Col-0 and overexpression lines L10, L12 and L16, the scale bar of the figure is 1cm), and it can be seen that after seedling transplantation, the plant height of the soil culture 27d, the transgenic lines L10, L12 and L16 has no obvious difference compared with Col-0, and the flowering time and the inflorescence size are basically consistent.

FIG. 11 is a phenotype analysis of ZmRCI2-8 overexpression lines (the overall growth vigor of the soil culture 47d Col-0 and the overexpression lines L10, L12 and L16, the scale bar of the figure is 1cm), and the plant heights of the transgenic lines L10, L12 and L16 are basically consistent with those of Col-0. The average plant height of ZmRCI2-8-10 is 17.5cm, the average plant height of L12 is 17.6cm, the average plant height of L16 is 17.1cm, and the average plant height of Col-0 is 17.9 cm. Compared with Col-0, the transgenic lines L10, L12 and L16 have no obvious difference in pod size and pod length. The average branch numbers of the whole plants of Col-0, L10, L12 and L16 are respectively 2.4, 2.3, 2.4 and 2.3, and the branch numbers of all the plants are similar and have no obvious difference. The transgenic line growth and development, flowering and pod bearing and Col-0 have no obvious difference under the normal nutrient soil culture condition, and the normal nutrient condition ZmRCI2-8 can be presumed to have no significant influence on the growth and development of Arabidopsis, but the expression of ZmRCI2-8 is strongly induced by low phosphorus.

FIG. 12 shows the length of the main root of the ZmRCI2-8 transgenic line after 7 days of phosphate-deficient culture (L10, L12 and L16 represent independent ZmRCI2-8 transgenic lines), and it can be seen that the length of the main root of the transgenic line is not different from that of the control Col-0 under normal nutrient conditions and phosphate-deficient conditions. After culturing for 7d under the condition of phosphorus deficiency, the length of the main root of the over-expression strain is not obviously different from that of Col-0.

FIG. 13 shows the number of visible side roots of the ZmRCI2-8 transgenic lines after phosphorus-deficient culture for 7 days (L10, L12 and L16 represent independent ZmRCI2-8 transgenic lines), and statistics on the number of visible side roots shows that the number of visible side roots of the three transgenic lines after phosphorus-deficient treatment is significantly different from that of Col-0. The increased number of lateral roots is a phenotype of plants responding to low phosphorus. It can be concluded that ZmRCI2-8 has a major effect on lateral roots, and that ZmRCI2-8 may affect phosphorus stress by regulating lateral root development.

FIG. 14 shows the germination rates of ZmRCI2-8 transgenic line and Col-0 treated with NaCl at different concentrations on the third day. L10, L12, L16 represent independent ZmRCI2-8 transgenic lines. "genes indicate significant differences between treatments (P < 0.05). Under the concentration of 120mmol/L NaCl, the germination rates of the transgenic lines L10, L12 and L16 are obviously higher than that of Col-0, under the concentration of 150mmol/L NaCl, the germination rates of the transgenic lines are greatly different, the germination rate of the transgenic line L12 is basically not obviously different from that of Col-0, but the germination rate of the line L16 is still obviously higher than that of Col-0. Therefore, the influence of ZmRCI2-8 on the salt stress tolerance of Arabidopsis can be presumed.

FIG. 15 is a schematic structural view of a vector pEZS-NL, in which "CaMV 35 promoter" represents CaMV35promoter and "ampicilin resistance" represents ampicillin resistance gene.

Detailed Description

As described above, the present invention provides in a first aspect the ZmRCI2-8 gene having a nucleotide sequence as shown in SEQ ID NO. 1.

In a second aspect, the invention provides a ZmRCI2-8 protein, the protein having an amino acid sequence as shown in SEQ ID No.2, wherein the amino acids at the 2 nd to 3 rd amino acid positions are all Ser (serine).

In a third aspect, the present invention provides the use of the gene of the first aspect of the present invention for increasing the tolerance of a plant to abiotic stress.

The inventor analyzes the sequence action element of the ZmRCI2-8 gene promoter by utilizing a PLACE database, and surprisingly discovers that MYC cis-element and PIBS cis-element exist in the gene promoter region, the core sequence of MYC is CANNTG, and the experiment proves that the gene promoter can be used for various stress-resistant gene promoters. The core sequence of the cis-element of P1BS is GNATATNC, and the experiment proves that the cis-element can be used for a phosphorus-induced gene promoter.

