CN111217898B - Application of protein OsZZW1 in regulation and control of drought resistance of rice - Google Patents

Abstract

The invention discloses application of protein OsZZW1 in regulation and control of drought resistance of rice, wherein the amino acid sequence of the protein OsZZW1 is shown as SEQ ID NO: 2, respectively. Experiments prove that the drought resistance of rice can be improved by reducing the expression level of protein OsZZW1 in Nipponbare; the drought resistance is improved by the following steps: increased survival, increased proline content and decreased malondialdehyde content. The protein OsZZW1 can regulate the drought resistance of rice. The invention has important application value.

Description

Application of protein OsZZW1 in regulation and control of drought resistance of rice

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of protein OsZZW1 in regulation and control of rice drought resistance.

Background

Drought is the most important environmental factor affecting plant growth and crop yield. Drought becomes a serious problem affecting agricultural production, and the key problem and the important problem which need to be solved urgently in new variety cultivation are that the drought resistance of crops is improved by using a genetic engineering means, and the adaptability of the crops and the economic crops to the adverse environment is improved. In recent years, a great deal of research is carried out on the mechanism of plants responding to adverse environmental stress such as drought and the like from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, abundant data are accumulated, particularly with the development of molecular biology, people can know the adverse resistance mechanism of the plants to the drought stress on the molecular level of gene composition, expression regulation, signal conduction and the like, and a new way is developed for improving the stress resistance of the plants by utilizing a gene engineering means. Due to the complexity of the stress resistance of plants, the traditional breeding method is very difficult to improve the stress resistance of the plants, with the development of molecular biology, a new approach for the stress resistance breeding of the plants is developed by a genetic engineering means, but the separation of high-efficiency stress resistance genes becomes a main factor for limiting the stress resistance genetic engineering of the plants.

Rice (Oryza sativa L.) is one of the most important food crops in the world, with rice being the staple food for more than about half of the global population. Cultivation of high-yield drought-resistant rice varieties is an important measure for guaranteeing national grain safety, and excavation of drought-resistant related genes is a basic stone for drought-resistant molecular breeding.

Disclosure of Invention

The invention aims to improve the drought resistance of rice.

The invention firstly protects the application of protein OsZZW1, which can be S1) or S2):

s1) regulating and controlling the drought resistance of the rice;

s2) cultivating the transgenic rice with changed drought resistance.

The invention also protects the application of a nucleic acid molecule for coding the protein OsZZW1, which can be S1) or S2):

s1) regulating and controlling the drought resistance of the rice;

s2) cultivating the transgenic rice with changed drought resistance.

In any of the above applications, the controlling of drought resistance of rice may be increasing drought resistance of rice or decreasing drought resistance of rice.

In any of the above applications, the cultivation of transgenic rice with altered drought resistance may be the cultivation of transgenic rice with increased drought resistance or the cultivation of transgenic rice with decreased drought resistance.

The invention also provides a method for cultivating the transgenic rice A, which comprises the following steps: improving the expression quantity and/or activity of protein OsZZW1 in the starting rice to obtain transgenic rice A; the drought resistance of transgenic rice plants A is reduced compared with the original rice plants.

In the method, the expression quantity and/or activity of the protein OsZZW1 in the starting rice can be improved by using methods known in the art such as transgenosis, multi-copy, promoter change, regulatory factor change and the like, so that the effect of improving the expression quantity and/or activity of the protein OsZZW1 in the starting rice is achieved.

In the above method, the "improvement in the expression level and/or activity of the protein OsZZW1 in the starting rice" may be achieved by introducing a nucleic acid molecule encoding the protein OsZZW1 into the starting rice.

In the above method, the "introducing a nucleic acid molecule encoding the protein OsZZW1 into the starting rice" may be carried out by introducing a recombinant vector into the starting plant; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein OsZZW1 into an expression vector. The expression vector can be a pCAMBIA1300 vector.

In the above method, the recombinant vector may be specifically a recombinant plasmid pCAMBIA1300-OsZZW 1. The recombinant plasmid pCAMBIA1300-OsZZW1 can specifically be a plasmid obtained by inserting a restriction enzyme BamHI recognition site of a pCAMBIA1300 vector into a plasmid shown in SEQ ID NO: 4 to obtain the recombinant plasmid.

The transgenic rice beetle can be specifically OE1, OE2, OE3 or OE4 mentioned in examples; at this time, the starting rice is the rice variety Nipponbare.

The invention also provides a method for cultivating the transgenic rice B, which comprises the following steps: inhibiting the expression quantity and/or activity of protein OsZZW1 in the starting rice to obtain transgenic rice B; the drought resistance of the transgenic rice B is increased compared with that of the starting rice.

