CN114480417A - Gene ZmSAG39 for regulating and controlling leaf senescence, encoding protein and application thereof - Google Patents

Abstract

The invention provides a gene ZmSAG39 for regulating leaf senescence, a coding protein and application thereof, belonging to the technical field of genetic engineering. The nucleotide sequence of the gene ZmSAG39 for regulating leaf senescence is shown in SEQ ID NO. 1. The invention provides a gene ZmSAG39 for regulating and controlling leaf senescence. Protein ZmSAG39 is regulating the plant senescence process, and overexpression of gene ZmSAG39 promotes leaf senescence. The gene is a new plant senescence related gene, the gene ZmSAG39 can participate in the senescence regulation process, and the growth period of a plant population is adjusted to achieve the purpose of increasing the yield, so the gene has very important application value, and can be used for cultivating anti-senescence transgenic plants, particularly anti-leaf senescence transgenic plants.

Description

Gene ZmSAG39 for regulating and controlling leaf senescence, encoding protein and application thereof

Technical Field

The invention relates to the technical field of gene engineering, in particular to a gene ZmSAG39 for regulating and controlling leaf senescence, a coding protein and application thereof.

Background

Aging is a biological process in which an organism gradually degrades over time at the cellular, tissue, organ, and individual level, which triggers the end of life. Plant senescence is an organ-level decline process often accompanied by death of mesophyll cells and individuals, such as leaf senescence during the filling of annual plants like rice, corn and soybean. For perennial plants such as trees, senescence is mainly reflected in the change of leaf color in autumn.

Plant senescence is not a passive disordered decline process, which is essentially a programmed death process of mesophyll cells. The leaves are the main place for photosynthesis of plants, and after a period of time, the leaves are photosynthetic and accumulate nutrients, and the mesophyll cells enter the senescence stage. During senescence, mesophyll cells undergo ordered changes in structure, metabolism and gene expression. Chloroplasts contain about 70% of the total protein of the mesophyll cells and are the first organelles to be degraded. The rapid decrease of the chlorophyll content of the aged leaves is an important index of the leaf aging rate. Subsequently, the activities of organelles such as peroxisomes are gradually decreased and degraded. The nucleus and mitochondria control gene transcription and energy supply, and their integrity is maintained until the end of senescence. In the aspect of cell metabolism, the carbon assimilation process of the aged leaves is gradually replaced by the degradation process of biological macromolecules such as chlorophyll, proteins, membrane lipids and RNA, and therefore nutrients are promoted to be transferred to organs such as young leaves, seeds and fruits. Leaf senescence is a physiological phenomenon that all plant life activities in nature must undergo, and is a biological process that is selected to be retained in the plant evolution process. In agricultural production, leaf senescence shortens the life cycle of crops, reduces the yield of crops, and causes leaf yellowing and nutrient loss of vegetable crops. Therefore, the digging of genetic control genes of leaf senescence is helpful for deepening the understanding of human beings on the physiological phenomenon, thereby providing a theoretical basis for improving senescence-related traits in the biotechnology.

Research shows that plant senescence is regulated and controlled by interaction of internal genetic factors, growth environment and other external factors. The external factors influencing the occurrence of senescence include various environmental stress factors such as high temperature, low temperature, drought, ozone, nutrient deficiency, pathogen infection and shading, and the internal factors mainly include fluctuation of the expression level of genes (such as photosynthesis regulatory gene CAB2, protein synthesis regulatory genes RPS and RBC, senescence-associated marker genes SAGs and the like) and changes of the content of phytohormones (such as cytokinin, ethylene, acetylsalicylic acid, jasmonic acid and the like).