In some preferred embodiments, the abiotic stress is low phosphorus stress and/or high salt stress.

In some preferred embodiments, the low phosphorus stress is a phosphorus concentration in the planting substrate of no more than 2.000 x 10^-3mol/L, preferably 2.000X 10^-3mol/L to 5.000X 10^-9mol/L。

In some preferred embodiments, the high salt stress is a salt concentration in the planting substrate of not less than 120mmol/L, preferably 120mmol/L to 150 mmol/L.

In some preferred embodiments, the plant has improved root growth under conditions of low phosphorus stress; and/or the germination rate of seeds of the plant is improved under the condition of high salt stress. The ability of the gene to improve plant tolerance to low phosphorus stress may be achieved by improving phosphorus utilization and more effective regulation of phosphorus.

In some embodiments, the gene of claim 1 is transferred into a plant to increase the expression of the gene in the plant, for example, to increase the expression of the gene in the roots or leaves.

The inventor surprisingly finds that the gene not only can improve the low-phosphorus stress resistance and high-salt stress resistance of the plant, but also plays a role in nutrient absorption, photosynthesis, transpiration, carbohydrate accumulation, kernel nutritional quality, biomass, kernel yield and the like. Therefore, the use of the present invention may further include the use of the gene of the first aspect of the present invention in: (1) regulating and controlling nutrient absorption capacity, photosynthesis or transpiration function; (2) increased carbohydrate accumulation; (3) improving the nutritional quality of the grains; and/or (4) increasing biomass and/or grain yield.

The inventor discovers that ZmRCI2-8 is strongly induced by phosphorus deficiency in the overground part and the root of the corn through analyzing the quantitative PCR result, and the up-regulation times of the root are higher. The expression level analysis of each tissue ZmRCI2-8 of the corn one week after the tassel stage shows that the expression level of ZmRCI2-8 is leaf, node, root, internode, tassel and female ear from high to low, and the expression level in the leaf and the root is obviously more than that in other tissues. Therefore, ZmRCI2-8 is supposed to play a certain role in the photosynthesis of corn leaves or the water and nutrient transportation process.

In some preferred embodiments, the plant is a graminaceous plant or a leguminous plant, preferably maize (Zea mays L.) or arabidopsis thaliana.

The ZmRCI2-8 gene is subjected to subcellular localization, is found to be expressed in a plasma membrane and accords with the localization prediction of RCI2 family genes.

The following examples further illustrate the present invention but are not intended to limit the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified.

Preparation of the Material

1. Plant material, strain and carrier

Materials used in the present invention include: the corn (Zea mays L.) material is inbred line B73; the arabidopsis material is wild Columbia Col-0; escherichia coli DH 5. alpha. bacterium competent cell (Code No. CB101, Tiangen Biochemical technology Co., Ltd.); agrobacterium-infected cells GV3301 (available from Tiangen Biotechnology Ltd.); ZmRCI2-8 gene TA cloning vector

19-T vector (Code No. D102, Takara Co.); ZmRCI2-8 Arabidopsis thaliana overexpression vector pSuper1300+ -Kanamycin (North Noro Biotech, Inc. of Shanghai); ZmRCI2-8 transgenic onion epidermal cell subcellular localization vector pEZS-NL (see FIG. 15).

2. Biochemical reagent and kit

MS medium (PhytoTech Co.); PrimeSTAR HS DNApolymerase (TaKaRa Co.); taq enzyme, 2 × PCR mix (Beijing Virginia Oriental Biotechnology Co.); RaNA (bio-technology company, dawn, east); DNA markers, RNAseA (Beijing Tiangen Biotech Co.); t4DNA ligase, restriction enzyme, Sal-HF, BamH1-HF (NEB Corp.); oligo d (T)₁₈Reverse transcriptase, dnase, RNase inhibitors (Invitrigen); SYBE GREEN (quantitative PCR reagent, TAKARA Co.); antibiotics (BBI Co., Ltd.) such as kanamycin (100mg/mL), rifampicin (25mg/mL), ampicillin (100mg/mL), hygromycin (50 mg/mL); other chemical reagents are analytically pure reagents (Beijing chemical reagent factory); a common agarose gel DNA recovery kit and a plasmid miniprep kit (Beijing Tiangen Biotech Co.); trizol kit (Invitrigen corporation); all other chemical reagents were analytical reagents (Beijing chemical reagent factory).

