CN104711260B - Recover the promoter Y8A for the special induced expression of nitrogen and application after paddy rice nitrogen stress - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering. In particular, it relates to a specific inducible expression promoter Y8A for restoring nitrogen supply after nitrogen deficiency in rice and its application. Using microarray technology, the information on nitrogen-supply-induced expression of promoter Y8A downstream genes was obtained, and the nitrogen-supply-induced expression patterns of Y8A downstream genes were verified by different rice varieties. The full length of the promoter Y8A and its truncated fragment Y8B at the 5' end were amplified by PCR method, and connected to the vector DX2181b to construct a vector expressing the fusion of the promoter and GUS. Then the constructed vector was transformed into the rice variety Zhonghua 11, and the regulation of the promoter on the expression of the GUS gene was verified in the transformed positive plants. Through the Realtime expression verification, it was further confirmed that Y8A and Y8B belonged to the specific induction of nitrogen supply recovery after nitrogen deficiency. The expressed promoter has a functional segment at 982bp upstream of ATG. The invention provides a new promoter resource for rice.

Description

Promoter Y8A and its application to restore nitrogen-supply-specific inducible expression after nitrogen deficiency in rice

technical field

The invention relates to the technical field of plant genetic engineering. In particular, it relates to a promoter Y8A that specifically induces the expression of nitrogen supply after nitrogen deficiency in rice and its application

Background technique

At present, transgenic research mainly uses constitutive promoters to drive the expression of target genes, the most typical is the CaMV35S promoter isolated from cauliflower mosaic virus (Hirt et al., 1990; Battraw et al., 1990), and some plant sources in recent years promoters, such as the promoter of the actin gene Actin1 cloned from rice and the ubiquitin gene promoter cloned from maize (Schledzewski et al., 1994). However, the constitutive expression of exogenous genes often causes unnecessary waste of resources, and the accumulation of a large number of heterologous proteins will also break the original metabolic balance of plants and hinder the normal growth of plants (Nie Lina et al., 2008; Rai et al., 2009). Kasuga et al. (1999) found in their research that when the strong constitutive expression promoter CaMV35S is used to promote the expression of DREB1A, it may hinder the growth of plants to some extent, while the use of an inducible promoter can alleviate this effect. situation. The inducible promoter can quickly and effectively induce the "on and off" of the transcribed gene: according to the needs of the specific developmental stage of the plant, the tissue organ or the growth environment, the induction signal can be induced to induce gene expression, and the stress can also be relieved at any time to stop the expression (Li Jie et al., 2006). Therefore, obtaining a promoter that induces expression in rice under nitrogen starvation can not only reduce the negative impact of massive expression of exogenous genes, but also provide safe and effective regulation for genetic engineering improvement of rice.

Among the various macroelements required for plant growth, nitrogen is the primary factor that limits plant growth and forms plant yield. Nitrogen is not only the basis of genetic material, but also an important component of proteins, nucleic acids, chlorophyll, enzymes, vitamins, alkaloids and plant hormones (Lu Jingling, 1994). For nearly half a century, countries all over the world have regarded increasing nitrogen fertilizer as an important agricultural measure to increase rice production. Especially after the first green revolution, with the popularization and application of high-yield and fertilizer-tolerant varieties, the amount of nitrogen fertilizer application in farmland has increased rapidly, greatly improving Increased the yield of rice (Zhong Daibin et al., 2001). The excessive application of nitrogen fertilizer in farmland is followed by the reduction of nitrogen use efficiency and a series of environmental problems. The massive loss of nitrogen directly leads to groundwater pollution and eutrophication of rivers and lakes (Wang Guanghuo et al., 2003).