The identified senescence regulatory genes greatly promote the human understanding of the senescence process, but the mining of new senescence regulatory genes still has important theoretical significance and application value. Although the art of isolating genes that identify SAGs regulation is mature, consideration remains for identifying a number of novel genes that regulate senescence independent of the SAGs pathway. Screening for senescence-associated mutants using forward genetics is one of the effective methods for identifying such genes. It is known that senescence initiation, occurrence and termination are a complex biological process that is finely regulated, and the existing genetic screening is far from being saturated, so it is important to select different senescence mutants to clone new senescence regulatory genes. Compared with T-DNA insertion mutant libraries, the chemical mutagenesis approach would be more valuable because it could provide new allelic variations, such as AHK3/ORE12, involved in the discovery process that regulates senescence. Screening the inhibitory mutation of the existing senescence mutant is helpful for further analyzing the molecular genetic network for regulating senescence. In addition, senescence is the last step in leaf development, and thus senescence-controlling genes may also be involved in other biological processes to play a role. To identify other biological pathways coupled to regulate the senescence process, analysis of senescent mutant transcriptome changes can provide important clues. It is worth pointing out that the molecular control mechanism of leaf senescence is mainly based on the analysis of gene expression level, which is only one aspect of the biological function of gene, and other control mechanisms (such as protein level, protein stability and protein subcellular localization) mediated senescence process should be considered. Integrating proteome and metabonomics analysis methods helps to further elucidate the mechanisms of regulation of aging molecules. Another significant challenge in the field of leaf senescence research is the evaluation of the application prospects of senescence-controlling genes. Methods for genetically modifying a single gene to directionally improve target traits (such as crop yield, nutritional quality, stress tolerance or disease resistance) by using bioengineering technology are becoming mature, and it is important to identify a practical and effective senescence control gene. For crops growing in fields, because various competitive inhibition and environmental stress are faced, the regulatory gene which essentially changes the crop senescence trait is screened, and the method has important theoretical significance and application value for breeding new varieties of anti-senescence crops. In consideration of the future faced significant problems of food shortage and energy shortage, the improvement of crop yield should be prioritized.

Disclosure of Invention

The invention aims to provide a gene ZmSAG39 for regulating and controlling leaf senescence, a coding protein and application thereof, wherein the protein ZmSAG39 positively regulates the plant senescence process, and the over-expression ZmSAG39 gene promotes the leaf senescence. The gene is a new plant senescence related gene, the gene ZmSAG39 can participate in the senescence regulation process, and the growth period of a plant population is adjusted to achieve the purpose of increasing the yield, so the gene has very important application value, and can be used for cultivating anti-senescence transgenic plants, particularly anti-leaf senescence transgenic plants.

The technical scheme of the invention is realized as follows:

the invention provides a gene ZmSAG39 for regulating leaf senescence, and the nucleotide sequence is shown in SEQ ID NO. 1.

The invention further protects the application of the gene ZmSAG39 for regulating the leaf senescence in regulating the leaf senescence performance.

The invention further protects the application of the gene ZmSAG39 for regulating and controlling leaf senescence in the improvement of maize germplasm resources.

The invention further protects the application of the gene ZmSAG39 for regulating the leaf senescence in transgenic corn for regulating the leaf senescence.

The invention further protects a protein coded by the gene ZmSAG39 for regulating leaf senescence, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention further protects the biological material containing the gene ZmSAG39 for regulating the leaf senescence, wherein the biological material is an expression cassette, a vector, an engineering bacterium or a cell.

As a further improvement of the invention, the vector is a pCAMBIA3301 vector, and the multiple cloning site region of the pCAMBIA3301 vector is sequentially connected with a 35S promoter, the gene ZmSAG39 as described above and a terminator.

As a further development of the invention, the cell is a host cell which contains a vector as described above and/or has integrated into its genome the foreign forward or reverse sequence of the gene ZmSAG39 as described above.

The invention further provides a method for delaying the leaf senescence of plants, which integrates the gene ZmSAG39 for regulating the leaf senescence into cells, tissues and organs of the plants and leads the cells, tissues and organs to be over-expressed.

As a further improvement of the invention, the plants include corn, rice, wheat and Arabidopsis.