3. PCR amplification primer

4. Culture medium formula

(1) Corn nutrient solution formula

Note: the components of each nutrient solution are shown in the table, and the pH is adjusted to 6.0 by using 1mol/L NaOH.

(2)1/2MS Medium (100 mL solids): MS 0.217g, sucrose 3g, agar powder 0.8g, adjusting pH to 5.8 with 1mol/L KOH, and sterilizing at 121 deg.C for 20min (min).

(3) Arabidopsis phosphorus-deficient medium (solid)

Note: the components of each nutrient solution are shown in the table, 1% of sucrose is added, the pH is adjusted to 5.8 +/-0.1 by using 1mol/L KOH, and then 1% of agar is added.

(4) Arabidopsis salt stress medium (solid): different concentrations of NaCl solution (0mmol/L, 120mmol/L, 150mmol/L) were added to 1/2MS medium.

(5) LB medium (100 mL solids): 1g of tryptone, 0.5g of yeast extract, 1g of NaCl and 1.5g of agar powder, and sterilizing at 121 ℃ for 20 min.

(6) Liquid culture medium: the formula of the medium is the same as that of 1/2MS or LB solid medium except that no agar powder is added.

(7) Screening a culture medium: 1/2 sterilizing MS or LB solid culture medium under high pressure, adding antibiotic when the temperature is reduced to about 50 deg.C, shaking, and spreading on a flat plate.

Example 1 method for culturing test Material

1. Arabidopsis culture and transgenic seedling screening

Vernalization of seeds: an appropriate amount of arabidopsis thaliana seeds are taken into a 1.5mL centrifuge tube, all the seeds are soaked by deionized water, and the arabidopsis thaliana seeds are placed in a refrigerator at 4 ℃ for vernalization for 2 days.

Seed disinfection: adding 75% ethanol into 1.5mL centrifuge tube containing seeds, sterilizing for 1min, washing with sterilized water, adding 2% sodium hypochlorite, sterilizing for 2min, and washing with sterilized water for 5-7 times.

Dibbling and culturing: the seeds were spotted evenly on 1/2MS +50mg/mL hygromycin medium using a 10 culture pipette and the seeded medium was placed in a culture chamber (photoperiod: 16h day/8 h night, temperature 22/20 ℃ C., humidity 100%) for culture. Transplanting the seedlings into a flowerpot, filling sterilized nutrient soil (the volume ratio of vermiculite to nutrient soil is 1:1) into the flowerpot, covering a layer of preservative film on the surface of the flowerpot after transplanting, pouring 400mL of water every 5 days, and uncovering after 10 days. After the preservative film is removed, 400mL of water is poured every 3 days.

Screening transgenic seedlings: vernalization and disinfection of T0 generation Arabidopsis seeds, and uniform seeding of seeds on 1/2MS +50mg/mL hygromycin medium. Wrapping a layer of black plastic film outside the culture dish, placing the culture dish in a culture room for 5-7 days in the dark, taking out high seedlings from seedling stems as transgenic seedlings with resistance, selecting 13 seedlings from each plant line in the transgenic plants with resistance, numbering, transferring to 1/2MS culture medium for 3-5 days, transferring to nutrient soil for culture after 4 young leaves grow out, and finishing the whole growth period. Harvesting seeds and continuously screening until T3 generation to obtain homozygous transgenic lines.

2. Corn culture and sample obtaining method

Corn water culture: selecting semen Maydis with plump and substantially consistent size, washing with deionized water for 3-5 times, and adding 10% H₂O₂Soaking for 30min, washing with deionized water, and saturating with CaSO₄Soaking in the solution for 5-6 hr, transferring to a tray paved with filter paper, wetting the filter paper with deionized water, placing endosperm face down on the filter paper, paving the wetted filter paper on the upper layer, and covering with black plasticAnd (5) carrying out tray and light-shielding germination acceleration. Spraying water once every morning, noon and evening to keep the filter paper moist. And when the buds grow to 1-2cm, rolling the seedlings by using a wet filter paper, vertically placing the rolled seedlings into a water culture tank containing deionized water, and performing covering culture by using black plastic. When the cotyledon emerged, the black plastic was removed and given appropriate light. When the corn is long enough to have two leaves and one heart, seedlings with regular seedlings are selected, the endosperm of the seedlings is removed, and the seedlings are transplanted into each treatment nutrient solution to grow.