With the rapid development of molecular biology and genetic engineering in the field of plants, the molecular mechanism of nitrogen uptake and transport in plants is becoming more and more clear. A large number of ammonium salt transporters and nitrate transporters were discovered, and the mechanism of GS/GOGAT (glutamate amine synthase/glutamate synthase) cycle assimilation of ammonium was revealed, providing a large amount of resources for genetic engineering to improve rice nitrogen uptake and utilization. research material. Although the absorption and transport functions of many genes have been verified by mutants or absorption experiments, the overexpression of these genes in rice has not achieved the purpose of improving the absorption and transport of ammonium. Mohammad et al. (2006) overexpressed the ammonium transporter OsAMT1-1 in rice with the maize ubiquitin promoter, which improved the ammonium absorption capacity in rice and the total ammonium content in roots, but at the same time produced ammonium poisoning, resulting in Biomass is greatly reduced. If it is driven by a nitrogen deficiency-induced promoter, it is possible to properly control the absorption of ammonium according to the needs of the plant and avoid poisoning. GS is the main enzyme involved in the synthesis of glutamine (Gln) by NH4 ⁺ assimilation. Cai et al. (2009) used the 35S promoter to overexpress rice glutamine synthetase genes GS1;1, GS1;2 and Escherichia coli glutamine synthetase gene glnA, GS activity, total nitrogen content, amino acid content, soluble protein, etc. have all been significantly improved, but the biological yield and grain yield per plant have been significantly decreased. If it is driven by a nitrogen-supply-induced promoter, it can not only reduce the "waste" caused by constitutive expression, but also ensure that the assimilation of GS is only promoted under the condition of sufficient nitrogen supply, avoiding the biological yield of a single plant due to insufficient nitrogen source reduce.

The present invention excavated a nitrogen-supply-induced gene expression promoter through chips, verified it in three different varieties by realtime quantitative realtime PCR, and further confirmed that the promoter was a nitrogen-supply-induced promoter through stable transformation of rice. It provides a new material for the genetic engineering improvement of rice's absorption and utilization of nitrogen.

Contents of the invention

The purpose of the present invention is to overcome the existing technical defects and provide a promoter for restoring nitrogen-supply-specific inducible expression after nitrogen deficiency in rice and its application. The promoter is Y8A and its truncated fragment Y8B. The use of the promoter in rice for nitrogen-supply-induced gene expression is as follows: chip results show that Nipponbare and Zhenshan 97 start when nitrogen is supplied after 7 days of nitrogen deficiency stress. The downstream genes controlled by sub-Y8A were all induced to express about 20 times in roots, but not in shoot tissues. In the other four conventional rice varieties: Zhonghua 11, Huajing 295, 9311, and MH63, it was found that the promoter Y8A controlled Downstream genes can also be induced to express more than 20 times after nitrogen deficiency for 7 days and then nitrogen supply for 2 hours. Amplify the full-length promoter Y8A sequence from Zhonghua 11 (or ZH11) materials and fuse the GUS gene to stably transform rice, and construct a series of 5'-end deletion vectors, and treat the transgenic positive materials with nitrogen deficiency stress and nitrogen supply. Before and after the GUS gene expression in the root was significantly induced expression. Further verified the nitrogen-inducible expression of the promoter Y8A, and the functional segment was within 982bp upstream of ATG.

The present invention is achieved like this:

The present invention is based on the rice variety Nipponbare (English name: Nipponbare, a well-known and public rice variety material, and a rice variety that has completed genome sequencing) and Zhenshan 97 (a well-known and public rice variety, from Jiangxi Academy of Agricultural Sciences) Materials, from hydroponic seedlings to the 5-leaf stage, samples were taken under nitrogen deficiency stress at different time points (nitrogen deficiency for 1 hour, nitrogen deficiency for 1 day, nitrogen deficiency for 3 days, nitrogen deficiency for 7 days, nitrogen deficiency for 7 days, nitrogen supply for 2 hours, and nitrogen supply for 1 day) , with the seedlings cultured in continuous normal plant cultivation nutrient solution (the conventionally reported plant cultivation nutrient solution) as a control, using the whole genome cDNA chip expression profile as a technical means (Yang Rong et al., 1999), obtained a comparison between the two materials According to the information of these genes, the upstream sequence of the gene, namely its promoter sequence, was obtained from the NCBI website (www.ncbi.nlm.nih.gov) after 2 hours of nitrogen supply in the root. At the same time, the applicant followed the same procedure Treatment methods Four other conventional varieties: Zhonghua 11, Huajing 295, 9311, and MH63 were treated, and the nitrogen-induced expression pattern of the gene was verified by realtime PCR. The promoter sequence was fused with the GUS reporter gene to construct the promoter vector DX2181b (the vector DX2181b was transformed by the researchers of the applicant's State Key Laboratory of Crop Genetics on the basis of the commercial vector pCAMBIA1381, a plasmid publicly reported and used in Australia, See Fig. 2), and then carry out Agrobacterium-mediated genetic transformation to transform the rice variety Zhonghua 11, and obtain positive transformed plants. Detecting the expression of GUS gene in the transformed positive plants found that the expression of GUS gene after nitrogen deficiency for 7 days increased significantly after nitrogen supply for 2 hours.