In the above method, the expression level and/or activity of the protein OsZZW1 in the starting rice can be inhibited by methods known in the art, such as DNA insertion, RNA interference, homologous recombination, gene site-directed editing, and the like, so as to achieve the purpose of inhibiting the expression level and/or activity of the protein OsZZW 1.

In the above method, the "inhibition of the expression level and/or activity of protein OsZZW1 in the starting rice" may be achieved by introducing into the starting rice a substance that inhibits the expression of a nucleic acid molecule encoding protein OsZZW 1.

In the above method, the substance inhibiting the expression of the nucleic acid molecule encoding the protein OsZZW1 can be a specific DNA molecule, an expression cassette containing the specific DNA molecule or a recombinant plasmid containing the specific DNA molecule;

the specific DNA molecule may include a sense fragment, an antisense fragment, and a spacer fragment located therebetween;

the sense fragment is a DNA molecule shown from 94 th to 474 th from the 5' end of a sequence 1 in a sequence table;

the antisense fragment is a reverse complementary sequence of a DNA molecule shown from 94 th to 474 th of the 5' end of the sequence 1 in the sequence table.

The specific DNA molecule may in particular consist of the sense fragment, the antisense fragment and a spacer fragment located between them.

The recombinant plasmid containing the specific DNA molecule can be specifically a recombinant plasmid pTCK303-OsZZW 1. The recombinant plasmid pTCK303-OsZZW1 specifically comprises a nucleotide sequence which is obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes SpeI and SacI of a pTCK303 vector with a nucleotide sequence shown as SEQ ID NO: 1 from the 5' end, 94 to 474 th, and a small fragment of DNA between the recognition sequences for the restriction enzymes KpnI and BamHI is replaced with a DNA fragment whose nucleotide sequence is SEQ ID NO: 3 to obtain the recombinant plasmid.

The transgenic rice B can be specifically RNAi1, RNAi2, RNAi3 or RNAi4 mentioned in the examples; at this time, the starting rice is the rice variety Nipponbare.

The invention also protects a rice breeding method A or a rice breeding method B.

The rice breeding method A protected by the invention can comprise the following steps: the content and/or activity of protein OsZZW1 in rice is increased, so that the drought resistance is reduced.

The rice breeding method B protected by the invention can comprise the following steps: the content and/or activity of protein OsZZW1 in rice is reduced, so that the drought resistance is increased.

The invention also relates to substances which inhibit the expression of the nucleic acid molecule coding for the protein OsZZW 1.

The substance for inhibiting the expression of the nucleic acid molecule of the coding protein OsZZW1 can be a specific DNA molecule, an expression cassette containing the specific DNA molecule or a recombinant plasmid containing the specific DNA molecule;

the specific DNA molecule may include a sense fragment, an antisense fragment, and a spacer fragment located therebetween;

the sense fragment is a DNA molecule shown from 94 th to 474 th from the 5' end of a sequence 1 in a sequence table;

the antisense fragment is a reverse complementary sequence of a DNA molecule shown from 94 th to 474 th of the 5' end of the sequence 1 in the sequence table.

The specific DNA molecule may in particular consist of the sense fragment, the antisense fragment and a spacer fragment located between them.

The recombinant plasmid containing the specific DNA molecule can be specifically a recombinant plasmid pTCK303-OsZZW 1. The recombinant plasmid pTCK303-OsZZW1 specifically comprises a nucleotide sequence which is obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes SpeI and SacI of a pTCK303 vector with a nucleotide sequence shown as SEQ ID NO: 1 from the 5' end, 94 to 474 th, and a small fragment of DNA between the recognition sequences for the restriction enzymes KpnI and BamHI is replaced with a DNA fragment whose nucleotide sequence is SEQ ID NO: 3 to obtain the recombinant plasmid.

The invention also protects the application of the specific DNA molecule, the expression cassette containing the specific DNA molecule or the recombinant plasmid containing the specific DNA molecule, which can be S1) or S2):

s1) increasing the drought resistance of the rice;

s2) cultivating the transgenic rice with increased drought resistance.

Any of the above rice plants may be a rice variety Nipponbare.

The amino acid sequence of any one of the above-mentioned proteins OsZZW1 can be shown as SEQ ID NO: 2, respectively. SEQ ID NO: 2 consists of 521 amino acid residues.

Any one of the proteins OsZZW1 can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Any one of the nucleic acid molecules for encoding the protein OsZZW1 can be a DNA molecule shown as b1) or b 2):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 1566 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Any of the above-described improvements in drought resistance may be manifested as an increase in survival (after drought treatment), an increase in proline content, and/or a decrease in malondialdehyde content.