Sampling: and (3) treating the low-nitrogen, low-phosphorus and low-potassium expression characteristic sample and a corn inbred line B73 contrast, low-nitrogen, low-phosphorus and low-potassium nutrient solution, and sampling for 1 time, wherein the sampling part is the overground part and the root of the transplanted 10d seedling. The tissue expression characteristic sample is taken 1 time in the male-drawing period of the maize inbred line B73 under the field condition, and the sampling parts are roots, nodes, internodes, female ears, leaves and male ears. The sample is taken from a maize inbred line B73 variety under field conditions. The collected samples were rapidly frozen with liquid nitrogen and stored in a-80 ℃ freezer.

Example 2 cloning of target Gene fragments

1. Target gene reaction system

Reaction procedure: at 98 deg.C for 5min (98 deg.C, 30s, 60 deg.C, 30s, 72 deg.C, 20s) for 30 cycles (cycle), 72 deg.C, 10 min. And after the reaction is finished, performing 1% agar gel electrophoresis on the reaction solution, then performing gel recovery on the product obtained by amplification, and performing gel recovery according to a Tiangen centrifugal column kit gel in the gel recovery step.

2. Construction of recombinant plasmid vector

Add Poly A tail, 200. mu.L EP tube, 10. mu.L system: DNA 7. mu. L, Taq buffer 1. mu.L, Taq dNase 1. mu. L, dNTPs 1. mu.L. After mixing, incubation was carried out at 72 ℃ for 30 min. T vector

19-T Vector connection, 200. mu.L EP tube, 5.0. mu.L system: DNA 2. mu. L, T vector 0.5. mu.L, solution I2.5. mu.L.

The connection was carried out at room temperature for about 3 hours. The carrier of the purpose is connected with the carrier,and (3) carrying out gel recovery after the connection T-loading sequencing is correct, constructing recombinant plasmid, and connecting for 3h at normal temperature. Reaction system: 10 Xligase buffer 0.5. mu.L, target gene a, vector (expression vector) b, T4DNA ligase 0.5. mu.L, sterilized ddH₂Make up to 5. mu.L of O.

3. Target gene detection and recovery

The ligation products are transformed into escherichia coli competent cells DH5 alpha, then spread on LB + antibiotic culture medium plates, and placed in a 37 ℃ constant temperature incubator for 12-16h in an inverted mode. A single colony was picked up as cDNA on a clean bench and subjected to PCR amplification. And adding the positive clone bacterial liquid into an LB + antibiotic liquid culture medium for activation for 12 hours. Sucking 1-1.5mL of bacterial liquid, and extracting plasmids according to the instructions of a small Tiangen plasmid extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.). Carrying out enzyme digestion inspection/gel recovery, wherein 10 mu L of reaction system is used for enzyme digestion inspection, and the bacterial liquid which is successfully inspected is sent to be sequenced; the digestion fragment was recovered by using 50. mu.L of the reaction system and recovering the target fragment. The reaction system is as follows:

10 XNEB buffer	1μL	5μL
			DNA	≤0.5μg	≤2μg
100×BSA	0.1μL	0.5μL
			Endonuclease
1	0.2μL	1μL
			Endonuclease
2	0.2μL	1μL
			In all	10μL	50μL