The advantages of the present invention are:

(1) The present invention provides an application of a nitrogen supply-induced gene expression promoter in rice. The expression level of genes downstream of the promoter was verified in six different cultivars after nitrogen deficiency treatment, and the expression pattern induced by high-fold nitrogen supply was restored. In positive rice plants stably transformed with promoter fusion GUS gene, the expression of GUS increased significantly after nitrogen treatment.

(2) The present invention discovered for the first time that the promoter Y8A is a promoter widely induced by nitrogen supply and high-magnification gene expression in various rice varieties, and the functional segment is within 982bp upstream of ATG, which is how to effectively control nitrogen in genetic engineering improved rice The assimilation of absorption provides new materials.

(3) The promoter and its downstream genes used in the present invention can provide support for the research on nutritional metabolism of cereal crops such as rice and other crops.

Description of drawings

SEQ ID NO: 1 is the nucleotide sequence of the promoter Y8A, and the full length of the sequence is 1936bp.

SEQ ID NO: 2 is the nucleotide sequence of one of the truncated promoter segments Y8B of the promoter Y8A, and the full length of the sequence is 982bp.

SEQ ID NO: 3 is the sequence of the CDS region of the promoter Y8A, the full length of the sequence is 414bp, encoding 137 amino acids.

SEQ ID NO: 4 is the sequence of the protein of the promoter Y8A.

Fig. 1. is the overall technical roadmap of the present invention.

Figure 2 is a schematic diagram of the structure of the vector DX2181b. Figure 2a is a structural diagram of the vector pCAMBIA1381 before DX2181b transformation; Figure 2b is a schematic structural diagram of the vector DX2181b; Figure 2c is the structure of the multiple cloning site of the vector DX2181b.

Figure 3 is a schematic diagram of the structure of the rice fragment stably transformed with the promoter fusion GUS expression vector. Among them: Figure 3a is a schematic diagram of the construction of the Y8A fusion GUS expression vector; Figure 3b is a schematic diagram of the construction of the Y8B fusion GUS expression vector.

Figure 4. Expression patterns of genes downstream of promoter Y8A in chip materials. A in Figure 4 is the microarray data, which shows that the gene is strongly induced by nitrogen supply in the microarray data of two varieties of Nipponbare (nip) and Zhenshan 97 (ZS); RNA was extracted for realtime verification of expression results, which also showed strong induction by nitrogen supply.

Figure 5 shows the results of realtime verification of the expression level in the rice variety Zhonghua 11 of the downstream genes of the promoter Y8A.

Fig. 6 is the Realtime verification result of Y8A downstream gene expression patterns in three conventional rice varieties selected in the embodiment of the present invention.

Fig. 7. After stably transforming rice with promoter Y8A fusion GUS, the expression level of GUS gene in different positive individual plants of each truncation pattern changes with nitrogen supply before and after. Among them: Fig. 7a and Fig. 7b are, from left to right, the expression detection results of full-length promoter Y8A and promoter truncated segment Y8B, and different numbers behind the line represent different rice transgenic positive lines.

detailed description

The following examples further define the present invention, and describe the mode of regulation of gene expression by the promoter Y8A and its truncated fragment Y8B, genetic transformation, and the assay method of expression level and GUS activity and the mode of regulation and expression of the promoter in rice . According to the following description and these examples, those skilled in the art can determine the basic features of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention, so that it is applicable to various uses and conditions.

