CN110885813B - Application of rice histone deacetylase gene HDA710 in delaying leaf senescence - Google Patents

Abstract

Translated fromChinese

本发明公开了水稻组蛋白去乙酰化酶基因HDA710在延迟叶片衰老中的应用。本发明提供了水稻HDA710蛋白的新用途，即在调控植物的叶片衰老进程中的应用。过表达HDA710基因的转基因植株，叶绿素含量升高、电导率降低，符合其叶片晚衰的特征。低表达HDA710基因的转基因植株，符合叶片早衰的特征。本发明为促进表观遗传和遗传变异在作物育种中的应用提供了新的方向。The invention discloses the application of rice histone deacetylase gene HDA710 in delaying leaf senescence. The present invention provides a new use of rice HDA710 protein, that is, the application in regulating the leaf senescence process of plants. The transgenic plants overexpressing the HDA710 gene had increased chlorophyll content and decreased electrical conductivity, which was in line with the characteristics of late leaf senescence. Transgenic plants with low expression of the HDA710 gene are in line with the characteristics of premature leaf senescence. The present invention provides a new direction for promoting the application of epigenetics and genetic variation in crop breeding.

Description

Application of rice histone deacetylase gene HDA710 in delaying leaf senescence

Technical Field

The invention relates to application of a rice histone deacetylase gene HDA710 in delaying leaf senescence.

Background

Epigenetic alterations can reprogram the transcriptome in a variety of biological processes. Deacetylation of histones plays an important regulatory role in plant development and response to various adversity stresses. In rice, 18 histone deacetylase genes have been reported. Wherein HDA710/OsHDAC2 belongs to type I histone deacetylase.

Leaf senescence is an important stage in plant development, and in agricultural production, early leaf senescence limits the plant photosynthesis cycle, thereby affecting crop yield. Rice, as a main grain crop in the world, mainly comprises two subspecies, and previous researches show that the leaf senescence in indica rice and japonica rice is different, and the leaf senescence of the indica rice is earlier than that of the japonica rice, but the specific mechanism research of the leaf senescence is not clear.

Genetic variation of different varieties provides valuable resources for improving agronomic traits of crops, and can widen the sources of phenotypic variation of crops. With the completion of 3K rice genome project, abundant genetic resources can help us to better establish the association between genes and phenotypes, and the method is greatly helpful for researching agronomic traits such as aging and the like.

Disclosure of Invention

The invention aims to provide application of a rice histone deacetylase gene HDA710 in delaying leaf senescence.

The HDA710 protein is also called HDA710/OsHDAC2 protein. The HDA710 protein is (a1) or (a2) or (a3) as follows:

(a1) protein shown as asequence 1 in a sequence table;

(a2) the protein shown in thesequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to the plant senescence process and derived from the protein;

(a3) a protein derived from rice, having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in (a1), and involved in the senescence process of plants.

The HDA710 gene is a gene encoding HDA710 protein. The HDA710 gene is also called HDA710/OsHDAC2 gene.

The HDA710 gene is (b1) or (b2) or (b3) or (b4) or (b5) as follows:

(b1) the coding region is shown as the 140 nd 1669 th nucleotide of thesequence 2 in the sequence table;

(b2) a DNA molecule shown as 140-position 2031 nucleotide of asequence 2 in a sequence table;

(b3) DNA molecule shown insequence 2 in the sequence table;

(b4) a DNA molecule derived from rice and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity to (b1) or (b2) or (b3) and encoding said protein;

(b5) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) or (b3) under stringent conditions and encodes the protein.

The stringent conditions are hybridization and washing of themembrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

The invention provides a method for preparing a transgenic plant, which comprises the following steps: introducing HDA710 gene into a receptor plant to obtain a transgenic plant; the transgenic plant senescence process is slowed compared to the recipient plant. The transgenic plant has a late senescence phenotype compared to the recipient plant.

The HDA710 gene may be specifically introduced into a recipient plant by a recombinant expression vector. The recombinant expression vector can be transformed into a recipient plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.