Any of the above-described reductions in drought resistance may be manifested as a reduction in survival (after drought treatment), a reduction in proline content, and/or an increase in malondialdehyde content.

Experiments prove that the drought resistance of rice can be improved by reducing the expression level of protein OsZZW1 in Nipponbare; the drought resistance is improved by the following steps: increased survival (after drought treatment), increased proline content and/or decreased malondialdehyde content. The protein OsZZW1 can regulate the drought resistance of rice. The invention has important application value.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of the full length of the cDNA of OsZZW1 gene, which is the PCR amplification product of example 1.

FIG. 2 shows the relative expression level of OsZZW1 gene in transgenic rice detected by real-time fluorescent quantitative PCR.

FIG. 3 shows the growth state of rice seedlings before, after and after rehydration.

FIG. 4 is the statistical result of the survival rate of rice seedlings after rehydration.

FIG. 5 shows the results of measurement of proline content in rice plants before and at 8d after drought treatment.

FIG. 6 shows the results of measurement of malondialdehyde content in rice plants before and at 8d after drought treatment.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The pTCK303 vector is described in the following documents: jiang J, Li J, Xu Y, Han Y, Bai Y, Zhou G, Lou Y, Xu Z, Chong K. RNAi knock down of Oryza sativa root mean curing gene led to altered root resolution and linking of lower ware medium characterized by jasmonic acid signalling in rice Cell Environ.2007, 30(6):690-9.

The pCAMBIA1300 vector is described in the following documents: gou M, Su N, Zheng J, Huai J, Wu G, ZHao J, He J, Tang D, Yang S, Wang G.an F-box gene, CPR30, functions as a negative regulator of the feedback response in Arabidopsis.plant J.2009Dec; 60(5): 757-70.

Specific methods for transforming recombinant Agrobacterium into Nipponbare are described in the following references: hiei Y, Ohta S, Komari T, Kumashiro T. efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the bases of the T-DNA plant J.1994; 6(2): 271-82.Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H.early introduction of cutellum tissue with Agrobacterium infection high-speed transformation of rice plant J.2006; 47(6): 969-76.

Methods for determining proline content are described in the following documents: cui Y, Li M, Yin X, Song S, Xu G, Wang M, Li C, Peng C, Xia x.osdssr1, alpha novel small peptide, enhances drop height in transient rice.plant sci.2018; 270:85-96.

The method for determining the malondialdehyde content is described in the following documents: ZHao Q, ZHou L, Liu J, Cao Z, Du X, Huang F, Pan G, Cheng F. invasion of CAT in the detoxification of HT-induced ROS burst in boundary and its correlation to polar transfer. plant Cell Rep.2018; 37(5):741-757.

Example 1 cloning of the Gene encoding the protein OsZZW1 (i.e., OsZZW1 Gene)

1. Total RNA of leaves of 7d rice variety Nipponbare (hereinafter abbreviated as Nipponbare) is extracted by a Trizo1 method, first strand cDNA is reversely transcribed by reverse transcriptase, and dscDNA is synthesized by a SMART method, namely ds-cDNA of the Nipponbare is obtained.

2. Taking the ds-cDNA of Nipponbare obtained in the step 1 as a template, and adopting a primer OsZZW 1-F: 5'-ATGCCGAGCGCCTTCCACT-3' and primer OsZZW 1-R: 5'-GAATGAGAACTTGAATCCTCCA-3' to obtain PCR amplification product.

The PCR amplification product was subjected to 1% agarose gel electrophoresis.

The result of 1% agarose gel electrophoresis is shown in FIG. 1(1 is PCR amplification product).

3. Recovering 1566bp DNA fragment from the PCR amplification product obtained in step 2.

Sequencing the DNA fragment recovered in the step 3. Sequencing results show that the nucleotide sequence of the DNA fragment (namely OsZZW1 gene) recovered in the step 3 is shown as SEQ ID NO: 1 is shown at positions 1 to 1563 from the 5' end.

Example 2 acquisition of transgenic Rice and identification of drought resistance

Construction of recombinant plasmid

1. Construction of recombinant plasmid pCAMBIA1300-OsZZW1

(1) Using the DNA fragment obtained in step 3 of example 1 as a template, primer F: 5' -TTCTGCAGCGGGATCCATGCCGAGCGCCTTCCAC-3' (recognition site for restriction enzyme BamHI is underlined) and primer R: 5' -CGTAACGCGTGGATCCTTGAATGAGAACTTGAATCCTCC-3' (recognition site for the restriction enzyme BamHI is underlined) was subjected to PCR amplification, and then a 1597bp DNA fragment A was recovered.

(2) The pCAMBIA1300 vector was digested with the restriction enzyme BamHI, and the 14.58kb vector backbone was recovered.