Example 3 extraction and testing of plant DNA and cDNA

1. Extraction of arabidopsis T0 generation leaf DNA by CTAB method

0.5g of fresh plant leaves are taken and placed in a mortar, and a proper amount of liquid nitrogen is added, and the mixture is quickly ground into uniform powder. The powder was transferred to a 1.5mL centrifuge tube, 800. mu.L CTAB separation buffer was added, the tube was inverted upside down, and mixed well. Placing the centrifuge tube containing the sample in a water bath at 65 deg.C, keeping the temperature for 30min, and shaking gently every 3-4 min. Centrifuge at 12000rpm for 5min and transfer the upper aqueous phase to a new centrifuge tube. Adding equal volume of Tris-phenol and chloroform isoamylol, turning the centrifuge tube upside down, and mixing uniformly; centrifuging at 12000rpm for 5min, and transferring the upper aqueous phase into a new centrifuge tube. Adding chloroform isoamyl alcohol with the same volume into the centrifuge tube, and mixing uniformly; centrifuging at 12000rpm for 5min, and transferring the upper aqueous phase into a new centrifuge tube. Adding 0.6 times volume of isopropanol, mixing, and precipitating at-20 deg.C for 30 min. Centrifuging at 12000rpm for 5min at low temperature to remove supernatant, adding 500 μ L70% ethanol, washing the precipitate at normal temperature at 12000rpm for 2min, removing supernatant, and drying the precipitate as much as possible. The precipitate was dissolved by adding an appropriate amount of TE (20. mu.L) containing RNase, and the RNA was digested by a water bath at 37 ℃ for 30 min. Taking a proper amount of genome DNA sample, and carrying out electrophoresis detection.

2. Trizol method for extracting corn total RNA

Grinding and sample preparation: a small amount of liquid nitrogen is added into the mortar, and the mortar is precooled when the liquid nitrogen is volatilized. Then, a proper amount of liquid nitrogen is poured into the mortar. The tissue was removed from the freezer at-70 ℃ and a small piece was removed from the vial with forceps and placed in a mortar. The tissue mass was rapidly ground and 1ml trizol was added as a powder, and the grinding was continued, where the powder was seen to be slurried, diluted from concentrate until the tissue sample was completely dissolved, and pipetted into an enzyme-free EP tube.

Extracting RNA by a Trizol method: standing at room temperature for 3-10min to denature protein; adding 1/4-1/5 volume of chloroform, shaking for 1min by reversing, standing at room temperature for 3-5min, standing for layering, and centrifuging at 4 deg.C at 12000rpm for 5 min. The supernatant was pipetted into an enzyme-free EP tube and an equal volume of phenol was added: and (3) shaking chloroform for 1min by reversing, standing at room temperature for 3-5min, standing for layering, then centrifuging at 4 ℃, and taking supernatant at 12000rpm for 5 min. If the cell RNA extraction is carried out, the chloroform extraction can be carried out only once in equal volume, but the steps are generally repeated for tissues until no protein layer exists, and the extraction is generally carried out twice. Adding equal volume of precooled isopropanol, reversing and mixing evenly, standing on ice for 30min, then centrifuging at 12000rpm at 4 ℃ for 30min, and discarding the supernatant. 100ul of 70% ethanol was added and the pellet was blown up, taking care not to blow it off, and then 4 ℃ 12000rpm for 5 min. The supernatant was discarded and ethanol evaporated in the air until no droplets were on the wall. An appropriate amount of DEPC water (typically 20. mu.L) was added and dissolved. Detecting the RNA concentration and OD value by a spectrophotometer, and diluting Total RNA to 1 mu g/mu L for later use.

3. Plant total RNA purification

Putting 5-10g of sample into a mortar, adding excessive liquid nitrogen, and quickly grinding into uniform powder (in the grinding process, ensuring that the sample is always immersed in the liquid nitrogen and is ground for about 30 min). After the liquid nitrogen naturally volatilizes completely, transferring the sample into a centrifuge tube of 100ml, adding 6-8 times of the volume of the lysate 1, stirring slightly, and carrying out ice bath at 0 ℃ for 20 min. Centrifuge at 12000rpm at 4 ℃ for 15 min. And (3) adding 1/3 volumes of 3mol/L sodium acetate and 1/5 volumes of chloroform isoamyl alcohol into the supernatant, gently shaking the mixture, and carrying out ice bath at 0 ℃ for 20 min. Centrifuge at 2000rpm at 4 ℃ for 15min (add 5 volumes of lysate 2 to dissolve the pellet to extract DNA). The supernatant was taken, added with iso-propanol cooled in an equal volume of ice bath, mixed well and ice-cooled for 10 min. The precipitate was centrifuged at 5000rpm at 4 ℃ for 10min, dissolved in 5ml of TE buffer containing 0.1% SDS, and 8mol/L LiCl was added to bring the final concentration to 2.5mol/L, and the mixture was ice-cooled for 3h or overnight at 4 ℃. Centrifuge at 12000rpm at 4 ℃ for 10 min. Washing the precipitate twice with 70% ethanol, air drying for 10min, dissolving in sterile water (DEPC diethyl pyrocarbonate, treatment), adding 1/10 vol 10% SDS, repeating steps 4-6, further removing protein, and storing with 70% ethanol at 20 deg.C for a long period.