The recombinant expression vector containing the HDA710 gene can be constructed by using the existing plant expression vector.

The slowed aging process is manifested by high chlorophyll content and/or low conductivity.

The slowing of the senescence process is manifested by high leaf chlorophyll content and/or low leaf conductivity.

The late senescence is manifested as high chlorophyll content and/or low conductivity.

The delayed senescence is manifested by high leaf chlorophyll content and/or low leaf conductivity.

The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of HDA710 protein in the plant, thereby promoting a slowing of the senescence process in the plant. Thereby promoting the late senescence of the plants.

The invention also protects the application of the HDA710 protein, which is (c1) or (c2) or (c 3):

(c1) regulating the senescence process of plants;

(c2) promoting the aging process of plants to slow down;

(c3) promoting the late senescence of plants.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: introducing a nucleic acid molecule for inhibiting the expression of the HDA710 gene into a receptor plant to obtain a transgenic plant; the transgenic plant senescence progresses earlier than the recipient plant. The transgenic plant has a premature senescence phenotype compared to the recipient plant.

The nucleic acid molecule that inhibits the expression of the HDA710 gene may specifically be an antisense gene of the HDA710 gene. The gene that produces antisense RNA by transcription is called antisense gene (antisense gene).

The nucleic acid molecule that inhibits the expression of the HDA710 gene can be specifically introduced into a recipient plant by a recombinant expression vector. The recombinant expression vector can be transformed into a recipient plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.

The recombinant expression vector containing the nucleic acid molecule that inhibits the expression of the HDA710 gene can be constructed using existing plant expression vectors.

The invention also provides a plant breeding method, which comprises the following steps: inhibit the expression of the HDA710 gene in the plant, thereby promoting the senescence process of the plant. Thereby promoting the premature senility of the plants.

The invention also provides a plant breeding method, which comprises the following steps: reduce the content and/or activity of HDA710 protein in the plant, thereby promoting the aging process of the plant to advance. Thereby promoting the premature senility of the plants.

The invention also protects the application of the nucleic acid molecule for inhibiting the expression of the HDA710 gene, which is (d1) or (d 2):

(d1) advancing the aging process of the plants;

(d2) promoting plant senilism.

The senescence process is manifested in advance as a low chlorophyll content and/or a high conductivity.

The senescence process is manifested in advance as a low chlorophyll content of the leaves and/or a high electrical conductivity of the leaves.

The premature senility is characterized by low chlorophyll content and/or high electrical conductivity.

The premature senility is characterized by low chlorophyll content and/or high leaf conductivity.

Any of the above recipient plants is a monocot or a dicot.

Any of the above plants is a monocot or a dicot.

The monocotyledon may be a gramineous plant.

The gramineous plant may be a plant of the genus oryza.

The plant of the genus oryza may be japonica rice, for example, nipponlily.

When constructing a recombinant expression vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter may be added in front of its transcription initiation nucleotide, either alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, may be used in the construction of recombinant expression vectors, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the expression vector used may be processed, for example, by adding a gene expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance, or a chemical-resistant marker gene, etc. From the viewpoint of transgene safety, the transformed plants can be directly screened for phenotypes without adding any selectable marker gene.

The plant expression vector can be specifically a vector pCAMBIA 1300.

The invention provides a new application of rice HDA710 protein, namely an application in regulating and controlling the leaf senescence process of plants. The aging of plant leaves is mainly embodied by chlorophyll content and electric conductivity. The transgenic plant over expressing the HDA710 gene has the advantages of high chlorophyll content and low conductivity, and accords with the characteristics of late leaf senescence. The transgenic plant of the low expression HDA710 gene (namely, the transgenic plant inhibiting the expression of the HDA710 gene by introducing the antisense gene) accords with the characteristics of the premature leaf senescence. Namely, the over-expression of the HDA710 gene can promote the senescence process of plant leaves to be obviously delayed.