The present invention will be further specifically described below in conjunction with the accompanying drawings.

Example 1: Determination of the regulatory mode of the Y8 gene promoter and acquisition of the sequence

In order to discover the promoters of nitrogen deficiency or high nitrogen supply inducing gene expression in rice, and to provide new promoter materials for improving rice nitrogen uptake by genetic engineering, the applicant designed the technical route in Figure 1 and selected two known and commonly used The conventional rice varieties of Nipponbare and Zhenshan 97 were used as experimental materials. Apply microarray technology to select new genes that have obvious responses to nitrogen stress. This technology can be used to quantitatively detect the expression levels of a large number of genes at different times (Yang Rong et al., 1999).

Soak the seeds of Nipponbare and Zhenshan 97 at 37°C for 3 days, accelerate the germination for 2 days, and transplant the seedlings to the rice full nutrient solution for hydroponic cultivation (the composition of the hydroponic nutrient solution: 1.44mM NH ₄ NO ₃ , 0.3mM NaH ₂ PO4, 0.5mM K ₂ SO ₄ , 1.0mM CaCl ₂ , 1.6mM MgSO ₄ , 0.17mM NaSiO ₃ , 50μM Fe-EDTA, 0.06μM(NH ₄ ) 6Mo ₇ O ₂₄ , _15μM H ₃ BO ₃ , 8μM MnCl ₂ , 0.12 μM CuSO ₄ , 0.12 μM ZnSO ₄ , 29 μM FeCl ₃ , 40.5 μM Citric acid, pH 5.5, see Yoshida et al., 1976 for details), culture to the 5-leaf stage, and move part of the rice seedlings to a nitrogen-deficient nutrient solution (above The nutrient solution does not need to add NH ₄ NO ₃ ) to carry out the nitrogen stress treatment, and the seedlings that continue to be cultured with the full nutrient solution are used as the control. According to the needs of the experiment, the treatment time points were designed as follows: nitrogen deficiency for 1 hour, nitrogen deficiency for 1 day, nitrogen deficiency for 3 days, nitrogen deficiency for 7 days, nitrogen deficiency for 2 hours after nitrogen deficiency, and nitrogen supply for 1 day. When sampling, the shoots and roots were separated, wrapped in tinfoil, and stored in liquid nitrogen. Extract the sample RNA and reverse transcribe it into cDNA (for specific steps, please refer to the SSIII reverse transcription kit manual of Invitrogen Company, Cat. Genome-wide cDNA chips were made (Yang Rong et al., 1999), and the feedback chip data were analyzed. From the chip results, it was found that the downstream genes activated by the promoter Y8A resumed nitrogen supply for 2 hours in the roots of these two varieties after 7 days of nitrogen deficiency. There is a high-power induction, but there is no such response in the shoot (see Figure 4 a, Figure 4 b). In order to confirm the response of the gene to nitrogen, the applicant used Zhonghua 11 to plant a batch of stress materials to verify the response of these genes to nitrogen. Realtime verification results (Realtime PCR method can be found in the instruction manual of the TAKARA commercial kit, article number code: DRR041A) showed that in the roots of Zhonghua 11, nitrogen deficiency for 7 days and nitrogen supply restored for 2 hours induced Y8A downstream genes to up-regulate about 20 times. As the nitrogen supply continued, the expression levels gradually returned to normal levels (see Figure 5). In order to further verify whether the gene has a similar expression pattern in different varieties, the applicant used another three rice varieties Huajing 295, 9311 and MH63 to plant a batch of stress materials (Huajing 295 is a well-known and public rice variety Japonica rice varieties, 9311 and MH63 are well-known indica rice varieties), the results were similar to those of Zhonghua 11 (see Figure 6). The applicant entered the gene sequence into the RGAP website (//rice.plantbiology.msu.edu/) to perform Blast to obtain the full gene sequence of the gene and accession number LOC_Os09g30490. On the softberry website, the full-length ORF is predicted to obtain the full-length genome sequence and promoter sequence of the downstream gene of Y8A. The gene is located on chromosome 9 of rice, the full-length genome sequence is 750bp, the full-length cDNA is 414bp, and encodes 137 amino acids (see the sequence table SEQ ID NO: 3). The applicant took the 1936 bp upstream of the ATG of the gene as the full length of the promoter of the gene (ie Y8A, see the sequence listing SEQ ID NO: 1).