4. RNA reverse transcription: the reaction system is as follows: DNTP mix (10 mmol/reaction each) 1. mu.L, Oligo Dt primer (Oligo thymine primer) (2.5. mu.M) 1. mu.L, total RNA 5. mu.L, using ddH₂Make up to 10. mu.L of O. Reaction procedure: 65 deg.C for 5 min.

Example 4 transgenic plant construction

1. Preparation of Agrobacterium-infected competent cells

A small amount of Agrobacterium LBA4404 was picked and inoculated into 5ml LB liquid medium (containing 50mg/L STR), cultured overnight at 28 ℃ and 200 rpm. 2ml of the culture was taken and cultured in LB liquid medium (containing 50mg/L STR) until OD800 was about 0.5. The culture was placed in an ice bath for 30min, centrifuged at 4 ℃ and 5000rpm for 5min, and the supernatant was discarded. The bacterial suspension was suspended in 10ml of cold 0.1mol/L NaCl. Centrifuging at 4 deg.C and 5000rpm for 5min, and discarding supernatant. With 1ml of cold CaCl₂(20mmol/L) were suspended, split-packed into 50. mu.L/tube, frozen in liquid nitrogen and stored at-80 ℃.

2. Agrobacterium electrotransformation

20 μ L of Agrobacterium and 0.5 μ L of plasmid were mixed; adding into a 4mm electric conversion cup by a gun, covering the electric conversion cup, and labeling; electric shock is carried out on the electric shock device at 2500V/6ms, so that plasmids enter agrobacterium; after electric shock, the liquid was transferred to a 2mL centrifuge tube containing 500. mu.L of LB medium by a gun; writing a label, and sealing by using a paraffin film; shake culturing in 28 deg.C biochemical incubator for about 4h to allow bacteria to recover under aerobic condition; centrifuging the centrifugal tube for 1min to make the thallus settle on the tube wall; preparing an LB flat plate, writing a label, wiping a triangular glass rod with alcohol, then burning on flame for about 10s for sterilization, and standing and cooling for later use; sucking 100 μ L of supernatant with a gun, pouring out the residual liquid, pumping the liquid in the gun into a centrifuge tube, repeatedly sucking with the gun, and flushing the bacteria on the wall to mix uniformly; sucking out the bacterial liquid, dripping the bacterial liquid on a flat plate, and uniformly coating the bacterial liquid by using a glass rod; covering, sealing with parafilm, and placing into 28 deg.C biochemical incubator for 48h (2 days) to obtain bacterial colony.

3. Arabidopsis thaliana dipping transformation

Marking lines on corresponding resistant plates of agrobacterium strains with expression vectors stored at low temperature, selecting a single colony, inoculating the single colony in 5ml of liquid LB culture medium added with corresponding antibiotics, and carrying out shaking culture at 250rpm at 28 ℃ for 18-24 h; then inoculating and expanding culture under the same condition according to the ratio of 1:100, wherein the volume of the total bacterial liquid is 200ml until the OD600 value is in the range of 0.8-1.0; centrifuging, precipitating, removing supernatant, resuspending the thallus with 5% sucrose solution with the same volume, adding 0.02-0.04% Silwet L-77 into the bacterial solution, and mixing; dipping the inflorescence of the arabidopsis into the bacterial liquid for 0.5-1min (before transformation, the horn and the fruit of the arabidopsis are cut off); then placing the arabidopsis seedlings in the dark under a humid condition for 16h, and repeatedly infecting the arabidopsis seedlings once after one week; after harvesting, progeny were planted and either sprayed with 54mg/ml herbicide onto 12d seedlings or the seeds were sown on resistant MS plates to screen for positive plants.

Example 5 Gene gun manipulation and confocal microscopy

1. Materials: inner layer skin of onion

2. Reagent: 2.5mol/L CaCl₂0.1mol/L spermidine, absolute ethanol

3. Early preparation: the inner layer of the onion is torn to about 2X 2cm, and the torn onion epidermis is placed on the prepared 1/2MS culture medium and cultured in the dark for 12 h.