The inventor of the invention checks sequence polymorphism of rice histone deacetylase gene HDA710/OsHDAC2 through a rice metagenome browser (RPAN), finds that the difference of the HDA710 gene in indica rice and japonica rice is extremely obvious, and two fragments of the HDA710 gene are deleted in a gene region and a gene downstream region in the indica rice. Further, the inventors of the present invention performed correlation analysis on the varieties (Nipponbare, 9311 and Teqing) and phenotypes using the corresponding phenotypic traits, and found that the varieties with deletions tend to have premature leaf senescence.

The invention provides a new direction for promoting the application of epigenetic and genetic variation in crop breeding.

Drawings

FIG. 1 is a schematic diagram of the elements ofrecombinant plasmid 35S:: HDA710-sense andrecombinant plasmid 35S:: HDA 710-antisense.

FIG. 2 shows the relative expression level results of HDA710 gene in example 1.

FIG. 3 shows the results of the plant phenotype in example 1.

FIG. 4 is the result of the chlorophyll content in example 1.

FIG. 5 shows the results of the conductivity in example 1.

FIG. 6 shows the sequence difference of rice deacetylase gene HDA710 in indica and japonica rice varieties.

FIG. 7 is a graph showing the senescence phenotype of the cultivars corresponding to the two HDA710 fragment deletions shown in FIG. 6.

FIG. 8 shows the results of the plant phenotype in example 2.

FIG. 9 shows the results of the chlorophyll content in example 2.

FIG. 10 shows the results of the conductivity in example 2.

Note: in fig. 4, 5, 9 and 10, p-value <0.001 is indicated by x, p-value <0.01 is indicated by x, and p-value <0.05 is indicated by x.

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. In the quantitative tests in the following examples, four replicates were set up and the results averaged. The rice Nipponbare belongs to japonica rice.Rice 9311 belongs to indica rice. The rice is specially green and belongs to indica rice.

Example 1 preparation and characterization of HDA710 Gene transgenic plants

Construction of recombinant plasmid

1. Extracting total RNA of the rice Nipponbare, and carrying out reverse transcription by taking the total RNA as a template to obtain cDNA.

2. And (3) taking the cDNA obtained in the step (1) as a template, carrying out PCR amplification by adopting a primer pair consisting of sense-vector-F and sense-vector-R, and recovering a PCR amplification product.

sense-vector-F：5’-gCTCTAgAATggACCCCTCgTCggC-3’；

sense-vector-R：5’-ggggTACCCTCATACATAAAAACgTAggAA-3’。

3. And (3) taking the PCR amplification product obtained in the step (2), carrying out double enzyme digestion by using restriction enzymes XbaI and KpnI, and recovering the enzyme digestion product.

4. The vector pCAMBIA1300 was digested with restriction enzymes XbaI and KpnI, and the vector backbone was recovered.

5. And (4) connecting the enzyme digestion product obtained in the step (3) with the vector skeleton obtained in the step (4) to obtain therecombinant plasmid 35S, HDA 710-sense. According to the sequencing results, the structure ofrecombinant plasmid 35S, HDA710-sense, is described as follows: a DNA molecule shown as 140 th-2031 st nucleotide of thesequence 2 in the sequence table is inserted between the XbaI and KpnI enzyme cutting sites of the vector pCAMBIA 1300. In thesequence 2 of the sequence table, the nucleotide at position 140-1669 is the open reading frame of the HDA710 gene.

6. And (3) taking the cDNA obtained in the step (1) as a template, carrying out PCR amplification by adopting a primer pair consisting of antisense-vector-F and antisense-vector-R, and recovering a PCR amplification product.

antisense-vector-F:5’-ggggTACCATggACCCCTCgTCg-3’；

antisense-vectot-R:5’-gCTCTAgACTCATACATAAAAACgTAggAA-3’。

7. And (4) taking the PCR amplification product obtained in the step (6), carrying out double enzyme digestion by using restriction enzymes KpnI and XbaI, and recovering the enzyme digestion product.

