CN111471692B - Application of AHL9 and AHL11 genes in regulation and control of plant leaf senescence - Google Patents

Abstract

The application belongs to the technical field of arabidopsis genome engineering, and particularly relates to two arabidopsis genes (AHL9，Also known as AT2G 45850;AHL11also known as AT3G 61310) in the aspect of regulating and controlling the leaf senescence of plants. The two genes are related to the senescence of plant leaves, and the encoded proteins can influence the expression of senescence-related genes and photosynthesis-related genes as transcription factors, so that the senescence process of the leaves is regulated by regulating ethylene accumulation and senescence signal pathways. The senescence-associated genes SAGs include:SAG12、SAG13、SAG113(ii) a The photosynthesis-related genes include:CAB1、RBCS1. After overexpression of both genes, the overexpression line material showed the typical pre-senescence phenotype. Based on the result, a certain technical basis can be established for the regulation and control of new plant species with short growth cycle or plant growth cycle.

Description

Application of AHL9 and AHL11 genes in regulation and control of plant leaf senescence

Technical Field

The application belongs to the technical field of arabidopsis genome engineering, and particularly relates to two arabidopsis genes (AHL9，Also known as AT2G 45850;AHL11also known as AT3G 61310) in regulating and controlling the aging of plant leaves.

Background

In the whole life cycle of a plant, senescence is the last stage of the development of plant leaves, is an important and unique development process, mainly relates to the ordered disintegration of macromolecules and the transfer of nutrients from the leaves to other organs, and a plurality of endogenous and exogenous environmental signals regulate and control the complex process of plant senescence. Currently, although dramatic advances have been made in understanding how aging signals are perceived and transmitted, how orderly degenerative processes are regulated, and how aging programs interact with environmental signals. However, the process of senescence regulation is still unclear, so further exploration of the mechanisms of senescence regulation of leaves is still of great significance.

Earlier studies have shown that proteins of the AT hook family, also known as AT-hook motif nuclear localized (AHL) proteins, contain an AT hook DNA binding motif and an unknown PPC domain (plants and promoters conserved domain), regulate the structure of chromosomal proteins and regulate transcription of target genes in conjunction with other transcriptional regulators, and are primarily involved in regulating many aspects of plant growth and development, including hypocotyl elongation, flower development, root growth, gibberellin biosynthesis, etc. Given the important role of this family of genes, it is clear that further research and discussion is needed for the specific role of different AHL proteins in the plant growth process.

Disclosure of Invention

Based on the genetic engineering of Arabidopsis thaliana, the present application aims AT providing two AT hook family proteins (AHL 9) in Arabidopsis thaliana、AHL 11) in the aspect of regulating and controlling the aging of plant leaves, thus laying a certain technical foundation for regulating and controlling the growth cycle of plants, breeding new varieties of plants and the like.

The technical solution adopted in the present application is detailed as follows.

AHL9AndAHL11the genes are applied to the aspect of regulating and controlling the senescence of plant leaves, the two genes are related to the senescence of the plant leaves, and the coded protein of the two genes, as a transcription factor, can influence the expressions of senescence-related genes and photosynthesis-related genes, so that the senescence process of the leaves is regulated and controlled by regulating ethylene accumulation and senescence signal pathways;

the senescence-associated genes SAGs include:SAG12、SAG13、SAG113；

the photosynthesis-related genes include:CAB1、RBCS1；

specifically, the method comprises the following steps:

these two genes (a)AHL9AndAHL11gene), compared with wild type, the transcription level of photosynthesis related genes in leaves of over-expression strain (or gene over-expression new variety) is obviously lower than that of wild type, while the transcription level of senescence related genes is obviously higher than that of wild type, and leaves are senescent in phenotype;

the senescence, in particular phenotypically expressed as: compared with the wild type, the chlorophyll content in the over-expression plant leaf is obviously lower and the etiolation area is obviously larger than that of the wild type in the process of the plant entering the aging stage of the growth cycle (life history) in the later period of growth.