4. Preparing a DNA-gold powder complex: preparing 2.5mol/L CaCl₂And 0.1mol/L spermidine 1mL each. The gold powder was vortexed and shaken for 5min to be suspended in sterile water. Draw 8.5. mu.L of the gold powder suspension into a 1.5mL EP tube and add 5. mu.g of plasmid DNA, 17. mu.L of CaCl in that order₂And 8. mu.L spermidine. Shaking for 5min, standing on ice for 10min, centrifuging at 3000rpm for 1min, and discarding the supernatant. Adding 100 μ L of anhydrous ethanol, washing without destroying precipitate, discarding supernatant, and repeating for 2 times. Adding 10 mu L of absolute ethyl alcohol for later use.

5. A gene gun step: and (3) uniformly coating 10 mu L of the prepared DNA-gold powder complex at the middle aperture of the bombardment membrane, placing a pressure membrane above the gene gun to bombard materials in the aperture, and opening the door to take out the materials when the pressure in the cabin is reduced to 0.

6. Culturing: after bombardment, placing onion epidermis in 1/2MS culture medium, culturing in dark at 28-30 deg.C for 22-24h, and observing with confocal microscope.

Results

The invention takes a maize inbred line B73 as a material, and uses molecular biology technology to preliminarily research the sequence characteristics, the evolution characteristics and the expression characteristics of ZmRCI2-8, the expression parts in cells and the action in the plant growth and development process, and the main results are as follows:

(1) ZmRCI2-8 contains the UPF0057 conserved domain, two transmembrane domains, and belongs to the RCI2 family (see FIGS. 2 and 4).

(2) As a result of analyzing the quantitative PCR, ZmRCI2-8 was strongly induced by phosphorus deficiency in both aerial parts and roots of maize, and the up-regulation of roots was high. The expression level analysis of each tissue ZmRCI2-8 of the corn one week after the tassel stage shows that the expression level of ZmRCI2-8 is leaf, node, root, internode, tassel and female ear from high to low, and the expression level in the leaf and the root is obviously more than that in other tissues. It is presumed that the ZmRCI2-8 gene may affect photosynthesis of leaves and establishment of root system and nutrient absorption (see FIG. 5).

(3) The subcellular localization of ZmRCI2-8 was performed and found to be expressed at the plasma membrane, consistent with the prediction of localization of RCI2 family genes (see FIG. 6).

(4) Under low phosphorus conditions, the lateral roots of the transgenic lines were significantly increased, indicating that the ZmRCI2-8 gene responded to phosphorus starvation induction (see FIGS. 7, 8, 11, 13).

(5) The germination rate of the transgenic line under the salt stress condition is obviously improved compared with that of the wild type, which shows that the ZmRCI2-8 gene participates in a salt stress regulation mechanism and can improve the tolerance of the transgenic line to the salt stress (see figure 14).

(6) The transgenic plants were cultured under normal conditions and showed no significant difference in overall growth vigor such as rosette leaf number, leaf size, leaf color, leaf shape, bolting time, and main root from the control plants (see FIGS. 9, 10 and 12).

Discussion of the related Art

Through sequence analysis and prediction of transmembrane structure, the ZmRCI2-8 gene is found to have 2 exons and 1 intron, comprises a 231bp open reading frame, encodes 76 amino acids, comprises two hydrophobic transmembrane domains and belongs to RCI2 family.

The expression level of ZmRCI2-8 is obviously up-regulated after low phosphorus stress, and the up-regulation multiple of roots is slightly higher than that of overground parts, which indicates that ZmRCI2-8 is strongly induced by phosphorus deficiency at the transcription level. In each tissue of the maize B73 in the tasseling period, the expression level of ZmRCI2-8 in a nutritive organ is higher, wherein the expression level of leaves is higher than that of other tissues, which shows that ZmRCI2-8 also plays a certain role in the photosynthesis of maize leaves or the transportation process of water and nutrients.

The transgenic arabidopsis line is obtained by using an agrobacterium flower dipping transformation method, the phenotype of the transgenic line is basically consistent with that of a wild type under the condition of normal nutrient soil culture, and the transgenic line has no obvious difference in different growth and development stages such as bolting time, flowering stage, aging degree and the like. This result indicates that the normal growth of the transgenic plants is not adversely affected.