8. The vector pCAMBIA1300 is subjected to double digestion by restriction enzymes KpnI and XbaI, and the vector backbone is recovered.

9. And (3) connecting the enzyme digestion product obtained in the step (7) with the vector skeleton obtained in the step (8) to obtain arecombinant plasmid 35S, namely HDA 710-antisense.

The components ofrecombinant plasmid 35S:: HDA710-sense andrecombinant plasmid 35S:: HDA710-antisense are shown in figure 1.

Preparation of pseudotransgenic plants

1. The recombinant plasmid (recombinant plasmid 35S:: HDA710-sense orrecombinant plasmid 35S:: HDA710-antisense) was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

2. Embryogenic callus was prepared from seeds of Nipponbare of rice.

3. Inoculating the recombinant agrobacterium obtained in thestep 1 to a liquid AAM culture medium, and culturing to OD_600nmApproximately equal to 0.1.

4. And (3) soaking the embryonic callus obtained in the step (2) in the bacterial liquid obtained in the step (3), slightly shaking for about 1.5min, then taking out, absorbing excess bacterial liquid by using sterile filter paper, and drying by blowing.

5. And (4) taking the callus obtained in the step (4), and sequentially carrying out co-culture, screening culture, induced differentiation and rooting culture to obtain a regenerated plant, also called a pseudotransgenic plant. The antibiotics and their concentrations used in the screening culture were: 50mg/L hygromycin B and 400mg/L carbenicillin. The culture medium contains 50mg/L hygromycin B and 200mg/L carbenicillin during induced differentiation and rooting culture.

Therecombinant plasmid 35S is a regenerated plant obtained by the HDA710-sense through the steps and is named as a quasi-overexpression plant.

Therecombinant plasmid 35S is a regenerated plant obtained by the steps of HDA710-antisense and is named as a pseudo-low expression plant.

6. And carrying out PCR identification on the pseudotransgenic plant. If the amplified product with the expected size is displayed, the PCR is identified as positive, and the plant is a T0 generation transgenic plant.

The PCR primers were as follows (target fragment located in hygromycin resistance gene, target fragment approximately 947 bp):

HPT-F：5’-TACTTCTACACAGCCATC-3’；

HPT-R:5’-CGTCTGTCGAGAAGTTTC-3’。

7. and (3) taking the T0 transgenic plants screened in the step (6), selfing, harvesting seeds (namely T1 seeds), and culturing the seeds into plants, namely T1 plants.

8. Taking T1 generation plants, selfing and harvesting seeds (the seeds are T2 generation seeds), and breeding the seeds into plants, namely T2 generation plants.

9. The T1 generation plants and the sampled T2 generation plants were subjected to PCR identification (the same method as the step 6). For a certain T1 generation plant, if the T2 generation plants obtained by selfing are all transgenic plants, the T1 generation plant is homozygous transgenic plant, and the selfed progeny of the plant is a transgenic line.

The transgenic strains obtained by the quasi-overexpression plants are strain S1 and strain S2.

The transgenic strains obtained by the pseudo-low expression plants are strain A1 and strain A2.

Three, real-time fluorescent quantitative RT-PCR identification

Test plants: nipponbare (Nipponbare), T3 generation plant of the strain S1, T3 generation plant of the strain S2, T3 generation plant of the strain A1, and T3 generation plant of the strain A2.

The test plants were cultured normally under parallel conditions.

1. Taking leaves of a test plant growing around, extracting total RNA, and carrying out reverse transcription by taking the total RNA as a template to obtain cDNA.

2. And (3) carrying out real-time fluorescence quantitative PCR by using the cDNA obtained in the step (1) as a template. The 18sRNA gene was used as an internal reference gene. The method reported by Livak KJ and Schmittgen TD (2001), 2^-ΔΔCTAnd calculating the relative expression amount. Three biological replicates were set and the results averaged.