(3) And (3) connecting the DNA fragment A with a vector skeleton by using infusion ligase to obtain a recombinant plasmid pCAMBIA1300-OsZZW 1.

The recombinant plasmid pCAMBIA1300-OsZZW1 was sequenced. The sequencing result shows that the recombinant plasmid pCAMBIA1300-OsZZW1 is a plasmid obtained by inserting the restriction enzyme BamHI recognition site of pCAMBIA1300 vector into the plasmid shown in SEQ ID NO: 4 to obtain the recombinant plasmid.

The recombinant plasmid pCAMBIA1300-OsZZW1 expresses SEQ ID NO: 2, OsZZW 1.

2. Construction of recombinant plasmid pTCK303-OsZZW1

(1) Using the DNA fragment obtained in step 3 of example 1 as a template, the DNA fragment was amplified using a primer pTCK303-OsZZW 1-F:

(recognition site for restriction enzyme KpnI is single underlined, recognition site for restriction enzyme SpeI is double underlined) and primer pTCK303-OsZZW 1-R:

(recognition site for restriction enzyme BamHI is single underlined, and recognition site for restriction enzyme SacI is double underlined) followed by PCR amplification of the primer pair, and then recovering DNA fragment 1 of about 409 bp.

(2) The DNA fragment 1 was ligated with a T vector (TAKARA) to obtain a recombinant plasmid T-OsZZW 1B.

(3) The recombinant plasmid T-OsZZW1B was digested with the restriction enzymes SpeI and SacI, and an about 390bp DNA fragment 2 was recovered.

(4) The pTCK303 vector was digested with the restriction enzymes SpeI and SacI, and the vector backbone 1 of about 14.6kb was recovered.

(5) And connecting the DNA fragment 2 with the vector framework 1 to obtain an intermediate vector.

(6) The recombinant plasmid T-OsZZW1B was digested with restriction enzymes KpnI and BamHI, and an about 400bp DNA fragment 3 was recovered.

(7) The intermediate vector was digested with restriction enzymes KpnI and BamHI, and the vector backbone 2 of about 15.0kb was recovered.

(8) The DNA fragment 3 and the vector framework 2 are connected to obtain a recombinant plasmid pTCK303-OsZZW 1.

The recombinant plasmid pTCK303-OsZZW1 was sequenced. The sequencing result shows that the recombinant plasmid pTCK303-OsZZW1 is obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes SpeI and SacI of a pTCK303 vector with a nucleotide sequence shown as SEQ ID NO: 1 from the 5' end, 94 to 474 th, and a small fragment of DNA between the recognition sequences for the restriction enzymes KpnI and BamHI is replaced with a DNA fragment whose nucleotide sequence is SEQ ID NO: 3 (the DNA molecule shown in the positions 7 to 387 from the 5 'end of SEQ ID NO: 3 is the reverse complementary sequence of the DNA molecule shown in the positions 94 to 474 from the 5' end of SEQ ID NO: 1), and obtaining the recombinant plasmid.

II, obtaining recombinant agrobacterium

1. The recombinant plasmid pCAMBIA1300-OsZZW1 is introduced into agrobacterium EHA105 to obtain recombinant agrobacterium, which is named as EHA105/pCAMBIA1300-OsZZW 1.

2. The recombinant plasmid pTCK303-OsZZW1 is introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which is named as EHA105/pTCK303-OsZZW 1.

Third, obtaining transgenic rice

1、T₃Generation of generation homozygous transgenic rice with OsZZW1 gene

EHA105/pCAMBIA1300-OsZZW1 was transformed into Nipponbare to obtain T₀Rice with OsZZW1 gene transferred; then selfing to obtain T₃Transgenic rice with OsZZW1 gene. The method for screening the positive seedlings comprises the following steps: extracting the genome DNA of the rice seedling to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of 5'-GTGCAGGAGGAGAGGCCATTGTACC-3' and 5'-GAATGAGAACTTGAATCCTCCA-3' to obtain a PCR amplification product; then, the following judgment is made: if a certain PCR amplification product only contains DNA fragments of about 726bp, the rice seedling corresponding to the PCR amplification product is a homozygote positive seedling.

2、T₃Obtaining of rice with OsZZW1 gene silenced generation

Converting EHA105/pTCK303-OsZZW1 into Nipponbare to obtain T₀A silent plant is substituted; then selfing to obtain T₃OsZZW1 gene-silenced rice. The method for screening the positive seedlings comprises the following steps: extracting the genome DNA of the rice seedling to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of 5'-GGTGCCGCCAGGAGTTCATC-3' and 5'-TCTTCTTGCGAGCAGCAGCCTTC-3' to obtain a PCR amplification product; then, the following judgment is made: if a certain PCR amplification product only contains about 407bp DNA fragments, the rice seedling corresponding to the PCR amplification product is a homozygote positive seedling.