Embodiment 2: Construction of promoter expression transformation vector

The present invention uses the total DNA of rice variety Zhonghua 11 as a template to amplify the full-length sequence of the Y8A promoter through the PCR method. At the same time, the full-length sequence of the promoter was truncated to 982 bp upstream of the ATG according to the 5'-end truncation mode, named: Y8B (see SEQ ID NO: 1 in the sequence listing), and the truncated fragment was obtained by PCR. The details are as follows: According to the known full-length sequence of the promoter Y8A (1936bp, see SEQ ID NO: 1 in the sequence listing) and its truncated promoter segment Y8B (see SEQ ID NO: 2 in the sequence listing), design two pairs of primers (The sequence of the primer pair is shown in Table 1). Using the total DNA of rice variety Zhonghua 11 as a template, the full-length sequence of promoter Y8A (1936bp) and the truncated Y8B (982bp) were amplified respectively. When amplifying Y8A and Y8B, the same restriction endonuclease site BamHI was added to the 5' ends of the two pairs of primers (see Table 1-1 for the sequence of the primer pair), so the amplified fragments can be used for restriction The endonuclease BamHI was digested, and then the fragments of Y8A and Y8B obtained by the enzyme digestion were respectively connected to the promoter vector DX2181b (the vector DX2181b is a commercial vector pCAMBIA1381 developed by researchers at the State Key Laboratory of Crop Genetics where the applicant works, a source from Transformed on the basis of the plasmids publicly reported and used in Australia. The vector contains the screening gene for hygromycin: hygromycin (R), the structure of the vector DX2181b is described in Figure 2), and the basic schematic diagram of the construction of these two vectors is shown in As shown in Figure 3 (Figure 3a and Figure 3b correspond to the construction of Y8A and Y8B fusion DX2181b, respectively), we named the constructed vectors as DX2181b-Y8A and DX2181b-Y8B, respectively. Then use the primer pairs shown in Table 1-2 to sequence the obtained clones, and confirm that the promoters Y8A and Y8B are connected to the DX2181b vector in the correct direction (see Figure 3).

Using the classic Agrobacterium-mediated transformation method (Agrobacterium EHA105, from CAMBIA Laboratory, Australia, for the specific operation method of transformation, see: Elizabeth et.al., 1993), Y8A and its truncation were used in the rice variety Zhonghua 11 The promoter drives the expression of the GUS gene. Investigating the transgenic positive plants, it was found that nitrogen supply after 7 days of nitrogen deficiency would lead to a significant increase in the expression of GUS.

Table 1-1 PCR primer pair designed by the present invention

Table 1-2 Sequencing primers designed by the present invention

Example 3: Rice transformation experiments after the full-length promoter Y8A and its truncated promoter fragments are fused to GUS

After connecting the full-length promoter Y8A and the truncated fragment Y8B to the vector DX2181b, the transformed rice positive plants were obtained by using the method of Agrobacterium-mediated transgenesis. The specific transformation steps are as follows:

The obtained correctly cloned plasmids (DX2181b-Y8A, DX2181b-Y8B) were introduced into the rice variety Zhonghua 11 through the rice genetic transformation system mediated by Agrobacterium (EHA105 provided by CAMBIA Laboratory, Australia), and pre-cultivated, infected , co-cultivation, selection of hygromycin-resistant calli, differentiation, rooting, training and transplanting of seedlings to obtain transformed plants. The Agrobacterium-mediated genetic transformation system of rice (subspecies japonica) mainly adopts the further optimized method based on the report of Hiei et al. (1994).