The primers used to identify the HDA710 gene in the over-expressed strain were as follows:

sense-F：5'-AGTACCAGAACCGATGGCAG-3'；

sense-R：5'-GTTGATGGCCATCGAATTTG-3'。

the primers used to identify the HDA710 gene in low-expression lines were as follows:

antisense-F：5'-AGGCAATACTGGACGTGGAG-3'；

antisense-R：5'-TTCGCCTGAAAGTTCCATCT-3'。

primers used to identify the 18sRNA gene were as follows:

18s-F：5'-GCTTTGGTGACTCTAGATAAC-3'；

18s-R：5'-GTCGGGAGTGGGTAATTTGC-3'。

the relative expression levels of the HDA710 gene are shown in figure 2. The expression level of the HDA710 gene of the over-expression strain (strain S1 and strain S2) is obviously higher than that of the Nipponbare of rice, and the expression level of the HDA710 gene of the low-expression strain (strain A1 and strain A2) is obviously lower than that of the Nipponbare of rice.

Fourth, character identification

Test plants: nipponbare (Nipponbare), T3 generation plant of the strain S1, T3 generation plant of the strain S2, T3 generation plant of the strain A1, and T3 generation plant of the strain A2.

The test plants were normally managed in parallel in the field.

1. And observing the phenotype of the plant.

Photographs of 104-day-old plants were shown in FIG. 3A.

Photographs of flag leaves of plants at 104 days of emergence are shown in fig. 3B.

Photographs of the inverted two leaves of the plants at 104 days of emergence are shown in FIG. 3C.

Compared with Nipponbare, the leaves of the over-expression strain (strain S1, strain S2) are greener and have a late senescence phenotype. Compared with Nipponbare, leaves of low-expression strain (strain A1, strain A2) are yellow and have a premature senescence phenotype.

2. Detection of chlorophyll content

And (3) sprouting for 104 days, taking flag leaf leaves of plants, weighing 20mg (fresh weight), grinding into powder, soaking in 80% acetone solution at 4 ℃ overnight, centrifuging at 12000rpm for 10min, collecting supernatant, and measuring the wavelengths of 646nm and 663nm by using an enzyme labeling instrument.

Chlorophyll a (mg/ml) is 12.21A_663nm-2.81_A646nm；

Chlorophyll b (mg/ml) ═ 20.13A_646nm-5.03A_646nm；

Chlorophyll content ═ chlorophyll a + chlorophyll b.

Calculating the chlorophyll content of each gram of fresh weight of the leaves, wherein the unit is mg/g FW.

Set up 6 biological replicates and average the results.

The results are shown in FIG. 4 and Table 1.

TABLE 1

	Repetition of 1	Repetition 2	Repetition of 3	Repetition of 4	Repetition 5	Repeat 6	Mean value of
								Strain A1	0.1009	0.2191	0.1732	0.1343	0.1691	0.1815	0.1915
Strain A2	0.1736	0.1672	0.1888	0.1734	0.2199	0.2258	0.163
								Nipponbare of rice	0.2749	0.2613	0.2986	0.3528	0.395	0.3947	0.3296
Strain S1	0.4853	0.67	0.7429	0.5669	0.6249	0.6312	0.6202
								Strain S2	0.5226	0.5527	0.5752	0.6047	0.6861	0.6807	0.6037

3. Detecting conductivity

After 114 days of emergence, flag leaf leaves of the plants were taken, soaked in 400mM mannitol solution for 3 hours with shaking at 70rpm, after which the initial conductivity S1 was measured, followed by boiling at 100 ℃ for 10 minutes, followed by measurement of the final conductivity S2, and then the relative conductivity REC was calculated. REC (%) (S1/S2) × 100.

Set 4 biological replicates and average the results.

The results are shown in FIG. 5 and Table 2.