Fourthly, detecting the relative expression level of OsZZW1 gene in transgenic rice by real-time fluorescent quantitative PCR

1. Respectively combine each T₃Seedlings of the rice with the generation homozygous OsZZW1 transgenic gene growing for 10 days are placed in liquid nitrogen for preservation, and corresponding samples to be detected are obtained. Respectively combine each T₃Seedlings of the generation-silenced OsZZW1 gene rice growing for 10 days are stored in liquid nitrogen to obtain corresponding samples to be detected. And (3) putting the seedlings growing to 10 days in Nipponbare into liquid nitrogen for preservation to obtain corresponding samples to be detected.

2. Extracting total RNA of a sample to be detected by adopting a Trizo1 method, and then reversely transcribing first strand cDNA by utilizing a reverse transcription kit (TaKala); the cDNA was diluted 10-fold with sterile water as a template, and the relative expression level of OsZZW1 gene (OsACTIN1 gene is an internal reference gene) was detected by real-time quantitative PCR.

Primers for detecting the OsZZW1 gene are 5'-TCGGCGTGGAATGCTGTGA-3' and 5'-CTTGGATGAACTCCTGGCGG-3'.

Primers for detecting the OsACTIN1 gene are 5'-CATCTCTCAGCACATTCCAGCAG-3' and 5'-AGGAGGACGGCGATAACAGC-3'.

The partial detection results are shown in FIG. 2(WT is Nipponbare, OE1, OE2, OE3 and OE4 are all T₃Transgenic rice with OsZZW1 gene homozygous for generation). The results show that each T is comparable to Nipponbare₃The relative expression level of OsZZW1 gene in generation homozygous transgenic OsZZW1 rice is obviously increased, wherein 4T genes₃The relative expression level of OsZZW1 gene in a generation homozygous transgenic OsZZW1 rice strain is the highest, and the generation homozygous transgenic OsZZW1 gene is sequentially named as OE1, OE2, OE3 and OE 4. Each T is compared with Nippon sunny₃The relative expression level of OsZZW1 gene in rice with generation-silenced OsZZW1 gene is remarkably reduced, wherein 4T genes₃The relative expression level of the OsZZW1 gene in the rice strain with the generation-silenced OsZZW1 gene is the lowest, and the rice strain with the generation-silenced OsZZW1 gene is sequentially named as RNAi1, RNAi2, RNAi3 and RNAi 4.

Fifth, drought resistance identification of transgenic rice

T with OE1 as rice seed to be detected₃Seed generation, T of OE2₃Seed generation, RNAi 2T₃Seed generation, RNAi 3T₃Seeds of the generations or Nipponbare seeds.

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. taking rice seeds to be detected, cleaning the rice seeds for 20min by using a 50% (v/v) sodium hypochlorite solution, and washing the rice seeds for 6 times by using clear water; and then culturing the rice seedlings for 10 days in a light-dark alternating mode at the temperature of 28 ℃ to obtain the rice seedlings to be detected.

2. And (3) after the step 1 is completed, transplanting 36 rice seedlings to be detected with basically consistent growth states to a flowerpot filled with nutrient soil, and normally culturing for two weeks to obtain the rice seedlings to be detected before drought treatment.

In order to ensure that the experimental conditions are consistent as much as possible, the weight of the nutrient soil in each flowerpot is the same, and the number of the rice seedlings to be tested transplanted in each flowerpot is also the same.

3. And (3) after the step 2 is completed, taking the rice seedlings to be detected before drought treatment, and carrying out drought treatment for 12d to obtain the drought-treated rice seedlings. Methods of drought treatment are described in the following documents: tang N, Ma S, Zong W, Yang N, Lv Y, Yan C, Guo Z, Li J, Li X, Xiaong Y, Song H, Xiao J, Li X, Xiong L.MODD media deactivation and deactivation of OsbZIP46 to novel regulation ABA signaling and reduction resistance in Rice.plant cell.2016; 28(9): 2161-2177.

4. And (4) after the step 3 is finished, taking the rice seedling to be detected after drought treatment, and obtaining the rice seedling to be detected after rehydration for 10 days.

5. Observing the growth states of the rice seedlings to be detected before drought treatment, the rice seedlings after drought treatment and the rice seedlings to be detected after rehydration; and (5) counting the survival rate of the rice seedlings to be detected after rehydration.

The growth state of the rice seedling to be tested is shown in figure 3.

The survival rate statistical result of the rice seedlings to be detected after rehydration is shown in figure 4.