The main steps of the genetic transformation of the present invention, the culture medium and the preparation method thereof are as follows:

(1) Abbreviations for reagents and solutions

The abbreviation of the plant hormone used in the culture medium among the present invention is as follows: 6-BA (6-BenzylaminoPurine, 6-benzyl adenine); CN (Carbenicillin, carbenicillin); KT (Kinetin, kinetin); NAA ( Napthalene acetic acid, naphthalene acetic acid); IAA (Indole-3-acetic acid, indole acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid); AS (Acetosringone, Acetosyringone); CH (Casein Enzymatic Hydrolysate, hydrolyzed casein); HN (Hygromycin B, hygromycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide); N6max (N6 macroelement solution); N6mix (N6 trace element composition solution); MSmax (MS macroelement composition solution); MSmix (MS trace element composition solution).

(2) Solution formula

1) N6 medium macroelement mother solution (prepared according to 10 times concentrated solution (10X)):

Dissolve the above reagents one by one, and then dilute to 1000 ml with distilled water.

2) N6 medium trace element mother solution (prepared according to 100 times concentrated solution (100X)

Dissolve the above reagents at 20-25 degrees Celsius and dilute to 1000 ml with distilled water.

3) Iron salt (Fe ₂ EDTA) stock solution (prepared according to 100X concentrated solution)

Dissolve 3.73 g disodium edetate (Na ₂ EDTA·2H ₂ O) and 2.78 g FeSO ₄ □7H ₂ O respectively, mix and dilute to 1000 ml with distilled water, incubate at 70°C for 2 hours, store at 4°C spare.

4) Vitamin storage solution (prepared according to 100X concentrated solution)

Add distilled water to make up to 1000 ml, and store at 4°C for later use.

5) MS medium macroelement mother solution (MSmax mother solution) (prepared according to 10X concentrated solution)

Dissolve the above reagents at a temperature of 20-25°C, and dilute to 1000 ml with distilled water.

6) MS medium trace element mother solution (MSmin mother solution) (prepared according to 100X concentrated solution)

Dissolve the above reagents at a temperature of 20-25°C, and dilute to 1000 ml with distilled water.

7) Preparation of 2,4--D stock solution (1 mg/ml):

Weigh 100 mg of 2,4-D, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water to dissolve completely, then set the volume to 100 ml, and store at 20-25°C.

8) Preparation of 6-BA stock solution (1 mg/ml):

Weigh 100 mg of 6-BA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water to dissolve completely, then set the volume to 100 ml, and store at 20-25°C.

9) Preparation of naphthaleneacetic acid (NAA) stock solution (1 mg/ml):

Weigh 100 mg of NAA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water to dissolve it completely, then dilute it to 100 ml, and store it at 4°C for later use.

10) Preparation of indole acetic acid (IAA) stock solution (1 mg/ml):

Weigh 100 mg of IAA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water to dissolve completely, then dilute to 100 ml, and store at 4°C for later use.

11) Preparation of glucose stock solution (0..5 g/ml):

Weigh 125 grams of glucose, then dissolve it in distilled water to 250 ml, and store it at 4°C after sterilization.

12) Preparation of AS stock solution:

Weigh 0.392 g of AS, add 10 ml of DMSO to dissolve, dispense into 1.5 ml centrifuge tubes, and store at 4°C for later use.

13) 1N potassium hydroxide stock solution

Weigh 5.6 grams of potassium hydroxide, dissolve it in distilled water to 100 ml, and store it at 20-25°C for later use.

(3) Medium formula for genetic transformation of rice

1) Induction medium

Add distilled water to 900ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and adjust the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize according to conventional methods (sterilized at 121°C) Sterilize for 25 minutes, the sterilization method of the following medium is the same as the sterilization method of this medium).