The describedAHL9AndAHL11the gene preparation method is characterized in that arabidopsis thaliana cDNA is used as a template, the gene is prepared by adopting a PCR amplification method, and during PCR amplification, a primer sequence is designed as follows:

AT2G45850-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGATCGAAGAGATGCAATGG-3’，

AT2G45850-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGACCGCGCATTAAATCAATATCA-3’，

AT3G61310-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGATCGAAGAGACGCAATG-3’，

AT3G61310-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGTCCACGCATTAGATCAATGTCC-3’。

based on the above gene action effect, a new plant species having a short growth cycle can be cultivated, that is, by using a genetic engineering techniqueAHL9AndAHL11after the gene is over-expressed, the growth cycle of the new variety can be shortened; the new plant variety is specifically a new arabidopsis variety.

In the prior art, although a series of researches on specific functions of AT hook family proteins are carried out, the family proteins are numerous and different in functions, so that the deep research on the specific functions of different proteins still has very important practical significance. In the present application, by specifyingAHL9AndAHL11the intensive research on the genes discovers and proves that the two genes influence the expression of senescence-associated genes and further influence a senescence signal pathway (influence the accumulation of plant senescence-associated hormones, namely, control the ethylene-mediated senescence signal pathway) to realize the regulation of the senescence process of plant leaves, and particularly after the two genes are overexpressed, the overexpressed strain material shows a typical premature senescence phenotype. Based on the result, a certain technical basis can be established for the regulation and control of new plant species with short growth cycle or plant growth cycle.

Drawings

FIG. 1 shows AHL9 and AHL11 subcellular localization; wherein:

(A) subcellular localization of AHL9 and AHL11 in arabidopsis protoplasts;

(B) subcellular localization of AHL9 and AHL11 in tobacco leaves;

FIG. 2 isAHL9AndAHL11constructing and identifying an over-expression plant; wherein:

(A) WT and four and five weeks around growthahl9Phenotypic observation, scale bar = 1 cm;

（B）AHL9andAHL11construction pattern diagram of the overexpression vector;

（C）AHL9andAHL11screening positive seedlings of over-expression plants, wherein the working concentration of kanamycin is 50 mug/mL;

(D) and (E)AHL9AndAHL11performing electrophoresis detection result of PCR identification of the over-expression plant;

FIG. 3 is a drawing showingAHL9AndAHL11quantitative analysis of overexpression transgenic lines. Wherein: (A) and (B) detecting WT after 12 days of germination,AHL9AndAHL11 multiple overexpressionThe relative expression amounts of the transgenic strains are respectively named as OE10-1, OE11-5, OE5-2 and OE6-4AHL9-OE10、AHL9-OE11、AHL11-OE5 andAHL11-OE6；

FIG. 4 is a drawing showingAHL9AndAHL11phenotypic analysis of the overexpressed transgenic plants; wherein:

(A) WT growing for about 32 days,AHL9-OE10 andAHL9-OE11 leaf phenotype analysis;

(B) WT growing for about 35 days,AHL9-OE10、AHL9-senescence phenotype of OE11 plants, with leaves ex vivo arranged in chronological order;

(C) WT growing for about 32 days,AHL11-OE5 andAHL11-OE6 leaf phenotype analysis;

(D) WT growing for about 35 days,AHL11-OE5 andAHL11senescence phenotype of OE6 plants, with leaves ex vivo arranged in chronological order. The rosette leaves are numbered from bottom to top, the 1 st leaf is the oldest, and the 15 th leaf is the youngest;

FIG. 5 is a drawing showingAHL11Leaf senescence analysis of the over-expression plants; wherein:

（A）WT、AHL11-OE5 andAHL11-senescence phenotype of fourth and fifth rosette leaves of OE6 plant at different growth stages; (B) chlorophyll content of the leaf shown in graph (A);

(C) qRT-PCR analysis of WT for 26 days,AHL11-OE5 andAHL11in leaves of the-OE 6 plantAHL11The level of transcription of (a);

(D-H) growth for 26 days WT,AHL11-OE5 andAHL11in leaves of the-OE 6 plantSAG12（D）、SAG13（E）、SAG113(F) And photosynthesis-related genesCAB1（G）、RBCS1(H) qRT-PCR transcript level analysis of (1);