6. And (3) taking the rice seedlings to be detected before the drought treatment or the rice seedlings to be detected at the 8 th day after the drought treatment, and determining the proline content and the malondialdehyde content.

The results of the proline content determination are shown in FIG. 5 and Table 1.

TABLE 1

The results of the assay for malondialdehyde content are shown in FIG. 6 and Table 2.

TABLE 2

Before drought treatment, T₃Generation homozygous OsZZW1 transgenic rice (OE1 and OE2) and T₃The generation silencing OsZZW1 gene rice (RNAi2 and RNAi3) has no significant difference from Nipponbare in growth state, proline content and malondialdehyde content; after drought treatment, T compares to Nipponbare₃The generation silencing OsZZW1 gene rice (RNAi2 and RNAi3) has the advantages of good growth state, obviously increased survival rate and proline content, and obviously reduced malondialdehyde content; t is₃The generation homozygous OsZZW1 transgenic rice (OE1 and OE2) has poor growth state (marked by wilting degree), remarkably reduced survival rate and proline content, and remarkably increased malondialdehyde content.

The results show that the drought resistance of the rice can be improved by reducing the expression level of the protein OsZZW1 in Nipponbare; the drought resistance is improved by the following steps: increased survival, increased proline content and decreased malondialdehyde content.

<110> university of northriver

Application of <120> protein OsZZW1 in regulation and control of drought resistance of rice

<160> 4

<170> PatentIn version 3.5

<210> 1

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<213> Oryza sativa L.

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atgccgagcg ccttccactc cctcctcctc cccgccattc gcaaccccaa acctagccgt 60

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ctcgctaggc tctccgcggc gtcgagggcg ctctacgtcg tcgcctccca cgacccgctc 420

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atcagggggt tctactccga ctacctcttc cagagctggc tctgcgccaa catggagatg 600

cggccggagt ggctccaccg ggacaccatc gatcgccgcc gcggcatgtc cgtcgagcaa 660

ttcgtctccg aattcgagga gcccaatagg ccggtgcttc tggaaggctg cctcgagagc 720

tggccagcat tgcagaagtg gaccagggag cacttgctga aggtctcggc cgggaaggag 780

ttcgccgtcg ggccggtgag catgacgctg gataggtacc tccagtatgc cgacaatgtg 840

caggaggaga ggccattgta cctgttcgat gccaagttca ccgagaaggt gccggagatg 900

gggagggact atgaggtgcc ggcgtacttc cgagaggacc tattcggggt gcttggggag 960

gaaaggccgg accaccgatg ggttatcatt gggccggcag gttcagggtc gtcgtttcat 1020

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gaaaagaggc ctgttgagtg tatatgccgg gctggagagg tggtttttgt gcctaatgga 1260

tggtggcatt tggttatcaa tctggaggaa tctattgcga ttactcagaa ttatgtgagc 1320

aggaggaatc tgttgaatgt tcttgacttt ctcaagaggc ccaatgcaag tgaacttgta 1380

tcagggacta cagacagggt aaacctgcat gacaagttcc gcaatgccat cgatatgact 1440

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aagaaagtct cgttctggga atctgcggta gatgccaata ctggaggatt caagttctca 1560

ttctga 1566

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<213> Oryza sativa L.

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Met Pro Ser Ala Phe His Ser Leu Leu Leu Pro Ala Ile Arg Asn Pro

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Asp Ala Thr Ala Phe His Leu Lys Thr Ser Ala Arg Ala Gly Pro Gly

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

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Leu Gly Asn Leu Leu Leu Leu Gly Gly Gly Gly Asn Leu Arg Asp Ala

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Gly Leu Gly Ala Leu Arg Pro Leu Pro Asp Asp Val Leu Leu Asp Val

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Leu Gly Leu Leu Ala Ala Arg Asp Leu Ala Arg Leu Ser Ala Ala Ser

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Arg Ala Leu Tyr Val Val Ala Ser His Asp Pro Leu Trp Arg Ala Leu

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Val Leu Asp Glu Leu Gly Gly Asp Phe Ala Phe Ser Gly Ser Trp Arg

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Ala Thr Tyr Ile Ala Ala Ala Ser Gly Gly Arg Ala His Leu Pro Pro

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Arg Gly Leu Glu Ile Arg Gly Phe Tyr Ser Asp Tyr Leu Phe Gln Ser

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Phe Asp Ala Lys Phe Thr Glu Lys Val Pro Glu Met Gly Arg Asp Tyr

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Glu Val Pro Ala Tyr Phe Arg Glu Asp Leu Phe Gly Val Leu Gly Glu

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Glu Arg Pro Asp His Arg Trp Val Ile Ile Gly Pro Ala Gly Ser Gly