The invention has the following beneficial effects: the invention provides a gene ZmSAG39 for regulating and controlling leaf senescence. Protein ZmSAG39 is regulating the plant senescence process, and overexpression of gene ZmSAG39 promotes leaf senescence. The gene is a new plant senescence related gene, the gene ZmSAG39 can participate in the senescence regulation process, and the growth period of a plant population is adjusted to achieve the purpose of increasing the yield, so the gene has very important application value, and can be used for cultivating anti-senescence transgenic plants, particularly anti-leaf senescence transgenic plants.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows the expression pattern of gene ZmSAG39 in different tissues of maize inbred line H8186;

FIG. 2 is a map schematic of recombinant expression vector pCAMBIA3301-SAG 39;

FIG. 3 shows the results of the identification of the expression level of gene ZmSAG39 in example 4;

FIG. 4 is the results of the phenotypic observation in example 4;

FIG. 5 shows the results of relative chlorophyll content measurement in example 4;

FIG. 6 is the results of the relative ion permeability test in example 4;

FIG. 7 shows H in example 4₂O₂The result of the content detection;

FIG. 8 is a graph showing the effect of dark treatment on leaf senescence in example 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The methods used in the examples are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used therein are commercially available products unless otherwise specified.

A novel protein was discovered from maize as described in sequence 2 of the sequence listing and designated ZmSAG39 protein. The full-length gene for coding the SAG39 protein is shown as a sequence 1 in a sequence table. In the experiment, roots, stems, leaves, female ears and male ears of a maize inbred line H8186 in the mature period are selected, the relative expression level of the gene ZmSAG39 detected by a qRT-PCR experiment method is shown in figure 1, and figure 2 is a map schematic diagram of a recombinant expression vector pCAMBIA3301-SAG 39. The transcriptional level of gene ZmSAG39 varied in each tissue, suggesting that gene ZmSAG39 may play an important role in certain tissues.

Example 1 cloning of SAG39 Gene

A maize inbred line H8186 is selected as an experimental material, and leaf tissue of maize in a three-leaf stage is extracted and is reversely transcribed for standby.

Biological information of SAG39 is retrieved from a corn database, and the transcript ID is GRMZM2G070011_ P01. Designing a specific amplification primer by taking a gene sequence as a template, wherein the specific amplification primer sequence is shown as follows:

SAG39-F：5’-AAACAACTGCACTAGACATCTC-3’；

SAG39-R：5’-AGCCTAATCTTATTTATTATTGAAA-3’。

the amplification reaction was carried out using cDNA as a template and SAG39-F and SAG39-R as primers and the high fidelity enzyme Primer STAR Max Premix (2X) manufactured by TAKARA, and the amplification reaction system was as follows:

TABLE 1

The PCR amplification procedure comprises pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 35s, extension at 72 ℃ for 30s, and finally, extension at 72 ℃ for 10min to complete the reaction, and the obtained PCR product is detected by agarose gel electrophoresis with the mass ratio of 2%.

The target gene is cut and recovered by a gel recovery kit of Axygen company, is connected with a pMD-18T vector and then is transformed into a DH5 alpha escherichia coli competent cell, and bacteria liquid PCR is carried out to screen positive clones and send the positive clones to sequencing.

Example 2 construction of SAG39 Gene overexpression vector

According to the multiple cloning sites of pCAMBIA3301 and the sequence characteristics of the target gene, BglII and BstEII enzyme cutting sites are added at two ends of the target gene pMD-18T-SGA39, and the sequences of amplification primers are shown as follows:

SAG39-F：5’-actcttgaccatggtagatctAAACAACTGCACTAGACATCTC-3’；

SAG39-R：5’-ggggaaattcgagctggtcaccAGCCTAATCTTATTTATTATTGAAA-3’。

the plasmid was double digested with BglII and BstEII to obtain pCAMBIA3301 large fragment. The enzyme was cleaved at 37 ℃ for 3 hours, and then ligated with the desired gene in the following manner:

TABLE 2

After the ligation reaction was carried out at 52 ℃ for 15min, the recombinant plasmid was transformed into E.coli competent cells, and after selection, shaking, plasmid extraction, and double restriction enzyme validation and company sequencing, the gene over-expression vector pCAMBIA3301-SAG39-Bar shown in FIG. 3 was obtained.