2) Subculture medium

Add distilled water to 900 ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and adjust the volume to 1000 ml, dispense into 50 ml Erlenmeyer flasks (25 ml/bottle), seal, and sterilize as above.

3) Pre-medium

Add distilled water to 250 ml, adjust the pH value to 5.6 with 1N potassium hydroxide, seal, and sterilize as above.

Heat to dissolve the medium before use, add 5 ml of glucose stock solution and 250 microliters of AS stock solution, and pour them into petri dishes (25 ml/dish).

4) Co-culture medium

Add distilled water to 250 ml, adjust the pH value to 5.6 with 1N potassium hydroxide, seal, and sterilize as above.

Heat to dissolve the medium before use, add 5 ml of glucose stock solution and 250 microliters of AS stock solution, and pour them into petri dishes (25 ml/dish).

5) Suspension medium

Add distilled water to 100 ml, adjust the pH value to 5.4, divide into two 100 ml Erlenmeyer flasks, seal, and sterilize as above.

Add 1 ml of sterile glucose stock solution and 100 µl of AS stock solution just before use.

6) Select medium

Add distilled water to 250 ml, adjust the pH to 6.0, seal, and sterilize as above.

Dissolve the medium before use, add 250 μl HN (50 mg/ml) and 400 μl CN (250 mg/ml) and aliquot into Petri dishes (25 ml/dish). (Note: The concentration of carbenicillin in the first selection medium is 400 mg/L, and the concentration of carbenicillin in the second and subsequent selection medium is 250 mg/L).

7) Pre-differentiation medium

Add distilled water to 250 ml, adjust the pH value to 5.9 with 1N potassium hydroxide, seal, and sterilize as above.

Dissolve the medium before use, 250 μl HN (50 mg/ml) and 250 μl CN (250 mg/ml), aliquot into Petri dishes (25 ml/dish).

8) Differentiation medium

Add distilled water to 900 ml, and adjust the pH to 6.0 with 1N potassium hydroxide.

Boil and dilute to 1000ml with distilled water, dispense into 50ml Erlenmeyer flasks (50ml/bottle), seal, and sterilize as above.

9) Rooting medium

Add distilled water to make up to 900 ml, and adjust the pH to 5.8 with 1N potassium hydroxide.

Boil and dilute to 1000ml with distilled water, dispense into rooting tubes (25ml/tube), seal, and sterilize as above.

(4) Agrobacterium-mediated genetic transformation step

callus induction

1) Shell the mature Zhonghua 11 rice seeds, then treat them with 70% ethanol for 1 minute, and disinfect the surface of the seeds with 0.15% mercury chloride (HgCl ₂ ) for 15 minutes;

2) Wash the seeds 4-5 times with sterilized water;

3) seeds are placed on the induction medium;

4) Culture the inoculated medium in a dark place for 4 weeks at a temperature of 25±1°C.

3..2 Callus subculture

Select bright yellow, compact and relatively dry embryogenic calli, and place them on the subculture medium for 2 weeks in the dark at a temperature of 25±1°C.

3..3 Pre-cultivation

Select compact and relatively dry embryogenic callus, put it on the pre-medium and culture it in the dark for 2 weeks at a temperature of 25±1°C.

3..4 Agrobacterium culture

1) Pre-cultivate Agrobacterium EHA105 (this strain comes from the Agrobacterium strain publicly used by CAMBIA Company) on LA medium with corresponding resistance selection (LA medium preparation refers to J. Sambrook et al., 1998) for two days , temperature 28°C;

2) Transfer the Agrobacterium to the suspension medium and culture on a shaker at 28°C for 2-3 hours.

3..5 Agrobacterium infection

1) transfer the pre-cultured callus to a sterilized bottle;

2) adjust the suspension of Agrobacterium to OD ₆₀₀ 0.8-1.0;

3) Soak the callus in the Agrobacterium suspension for 30 minutes;

4) Transfer the callus to a sterilized filter paper and blot dry; then place it on a co-culture medium for 3 days at a temperature of 19-20°C.