TABLE 2

REC(％)	Repetition of 1	Repetition 2	Repetition of 3	Repetition of 4	Mean value of
						Strain A1	15.0277	22.04424	15.29988	25.34364	19.42887
Strain A2	14.31818	13.63095	16.90476	30.26397	14.9513
						Nipponbare of rice	11.2945	12.64456	13.17104	11.34492	12.11376
Strain S1	5	5.171806	10.36154	11.76318	8.074131
						Strain S2	8.248276	9.021497	7.348259	10.73016	8.837048

Examples 2,

Firstly, digging indica rice and japonica rice difference key genes by using RPAN website and associating with phenotype

The deacetylase gene HDA710 is located on the website RPAN (http://cgm.sjtu.edu.cn/3kricedb/) Searching the website, and finding out five subspecies of rice [ JAP (japonica rice), IND (indica rice), AUS (indica rice similar variety), ARO (japonica rice similar variety), ADM (indica rice and japonica rice intermediate variety)]The frequency of occurrence of (a). The two segments of the HDA710 gene were found by manual review using the UCSC browser of the RPAN website in the absence of different species, see fig. 6. From Rice SNP-Seek Database (https://snp-seek.irri.org/_download.zul) Downloading 2266 varieties on websitePhenotypic data, phenotypic associations with hypergeometric distributions for varieties with deletion of the HDA710 gene segment, are shown in fig. 7.

Second, the comparison of the characteristics of Nipponbare,rice 9311 and extra-green

The rice Nipponbare belongs to japonica rice.Rice 9311 belongs to indica rice. The rice is specially green and belongs to indica rice. Compared with japonica rice Nipponbare, the HDA710 genes ofindica rice 9311 and indica rice ultragreen have two fragment deletions which are respectively positioned in an intron region and a 3' end non-transcription region.

Test plants: nipponbare (Nipponbare), 9311(9311) and TQ (TQ).

The test plants were normally managed in parallel in the field.

1. And observing the phenotype of the plant.

Photographs of flag leaves of 104-day-old plants were shown in fig. 8A.

Photographs of the inverted two leaves of the plants at 104 days of emergence are shown in FIG. 8B.

2. Detection of chlorophyll content

And (4) seedling emergence for 108 days, and directly measuring the chlorophyll content of flag leaf blades of the plants by using a SPAD-502 chlorophyll meter. The SPAD-502 chlorophyll meter determines the relative content of chlorophyll in the leaves at present by measuring the difference between optical concentrations of the leaves at two wavelengths, namely 650nm and 940nm, and automatically calculates the value, wherein the unit is SPAD.

16 biological replicates were set for rice Nipponbare and the results were averaged.

Rice 9311 were set with 7 biological replicates and the results averaged.

15 biological replicates of the ultra-green rice are set, and the results are averaged.

The results are shown in Table 3 and FIG. 9.

TABLE 3

3. Detecting conductivity

After 114 days of emergence, flag leaf leaves of the plants were taken, soaked in 400mM mannitol solution for 3 hours with shaking at 70rpm, after which the initial conductivity S1 was measured, followed by boiling at 100 ℃ for 10 minutes, followed by measurement of the final conductivity S2, and then the relative conductivity REC was calculated. REC (%) (S1/S2) × 100.

Set 4 biological replicates and average the results.

The results are shown in FIG. 10 and Table 4.

TABLE 4

	Repetition of 1	Repetition 2	Repetition of 3	Repetition of 4	Mean value of
						Nipponbare	11.2945	12.64456	13.17104	11.34492	12.11376
9311	38.13853	33.62989	37.29323	49.66346	39.68128
						TQ	80.5	81.47208	74.13636	78.52792	78.65909

SEQUENCE LISTING

<110> university of agriculture in China

<120> application of rice histone deacetylase gene HDA710 in delaying leaf senescence