EXAMPLE 3 Arabidopsis genetic transformation of the Gene ZmSAG39

(1) Cleaning of Arabidopsis seeds

50ml of sodium hypochlorite solution is firstly taken and put into 50ml of water to prepare 5 percent sodium hypochlorite solution. And (3) putting seeds to be cleaned in a sterile centrifuge tube, adding 1ml of 5% sodium hypochlorite solution into each tube, soaking and cleaning for 10-15min, shaking at intervals and then rinsing with sterile water, shaking and uniformly mixing, so as to clean the sodium hypochlorite solution and impurities on the surfaces of the seeds, and treating the cleaned and sterilized Columbia wild type Arabidopsis seeds in a refrigerator at 4 ℃ for 2-3 days for later use.

(2) Cultivation of Arabidopsis thaliana

Taking out the cleaned seeds, uniformly spreading the seeds on a culture dish of an MS solid culture medium by a liquid transfer gun, transferring the seedlings growing for about 15 days on the culture medium into mixed nutrient soil for growth in an arabidopsis thaliana culture room, wherein the volume ratio of the nutrient black soil to the frog stone is 3:8, continuously culturing in a greenhouse, keeping the moisture of the seedlings which are just transplanted, and uncovering the seedlings 2-3 days later by adopting a light-transmitting plastic film covering method.

(3) Agrobacterium-mediated transformation of Arabidopsis thaliana

The constructed pCAMBIA3301-SAG39-Bar overexpression vector is introduced into the GV3101 Agrobacterium infected cell to obtain the infectious bacterium. Most of the arabidopsis thaliana to be transplanted is prepared for the first flowering, namely the optimal period of arabidopsis thaliana infection. In order to promote the multiplication of flowering branches of arabidopsis thaliana, the buds flowering in advance can be trimmed, and existing siliques can be cut off at the same time, so that the positive rate of transgenic seeds is improved. The transformation method comprises the following steps: carrying out amplification culture on the infected bacteria to OD₆₀₀The value is 0.6-0.8. While preparing the Buffer solution, 1L of Buffer solution needs 2.1g MS, 50g sucrose, 1ml B5 vitamin, and pH is adjusted to 5.8 by NaOH. The treating fluid is added with a surfactant before use. And (3) suspending and centrifuging the stirred mixed Buffer solution to obtain a bacterium block, thus obtaining the infection solution.

Dip-dyeing arabidopsis inflorescences: when the height of the arabidopsis is about 10 cm, soaking the arabidopsis inflorescence by using the agrobacterium-mediated staining solution, repeating for 2-3 times, wrapping the overground part of the plant by using a preservative film, and culturing for 3 days in the dark. After one worship, the exhaust was repeated again. Harvesting T from mature Arabidopsis thaliana in about two months₀Seed generation, drying, and storing at 4 deg.C.

(4) Screening of ZmSAG39 transgenic Arabidopsis

The above-harvested Arabidopsis thaliana T₀The seeds were sterilized (rinsed with 5% sodium hypochlorite solution and then rinsed with sterilized distilled water 2-3 times, each for about 1 minute), vernalized at 4 ℃ for 1 day (protected from light), and spread evenly on a medium plate containing herbicide while using wild type Arabidopsis seeds as a control. If the wild type Arabidopsis seeds can normally germinate and grow on the plate without resistance, the wild type Arabidopsis seeds can not normally germinate and grow on the plate with resistance, but the transgenic Arabidopsis seeds can normally germinate and grow on the plate with resistanceIf the plants grow frequently, the screening of positive Arabidopsis plants is considered to be effective.

And (3) performing molecular detection on the screened positive plants by taking the gene ZmSAG39 as a target gene, and preliminarily identifying whether the transgenic arabidopsis thaliana is successfully transformed.

(5) Acquisition of transgenic Arabidopsis progeny ZmSAG39

According to the procedure, the seed (T) was preliminarily identified as a transgenic Arabidopsis plant₁Generation) screening with a medium plate containing herbicide to obtain T₁Plant generation, wait for T₁When the generation plant pod is mature, the transgenic T is harvested₂Seeds are generated, and the analogy is repeated, and finally two groups of stably inherited transgenic arabidopsis T are obtained₂Lines of generations, OE1 and OE6, respectively. The transgenic strain plays an important role in coordination in the whole physiological process.