325 330 335

Ser Ser Phe His Val Asp Pro Asn Ser Thr Ser Ala Trp Asn Ala Val

340 345 350

Ile Lys Gly Ala Lys Lys Trp Val Met Phe Pro Pro Glu Val Val Pro

355 360 365

Pro Gly Val His Pro Ser Ala Asp Gly Ala Glu Val Thr Ser Pro Val

370 375 380

Ser Ile Met Glu Trp Phe Met Asn Phe Tyr Gly Ala Cys Lys Thr Trp

385 390 395 400

Glu Lys Arg Pro Val Glu Cys Ile Cys Arg Ala Gly Glu Val Val Phe

405 410 415

Val Pro Asn Gly Trp Trp His Leu Val Ile Asn Leu Glu Glu Ser Ile

420 425 430

Ala Ile Thr Gln Asn Tyr Val Ser Arg Arg Asn Leu Leu Asn Val Leu

435 440 445

Asp Phe Leu Lys Arg Pro Asn Ala Ser Glu Leu Val Ser Gly Thr Thr

450 455 460

Asp Arg Val Asn Leu His Asp Lys Phe Arg Asn Ala Ile Asp Met Thr

465 470 475 480

Tyr Pro Gly Met Ile Lys Gln Leu Glu Leu Glu Ala Gln Gln Lys Ala

485 490 495

Ala Ala Arg Lys Lys Lys Val Ser Phe Trp Glu Ser Ala Val Asp Ala

500 505 510

Asn Thr Gly Gly Phe Lys Phe Ser Phe

515 520

<210> 3

<211> 393

<212> DNA

<213> Artificial sequence

<400> 3

gagctccgag cccgagaagg cgaagtcccc gccgagctcg tcgaggacga gcgcccgcca 60

gagcgggtcg tgggaggcga cgacgtagag cgccctcgac gccgcggaga gcctagcgag 120

gtcgcgcgcc gcgagcaggc cgagcacgtc gaggaggacg tcgtcgggga gcgggcggag 180

cgcgccgagc cccgcgtcgc ggaggttgcc gccgccgccg aggaggagga ggttgccgag 240

cggctgcacg aggcatcccc catcgcctcg ccggccgctc ccggcacccc ccggaccggc 300

gcgcgcggag gtcttcaggt ggaaggcggt ggcgtctccg gcggcggcgt cggcgaggcg 360

gttcttggat ttggtggtct tcttgggact agt 393

<210> 4

<211> 1565

<212> DNA

<213> Artificial sequence

<400> 4

atgccgagcg ccttccactc cctcctcctc cccgccattc gcaaccccaa acctagccgt 60

cgccgcggcc gcggccgcgg cggcagcaaa cgccccaaga agaccaccaa atccaagaac 120

cgcctcgccg acgccgccgc cggagacgcc accgccttcc acctgaagac ctccgcgcgc 180

gccggtccgg ggggtgccgg gagcggccgg cgaggcgatg ggggatgcct cgtgcagccg 240

ctcggcaacc tcctcctcct cggcggcggc ggcaacctcc gcgacgcggg gctcggcgcg 300

ctccgcccgc tccccgacga cgtcctcctc gacgtgctcg gcctgctcgc ggcgcgcgac 360

ctcgctaggc tctccgcggc gtcgagggcg ctctacgtcg tcgcctccca cgacccgctc 420

tggcgggcgc tcgtcctcga cgagctcggc ggggacttcg ccttctcggg ctcgtggcgc 480

gccacctaca tcgccgccgc gtcgggcggc cgcgcccacc tccccccgcg gggcctagag 540

atcagggggt tctactccga ctacctcttc cagagctggc tctgcgccaa catggagatg 600

cggccggagt ggctccaccg ggacaccatc gatcgccgcc gcggcatgtc cgtcgagcaa 660

ttcgtctccg aattcgagga gcccaatagg ccggtgcttc tggaaggctg cctcgagagc 720

tggccagcat tgcagaagtg gaccagggag cacttgctga aggtctcggc cgggaaggag 780

ttcgccgtcg ggccggtgag catgacgctg gataggtacc tccagtatgc cgacaatgtg 840

caggaggaga ggccattgta cctgttcgat gccaagttca ccgagaaggt gccggagatg 900

gggagggact atgaggtgcc ggcgtacttc cgagaggacc tattcggggt gcttggggag 960

gaaaggccgg accaccgatg ggttatcatt gggccggcag gttcagggtc gtcgtttcat 1020

gttgatccaa actcgacatc ggcgtggaat gctgtgatca agggagccaa gaagtgggtg 1080

atgttcccac cggaggtggt gccgccagga gttcatccaa gtgcggatgg agcagaagtc 1140

actagccctg tatctatcat ggaatggttc atgaattttt atggggcatg taagacctgg 1200

gaaaagaggc ctgttgagtg tatatgccgg gctggagagg tggtttttgt gcctaatgga 1260

tggtggcatt tggttatcaa tctggaggaa tctattgcga ttactcagaa ttatgtgagc 1320

aggaggaatc tgttgaatgt tcttgacttt ctcaagaggc ccaatgcaag tgaacttgta 1380

tcagggacta cagacagggt aaacctgcat gacaagttcc gcaatgccat cgatatgact 1440

tatcctggga tgattaaaca gcttgaactt gaagctcagc agaaggctgc tgctcgcaag 1500

aagaaagtct cgttctggga atctgcggta gatgccaata ctggaggatt caagttctca 1560

ttctt 1565

Claims (11)

1. Application of protein OsZZW1, S1) or S2):

s1) regulating and controlling the drought resistance of the rice;

s2) cultivating the drought resistance-changed transgenic rice;

the amino acid sequence of the protein OsZZW1 is shown in SEQ ID NO: 2, respectively.

2. The use of claim 1, wherein:

the drought resistance of the rice is adjusted to increase the drought resistance of the rice or reduce the drought resistance of the rice;