FIG. 6 is a schematic view ofAHL9Leaf senescence analysis of the over-expression plants; wherein:

（A）WT、AHL9-OE10 andAHL9-senescence phenotype of fourth and fifth rosette leaves of OE11 plants at different growth stages; (B) chlorophyll content of leaves shown in fig. (A);

(C) qRT-PCR analysis of WT grown for 26 days,AHL9-OE10 andAHL9-OE11 plant leavesAHL9The level of transcription of (a);

(D-H) growth for 26 days WT,AHL9-OE10 andAHL9in leaves of the-OE 11 plantSAG12（D）、SAG13（E）、SAG113(F) And photosynthetic related genesCAB1（G）、RBCS1(H) qRT-PCR transcript level analysis of (1);

FIG. 7 is a schematic view ofAHL9AndAHL11regulating and controlling leaf senescence induced by darkness; wherein:

(A) and (C)AHL9/AHL11The senescence phenotype of the overexpression plant and the wild type in vitro leaf blade under the dark induction for about three weeks, wherein the scale bar = 1 cm;

(B) and (D) after the dark treatmentAHL9AndAHL11and analyzing the chlorophyll content of the over-expression plants and wild plants, wherein n = 3.

Detailed Description

The present application is further explained below with reference to the drawings and examples.

Example 1

Based on the research foundation of the prior AHL family protein, the inventor firstly clones to obtainAHL9AndAHL11these two gene sequences. Specifically, the inventors firstly extract and obtain Arabidopsis RNA by TRIZol method, then design PCR amplification primer by referring to Arabidopsis cDNA database, and finally prepare and obtain the Arabidopsis RNA by PCR amplification methodAHL9AndAHL11PCR amplification products of both genes.

AHL9AndAHL11the coding frame base sequences of the genes are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the coding frame base sequences are as follows:

AHL9 (i.e., AT2G45850, 1047bp)

ATGGATCGAAGAGATGCAATGGGATTATCCGGGTCAGGTTCTTACTATATCCATAGAGGATTATCCGGGTCGGGTCCTCCAACGTTTCATGGATCACCACAGCAACAGCAAGGTCTTCGTCACTTACCTAATCAAAACTCTCCATTCGGGTCAGGCTCCACTGGTTTCGGATCTCCTTCTTTACACGGTGATCCTTCTCTGGCAACAGCAGCCGGAGGAGCCGGAGCTCTTCCTCATCATATCGGCGTTAATATGATTGCTCCTCCTCCACCTCCCAGTGAAACTCCGATGAAACGAAAGAGAGGACGGCCTAGAAAATACGGTCAAGACGGCTCTGTTTCTTTGGCTCTGTCGTCTTCCTCTGTTTCGACCATTACTCCCAACAACTCTAACAAACGCGGCCGTGGTCGACCTCCGGGCTCCGGCAAGAAACAGAGAATGGCTTCCGTTGGTGAACTGATGCCTTCATCTTCTGGAATGAGCTTCACGCCACATGTTATCGCGGTTTCAATAGGAGAAGATATTGCATCAAAGGTTATAGCTTTCTCTCAACAAGGTCCGAGAGCCATTTGCGTTTTATCTGCAAGTGGTGCAGTCTCTACTGCAACACTTATTCAACCATCAGCATCTCCCGGAGCCATTAAATACGAGGGCCGGTTTGAAATCCTAGCGTTATCAACATCTTATATAGTGGCAACTGATGGAAGCTTCCGTAACCGAACTGGAAACTTATCGGTTTCGCTTGCTAGCCCCGATGGGCGTGTGATTGGCGGTGCCATTGGTGGGCCTTTAATAGCTGCAAGTCCTGTTCAGGTTATTGTAGGGAGCTTTATATGGGCAGCTCCAAAGATCAAGAGCAAGAAACGAGAAGAAGAAGCTTCTGAAGTTGTTCAAGAAACTGATGATCACCACGTTCTGGACAATAATAACAACACGATTTCGCCTGTCCCTCAGCAGCAGCCAAACCAAAACCTGATTTGGTCAACAGGTTCAAGGCAAATGGATATGCGTCATGCTCATGCTGATATTGATTTAATGCGCGGTTGA