Example 4 identification of plants

The test plants were cultured under the same conditions. The culture conditions are as follows: 16 h light/8 h dark, 24 ℃.

Identification of expression level of gene ZmSAG39

Test plants: t of wild type Arabidopsis thaliana, OE1 strain₃Generation plant, OE6 strain T₃And (5) plant generation.

The test plants were cultured under the same normal conditions. Taking the leaf of the tested plant growing for 4 weeks, extracting total RNA, and carrying out reverse transcription to obtain cDNA. And carrying out real-time fluorescence quantitative PCR by taking the cDNA as a template. The reference gene is Actin. One parallel experiment was set to 3 replicates. By use of 2^-ΔΔCTAnd calculating the relative expression amount.

qSAG39-F：5'-ACATGAACCATGCAGTGACG-3'；

qSAG39-R：5'-AGCTGCATGAAACCGTTCTC-3'；

Actin-F:5'-CTACGAGCAGGAACTCGAGA-3'；

Actin-R:5'-GATGGACCTGACTCGTCATAC-3'。

The results are shown in FIG. 3. The relative expression quantity of the gene ZmSAG39 of the transgenic line is obviously higher than that of the ecotypic Arabidopsis.

Second, character identification

1. Phenotypic observations

Photographs of plants grown to 6 weeks are shown in FIG. 4. The senescence degree of the transgenic line is higher than that of the wild arabidopsis, and the high senescence degree is embodied in that the lotus throne leaves have high yellowing degree and the number of the yellowing lotus throne leaves is large.

2. Relative chlorophyll content detection

Plants up to 6 weeks old were harvested from rosette leaves and tested for relative chlorophyll content (3 plants per line). The relative chlorophyll content was determined as follows: 5ml of 80% acetone extract was added to the sample and extracted in the dark for 24 hours to completely de-green the leaves. The chlorophyll content was determined by measuring absorbance at 665nm and 645nm with a UV2400 UV/VIS spectrophotometer, using 80% acetone as a control.

The results are shown in FIG. 5 and Table 2. The relative chlorophyll content in the rosette leaves of the transgenic line is obviously lower than that of the wild arabidopsis.

TABLE 3

	Relative chlorophyll content (SPAD)
		Ecotypic arabidopsis thaliana	14.2531±1.4021
Strain OE1	6.9455±1.4234
		Strain OE6	5.8835±0.8994

3. Relative ion permeability detection

And (5) taking rosette leaves of plants growing to 5 weeks, and detecting the relative ion permeability. The relative ion permeability is determined as follows: putting the leaves into a 15ml centrifuge tube, adding 10ml distilled water, shaking up, oscillating at 180rpm at room temperature for 12h, and detecting the conductivity to obtain the initial conductivity (S1); and then putting the centrifuge tube into a water bath kettle, reacting for 10min at 100 ℃, cooling to room temperature, and detecting the conductivity to obtain the final conductivity (S2). The relative conductivity (REC) of the blade is calculated as: REC (%) ═ S1/S2 × 100. The conductivity was measured using a DDSJ-308A conductivity meter, Shanghai point science instruments, Inc.

The results are shown in FIG. 6 and Table 3. The relative ion permeability of the transgenic plant rosette leaves is obviously higher than that of wild arabidopsis thaliana.

TABLE 4

4、H₂O₂Content detection

Taking rosette leaves of plants growing to 4 weeks, and detecting H by adopting Red hydrogen peroxide/catalase detection kit₂O₂Content (3 plants tested per line).

The results are shown in FIG. 7 and Table 4. Transgenic line rosette leaves H₂O₂The content is obviously higher than that of wild arabidopsis.

TABLE 5

	H2O2Content (μ M/g fresh weight)
		Ecotypic arabidopsis thaliana	0.1895±0.0047
Strain OE1	0.2836±0.0034
		Strain OE6	0.3194±0.0028

5. Dark treatment

Referring to FIG. 8, for dark treatment of detached leaves, the 5 th or 6 th leaf of 30-day-old Arabidopsis thaliana was taken and placed on filter paper soaked with 3mM MES, 0.5 × MS mixed buffer. For the leaves to be dark-treated, they were wrapped with aluminum foil and dark-treated for 6 days.