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<170> PatentIn version 3.5

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cgaggactgc cccgtcttcg acggcctcta cgcctactgc cagacctacg cgggggcctc 540

cgtcggcgcc gccgtcaagc tcaaccacgg cacccacgac atcgccatca actggtccgg 600

cgggttgcac cacgccaaga agtccgaggc ctccggcttc tgctacgtca acgacatcgt 660

cctcgccatc ctcgagctcc tcaagctcca tgagcgagtt ctgtatattg atattgatat 720

ccatcatgga gatggagttg aggaggcatt ctacacaaca aacagggtta tgacagtctc 780

atttcacaag tttggggatt atttcccggg aacaggggac atccgcgata ttgggtattc 840

agaagggaag tattactgcc tgaatgtccc gctggatgat ggaattgatg atgacagcta 900

ccagtccatc ttcaagccga tcatcagcaa agtcatggag atgtatcgtc ctggtgcagt 960

cgtgcttcag tgcggcgctg attcgttgtc cggtgatagg ttgggctgtt tcaatctctc 1020

aggcaaaggt catgctgaat gtgttaagtt catgaggtct ttcaatgttc cgttgcttct 1080

tcttggtggt ggtggatata ccataagaaa tgttgcacgc tgctggtgtt acgagacagg 1140

agttgcactt ggtgaagagc tacgggagaa gttgccttat aacgagtatt atgaatattt 1200

tggtccagaa tacagtcttt acgttgcagc aagtaacatg gagaacagaa atacaaacaa 1260

gcaattggag gaaataaaat gcaacattct ggacaatctc tcaaaacttc aacatgctcc 1320

tagtgtccaa tttgaagagc gaattcctga aacaaagcta cctgagccag atgaagatca 1380

agatgatcca gatgaaaggc acgaccctga ctctgatatg ctgttggatg atcacaaacc 1440

tatgggacac tcagcaagaa gccttattca caacatcgga gttaagagag aaattactga 1500

aacagagacc aaagatcagc atggtaagag attaacaact gaacataaag taccagaacc 1560

gatggcagac gatcttggtt cctccaagca agttcctact gcggatgcaa attcgatggc 1620

catcaacgcg ccaggcaacg ccaagaatga accgggaagc tcactatgaa ctaacccact 1680

tcaggccacc agctcttatg ccacggattc ctgcatgcta ttagttactt actccgaggt 1740

gttgtatgac tgtttcttct gtaggagttt acaaattaca actgtttctt ttgttgaaga 1800

attccaaagt gtccaggcaa tactggacgt ggagacatta gtctgatcaa tgtactgcag 1860

agtacagaac atgtaacatg aaccaagtta cagtgtatag tcttttagac tgttagatgg 1920

aactttcagg cgaaatgcct gtgcctactt gtgtgtgctc tctgttggtt gtttttcgtt 1980

aaaatatttt gttccggttt atcatgtact tcctacgttt ttatgtatga gttgttttaa 2040

agagacaaat aaatctattt tttaatttat aatagttaat acttaattaa ttatgtgcta 2100

gtaaa 2105

Claims (4)

1. A method of making a transgenic plant comprising the steps of: introducing a gene coding HDA710 protein into a receptor plant to obtain a transgenic plant; the transgenic plant senescence process is slowed, as compared to the recipient plant; the slowing of the senescence process is reflected in high leaf chlorophyll content and/or low leaf conductivity; the HDA710 protein is a protein shown in a sequence 1 in a sequence table; the plant is a plant of the genus oryza.

2. The method of claim 1, wherein: the gene encoding the HDA710 protein is (b1) or (b2) or (b3) as follows:

(b1) the coding region is shown as the 140 nd 1669 th nucleotide of the sequence 2 in the sequence table;

(b2) a DNA molecule shown as 140-position 2031 nucleotide of a sequence 2 in a sequence table;

(b3) DNA molecule shown in sequence 2 in the sequence table.

The use of HDA710 protein to slow the senescence process in plants; the slowing of the senescence process is reflected in high leaf chlorophyll content and/or low leaf conductivity; the HDA710 protein is a protein shown in a sequence 1 in a sequence table; the plant is a plant of the genus oryza.

4. The use of a nucleic acid molecule which inhibits the expression of the HDA710 gene to promote the senescence process of plants in advance; the senescence process is manifested in advance as low chlorophyll content and/or high leaf conductivity; the HDA710 gene is a gene encoding HDA710 protein; the HDA710 protein is a protein shown in a sequence 1 in a sequence table; the plant is a plant of the genus oryza.

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