3..6 Callus washing and selection culture

1) Wash the callus with sterilized water until the Agrobacterium cannot be seen;

2) Soak in sterilized water containing 400 mg/L carbenicillin (CN) for 30 minutes;

3) transfer the callus to the sterilized filter paper and blot dry;

4) Transfer the callus to the selection medium for selection and culture for 2-3 times, each time for 2 weeks.

3..7 Differentiation

1) Transfer the resistant callus to the pre-differentiation medium and cultivate it in the dark for 5-7 days;

2) Transfer the pre-differentiation cultured callus to the differentiation medium, culture under light, and the temperature is 26°C.

3..8 Rooting

1) cutting off the roots produced during differentiation;

Then transfer it to the rooting medium and cultivate it under light for 2-3 weeks at a temperature of 26°C.

3..9 Transplanting

Wash off the residual medium on the roots and transfer the seedlings with a good root system to the greenhouse while keeping them moist for the first few days.

The japonica rice variety Zhonghua 11 was transformed to obtain transgenic single plant T ₀ generation plants (28 positive lines were obtained for Y8A, and 24 positive lines were obtained for Y8B). The T ₀ generation positive lines were harvested as T ₁ generation transgenic materials.

Example 4: Verification of the regulation mode of the promoter Y8A and its truncated Y8B transgenic plants

The full length of the Y8 promoter Y8A and the truncated promoter Y8B of the roots of the transgenic plants of the T ₀ generation were measured to regulate the expression of the GUS gene and the activity of the encoded GUS protein. It was found that the transgenic plants of each segment of the T ₀ generation showed similar The induction mode consistent with the chip, that is, the expression level of GUS gene and the activity of GUS were obviously induced 2 hours after nitrogen supply was restored. At the same time, the transgenic plants of the T1 generation _were measured, and the same induction results were also detected. This also proves that this promoter can improve rice quality through genetic transformation.

Realtime PCR verification method

For the method of Realtime PCR, please refer to the instruction manual of TAKARA commercial kit (product number: DRR041A) of Takara Bioengineering (Dalian) Co., Ltd. In the experiment, a 10 μl reaction system was used, including: 1.5 μl of cDNA template (rice material sample whose expression level is to be detected), 0.2 μM each of the left and right primers of the gene (the left and right primers used for GUS detection in this experiment are qGUS-F and qGUS-R respectively , the left and right primers of the internal reference gene Ubi are qUbi-F and qUbi-R, respectively, see Table 3) and 1U TaqDNA polymerase. The conditions of PCR amplification were: pre-denaturation at 95°C for 30s; 45 cycles at 95°C for 5s and 60°C for 34s; signals were collected at 60°C.

Table 3 Realtime expression detection primer list

In the test results (as shown in Figure 7), when the nitrogen supply was restored for 2 hours after 7 days of nitrogen deficiency stress, each of the 4 positive lines randomly selected for transformation of each promoter fragment showed a significant induction response to the restoration of nitrogen supply after nitrogen deficiency , proving that the promoter series are indeed promoters that regulate the expression of genes induced by nitrogen supply after nitrogen deficiency. And the regulatory element induced by nitrogen deficiency is within 982bp upstream of ATG.

Note: Figure 7a (GUS expression detection of different transgenic strains of Y8A) and Figure 7b (GUS expression detection of different Y8B transgenic strains), in which different numbers represent different transgenic strains, and CK represents normal nutrient solution For cultivation, -N7d means 7 days of nitrogen deficiency treatment, +N2h means 2 hours of nitrogen supply after 7 days of nitrogen deficiency treatment, +N1d means 1 day of nitrogen supply after 7 days of nitrogen deficiency treatment. All data are results from root samples.

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Claims (1)

1. applications of the promoter Y8A under recovering for nitrogen after paddy rice nitrogen stress in specific inducible gene expression, it is characterised in that should The nucleotide sequence of promoter such as sequence table SEQ ID NO：Shown in 1.