In conclusion, the over-expression of the gene ZmSAG39 results in the reduction of chlorophyll content, the increase of relative ion permeability and the increase of hydrogen peroxide content in the rosette leaves of the plants. The ZmSAG39 protein has the capacity of promoting the reduction of chlorophyll content and the accumulation of hydrogen peroxide, thereby promoting the senescence of plant leaves. Therefore, the process of plant leaf senescence can be regulated by regulating the expression level of the gene ZmSAG39 in the plant.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Jilin university of agriculture

<120> leaf senescence regulating gene ZmSAG39, encoding protein and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1477

<212> DNA

<213> corn (Zea mays)

<400> 1

aaacaactgc actagacatc tcaattaatt agctaatgag caagcggctg agctgcactc 60

aagacaaact accatggatc agtcaaacat tagcaacaag cacatgacga tgacaaccct 120

aatgctcctc ctctgtgtca tagccattgc agattgcatt tgccacgccg cagtggcagc 180

ccgggtggag ccatccacca ccgtcggcag aactacagga ggagacgagg cgatgatgat 240

ggcgaggtac aagaagtgga tggcgcagta tcgccggaag tacaaggacg acgccgagaa 300

agcacaccgt ttccaggtat tcaaggcgaa cgcggagttt attgacaggt ctaacgctgg 360

aggaaagaag aagtacgtcc tagggaccaa ccagttcgcc gacctgacca gcaaagagtt 420

cgcggccatg tacaccggtt tgaggaaacc ggcggcggtg ccttccgggg ccaagcagat 480

ccctgcagct ggttccaagt accagaattt tacgcgccta gatgatgatg tccaggttga 540

ttggaggcag cagggtgctg tcactcctgt caagaaccaa ggccaatgtg gtaaagttaa 600

ctgctccgtc tacgtctatc attattaact attgagtaac tggctaatgg catctaaatg 660

agatcaatat ttgcaggctg ttgctgggcg ttctctgcag taggtgccat ggaaggtttg 720

atcatgataa cgacaggaaa cctggtctcc ctgtcggagc agcagattct agactgcgac 780

gagtcagacg ggaaccaggg ctgcaacggt ggctacatgg acaacgcctt ccagtacgtc 840

atcaacaatg gcggcgtcac cactgaggac gcctaccctt actctgcagt ccaagggacg 900

tgccaaaacg tccagccagc cgccaccatc agcggcttcc aggacctgcc cagcggcgac 960

gagaacgcgc tcgccaacgc agtcgccaac cagccggtgt ctgttggcgt cgacggcgga 1020

tcgagtcctt tccagtttta ccagggtggc atatacgacg gggatggctg tggcacggac 1080

atgaaccatg cagtgacggc gatcggctac ggcgccgatg accagggaac ccagtactgg 1140

atcctcaaga actcctgggg cacaggatgg ggtgagaacg gtttcatgca gctacagatg 1200

ggcgtcggcg cctgtggtat ctccacgatg gcctcctacc caactccatg aacacaaatc 1260

ttcagataaa gcgtagtata taccagtcca tgcatatgac tgcagaataa agcatggaag 1320

attaattaca tgcgtgcatc aaggcatgct acgtactgta ttaaaacttc ggtactggcc 1380

agatatatac acaaaggtag aatgtttgtg tgataatata gtaatgcttg tacgaatgca 1440

aagaacatga cctttcaata ataaataaga ttaggct 1477

<210> 2

<211> 363

<212> PRT

<213> corn (Zea mays)