Analyzing the promoter cis-element of the ZmRCI2-8 gene to find that MYC cis-element and PIBS cis-element exist in the promoter region, the core sequence of MYC is CANNTG, and the promoter can be used for various stress-resistant gene promoters. The core sequence of the cis-element of P1BS is GNATATNC, and can be used for inducing gene promoter by phosphorus. In order to investigate whether MYC cis-element and PIBS cis-element in ZmRCI2-8 gene promoter induce expression to salt damage and phosphorus starvation, a low-phosphorus and salt damage treatment test is carried out. As can be seen from the low-phosphorus treatment, the number of lateral roots of the ZmRCI2-8 transgenic plants is increased under the low-phosphorus condition, which indicates that the ZmRCI2-8 gene has an influence on the root system configuration. As shown by the results of salt stress treatment experiments, ZmRCI2-8 also improves the tolerance of plants to salt stress.

Sequence listing

<110> university of agriculture in China

<120> ZmRCI2-8 gene and application thereof in promoting plant germination and lateral root growth under abiotic stress conditions

<160> 12

<170> PatentIn version 3.5

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<211> 1067

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tgtgtggcct cggaggtccg ggatgagctc cggcggctgc tcgacgtgcc tcgaggtcat 180

cttcgccgcc atcctcccgc cgctcggcgt cttcttccgc tacggctgct gcagctccga 240

gttcttcgtc tccctcctcc tgacgctgct gtgctacgtc cccggcgtcg cgtactccct 300

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ctcaggacat tcctgactgt gttctccagc gtcacgtacg tccctgttcc tcgtcgcgtg 540

tgatgactgt gcaattgttg gtcattggtt tgctgattct ttcgatcagt ttatcacgaa 600

cagtagggtg ctgccgtgct ggactgctgc tcgtctgttt gccgatcacg cagcagttgg 660

tggtaattgg caactgatat aaaatttatg cgtgtactca tgtataaatc ccaaccagta 720

atgcggttgg aattaaaaga aaggtattgt ttggatatgg tttacggcta cgctattccc 780

actaaggtct tgttcggtta atcccattac ccatggattg tacgggattg gaaaaattta 840

agaagaagtt tgacttgctt gggattcaaa cccatccaat cccactcaat ccacatggat 900

tgggagctaa ccgaacaagc cctaataatg atatttgtag acaccgttag gtcttcccat 960

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Met Ser Ser Gly Gly Cys Ser Thr Cys Leu Glu Val Ile Phe Ala Ala

1 5 10 15

Ile Leu Pro Pro Leu Gly Val Phe Phe Arg Tyr Gly Cys Cys Ser Ser

20 25 30

Glu Phe Phe Val Ser Leu Leu Leu Thr Leu Leu Cys Tyr Val Pro Gly

35 40 45

Val Ala Tyr Ser Leu Tyr Val Ile Leu Arg Thr Pro Pro Glu Pro Pro

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<213> Artificial sequence

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gggcccatga gctccggcgg ctgctc 26

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actagttcag gcgagcatgt cgtagggc 28

<210> 5

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<212> DNA

<213> Artificial sequence

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cgaggtcatc ttcgccgcca t 21

<210> 6

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<212> DNA

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ccgctccccg tcgatgcc 18

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gtcgacatga gctccggcgg ctgctc 26

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ggatccgggg cgagcatgtc gtaggg 26

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taagctgccg atgtgcctgc gtcg 24

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ctgaaagaca gaacataatg agcacag 27

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Claims (5)

1. The application of ZmRCI2-8 gene in promoting the growth of roots of plants under low-phosphorus stress condition or increasing the germination rate of seeds of plants under high-salt stress condition, wherein the nucleotide sequence of the ZmRCI2-8 gene is shown as SEQ ID No. 1; the plant is corn or arabidopsis;

   the use is achieved by transferring the ZmRCI2-8 gene into the plant to increase the expression of the ZmRCI2-8 gene in the plant.
2. 2. The use of claim 1, wherein the low phosphorus stress is a phosphorus concentration in the planting substrate of no more than 2.000 x 10^-3mol/L。
3. 3. The use of claim 1, wherein the low phosphorus stress is a phosphorus concentration of 2.000 x 10 in the planting substrate^-3mol/L to 5.000X 10^-9mol/L。
4. 4. The use according to claim 1, wherein the high salt stress is a salt concentration in the planting substrate of not less than 120 mmol/L.
5. 5. The use according to claim 1, wherein the high salt stress is a salt concentration in the planting substrate of 120 to 150 mmol/L.

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