the cultivation of the transgenic rice with changed drought resistance is to cultivate the transgenic rice with increased drought resistance or cultivate the transgenic rice with reduced drought resistance.

3. Use of a nucleic acid molecule encoding the protein OsZZW1 according to claim 1, S1) or S2):

s1) regulating and controlling the drought resistance of the rice;

s2) cultivating the transgenic rice with changed drought resistance.

4. Use according to claim 3, characterized in that:

the drought resistance of the rice is adjusted to increase the drought resistance of the rice or reduce the drought resistance of the rice;

the cultivation of the transgenic rice with changed drought resistance is to cultivate the transgenic rice with increased drought resistance or cultivate the transgenic rice with reduced drought resistance.

5. A method for breeding transgenic rice beetles comprises the following steps: improving the expression quantity and/or activity of protein OsZZW1 in the starting rice to obtain transgenic rice A; the drought resistance of the transgenic rice A is reduced compared with that of the starting rice; the amino acid sequence of the protein OsZZW1 is shown in SEQ ID NO: 2, respectively.

6. The method of claim 5, wherein: the expression quantity and/or activity of the protein OsZZW1 in the starting rice is improved by introducing nucleic acid molecules for coding the protein OsZZW1 into the starting rice.

7. A method for breeding transgenic rice B comprises the following steps: inhibiting the expression quantity and/or activity of protein OsZZW1 in the starting rice to obtain transgenic rice B; the drought resistance of the transgenic rice B is increased compared with that of the starting rice; the amino acid sequence of the protein OsZZW1 is shown in SEQ ID NO: 2, respectively.

8. The method of claim 7, wherein: the expression level and/or activity of the protein OsZZW1 in the starting rice is inhibited by introducing a substance which inhibits the expression of a nucleic acid molecule encoding the protein OsZZW1 into the starting rice.

9. The method of claim 8, wherein:

the substance for inhibiting the expression of the nucleic acid molecule of the coding protein OsZZW1 is a specific DNA molecule, an expression cassette containing the specific DNA molecule or a recombinant plasmid containing the specific DNA molecule;

the specific DNA molecule comprises a sense segment, an antisense segment and a spacing segment positioned between the sense segment and the antisense segment;

the sense fragment is SEQ ID NO: 1 from the 94 th to the 474 th position from the 5' end;

the antisense fragment is SEQ ID NO: 1 from the 94 th to the 474 th position from the 5' end.

10. A rice breeding method A or a rice breeding method B;

the rice breeding method A comprises the following steps: increasing the content and/or activity of protein OsZZW1 in rice, thereby reducing drought resistance;

the rice breeding method B comprises the following steps: reducing the content and/or activity of the protein OsZZW1 in the rice, thereby increasing drought resistance;

the amino acid sequence of the protein OsZZW1 is shown in SEQ ID NO: 2, respectively.

11. The specific DNA molecule, the expression cassette containing the specific DNA molecule or the application of the recombinant plasmid containing the specific DNA molecule are S1) or S2):

s1) increasing the drought resistance of the rice;

s2) cultivating transgenic rice with increased drought resistance;

the specific DNA molecule comprises a sense segment, an antisense segment and a spacing segment positioned between the sense segment and the antisense segment;

the sense fragment is SEQ ID NO: 1 from the 94 th to the 474 th position from the 5' end;

the antisense fragment is SEQ ID NO: 1 from the 94 th to the 474 th position from the 5' end.

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