<400> 2

Met Asp Gln Ser Asn Ile Ser Asn Lys His Met Thr Met Thr Thr Leu

1 5 10 15

Met Leu Leu Leu Cys Val Ile Ala Ile Ala Asp Cys Ile Cys His Ala

20 25 30

Ala Val Ala Ala Arg Val Glu Pro Ser Thr Thr Val Gly Arg Thr Thr

35 40 45

Gly Gly Asp Glu Ala Met Met Met Ala Arg Tyr Lys Lys Trp Met Ala

50 55 60

Gln Tyr Arg Arg Lys Tyr Lys Asp Asp Ala Glu Lys Ala His Arg Phe

65 70 75 80

Gln Val Phe Lys Ala Asn Ala Glu Phe Ile Asp Arg Ser Asn Ala Gly

85 90 95

Gly Lys Lys Lys Tyr Val Leu Gly Thr Asn Gln Phe Ala Asp Leu Thr

100 105 110

Ser Lys Glu Phe Ala Ala Met Tyr Thr Gly Leu Arg Lys Pro Ala Ala

115 120 125

Val Pro Ser Gly Ala Lys Gln Ile Pro Ala Ala Gly Ser Lys Tyr Gln

130 135 140

Asn Phe Thr Arg Leu Asp Asp Asp Val Gln Val Asp Trp Arg Gln Gln

145 150 155 160

Gly Ala Val Thr Pro Val Lys Asn Gln Gly Gln Cys Gly Cys Cys Trp

165 170 175

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

180 185 190

Gly Asn Leu Val Ser Leu Ser Glu Gln Gln Ile Leu Asp Cys Asp Glu

195 200 205

Ser Asp Gly Asn Gln Gly Cys Asn Gly Gly Tyr Met Asp Asn Ala Phe

210 215 220

Gln Tyr Val Ile Asn Asn Gly Gly Val Thr Thr Glu Asp Ala Tyr Pro

225 230 235 240

Tyr Ser Ala Val Gln Gly Thr Cys Gln Asn Val Gln Pro Ala Ala Thr

245 250 255

Ile Ser Gly Phe Gln Asp Leu Pro Ser Gly Asp Glu Asn Ala Leu Ala

260 265 270

Asn Ala Val Ala Asn Gln Pro Val Ser Val Gly Val Asp Gly Gly Ser

275 280 285

Ser Pro Phe Gln Phe Tyr Gln Gly Gly Ile Tyr Asp Gly Asp Gly Cys

290 295 300

Gly Thr Asp Met Asn His Ala Val Thr Ala Ile Gly Tyr Gly Ala Asp

305 310 315 320

Asp Gln Gly Thr Gln Tyr Trp Ile Leu Lys Asn Ser Trp Gly Thr Gly

325 330 335

Trp Gly Glu Asn Gly Phe Met Gln Leu Gln Met Gly Val Gly Ala Cys

340 345 350

Gly Ile Ser Thr Met Ala Ser Tyr Pro Thr Pro

355 360

Claims (10)

1. A gene ZmSAG39 for regulating leaf senescence is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. The use of the gene ZmSAG39 for regulating leaf senescence according to claim 1 for regulating leaf senescence performance.

3. The use of the gene ZmSAG39 for regulating leaf senescence according to claim 1 in maize germplasm resource improvement.

4. The use of the gene ZmSAG39 for regulating leaf senescence according to claim 1 in transgenic maize for regulating leaf senescence.

5. The protein encoded by the gene ZmSAG39 for regulating leaf senescence of claim 1, wherein the amino acid sequence is as shown in SEQ ID No. 2.

6. The biomaterial containing the gene ZmSAG39 for regulating leaf senescence according to claim 1, wherein the biomaterial is an expression cassette, a vector, an engineered bacterium or a cell.

7. The biomaterial according to claim 6, wherein the vector is a pCAMBIA3301 vector, and the multiple cloning site region of the pCAMBIA3301 vector is sequentially linked to a 35S promoter, the gene ZmSAG39 according to claim 1, and a terminator.

8. The biological material according to claim 6, wherein the cell is a host cell comprising the vector according to claim 7 and/or having integrated into its genome the exogenous forward or reverse sequence of gene ZmSAG39 according to claim 1.

9. A method for delaying senescence of leaves in a plant, comprising integrating the gene ZmSAG39 for regulating senescence of leaves of claim 1 into cells, tissues and organs of the plant and overexpressing the gene.

10. The method of claim 9, wherein the plant comprises maize, rice, wheat, arabidopsis.

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