AHL11(i.e., AT3G61310, 1065bp)

ATGGATCGAAGAGACGCAATGGCGTTATCCGGGTCGGGTTCTTACTATATCCAAAGAGGAATCCCCGGTTCTGGTCCTCCTCCTCCTCAAACTCAACCAACGTTTCACGGATCACAAGGATTTCATCATTTCACCAATTCCATCTCTCCTTTTGGGTCAAACCCAAACCCAAATCCAAACCCTGGAGGTGTCTCTACTGGATTCGTGTCTCCTCCTTTACCCGTTGACTCTTCTCCGGCTGATTCGTCAGCGGCGGCGGCGGGAGCTTTGGTTGCTCCTCCTTCAGGTGACACGTCTGTGAAGCGGAAGAGAGGACGGCCTAGAAAATATGGACAAGATGGTGGTTCTGTTTCGTTGGCATTGTCTCCTTCTATCTCCAACGTTTCCCCGAACTCTAACAAACGTGGCCGTGGAAGACCTCCTGGCTCCGGCAAGAAGCAACGGCTATCTTCCATTGGTGAAATGATGCCTTCATCAACTGGGATGAGCTTCACACCGCATGTAATCGTAGTTTCCATTGGTGAAGACATTGCTTCAAAGGTTATATCGTTCTCGCATCAAGGTCCACGAGCGATATGTGTCTTATCCGCAAGTGGTGCTGTCTCTACTGCAACTCTTCTTCAGCCAGCACCTTCTCATGGAACTATTATATACGAGGGTCTATTCGAGCTCATATCTCTCTCAACTTCTTATCTGAACACAACTGACAATGACTACCCAAACCGCACTGGAAGTCTAGCGGTCTCACTTGCTAGCCCCGATGGTCGTGTCATTGGTGGTGGAATTGGAGGTCCTCTAATAGCAGCAAGCCAAGTCCAGGTCATTGTTGGCAGCTTCATTTGGGCAATTCCGAAAGGGAAGATTAAAAAACGTGAAGAAACTTCTGAAGATGTCCAAGATACTGATGCTTTGGAAAACAACAACGATAACACAGCAGCAACGTCACCTCCTGTTCCTCAGCAAAGTCAGAACATTGTTCAGACTCCTGTAGGCATTTGGTCAACTGGTTCAAGGTCAATGGATATGCATCACCCCCATATGGACATTGATCTAATGCGTGGATGA。

In the specific PCR amplification, the primer sequence is designed as follows:

AT2G45850-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGATCGAAGAGATGCAATGG-3’，

AT2G45850-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGACCGCGCATTAAATCAATATCA-3’，

AT3G61310-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGATCGAAGAGACGCAATG-3’，

AT3G61310-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGTCCACGCATTAGATCAATGTCC-3’。

based on PCR amplification products, after the PCR amplification products are further purified and sequenced, the PCR amplification products are analyzed to obtainAHL9AndAHL11the AHL9 and AHL11 proteins encoded by the two genes respectively contain two AT-hook motifs and a PPC/DUF296 domain, and based on the research of other AHL family proteins, the following results can be found: the AT-hook DNA binding motif and the PPC/DUF296 domain are very important for the nuclear localization of AHL proteins.

To further determine the subcellular localization of AHL9 and AHL11, the inventors used PEG-mediated transient transformation of arabidopsis protoplasts to transiently transfer plasmids (pGWB 405) fused with GFP expression vectors into arabidopsis protoplasts cells for expression, followed by fluorescence detection using a laser scanning confocal microscope, in accordance with prior art procedures.

The results are shown in FIG. 1 (A). Analysis can show that AHL9 and AHL11 are both localized to the nucleus. Further, by using an agrobacterium-mediated tobacco transient transformation technology, the plasmid (pGWB 405) fused with the GFP expression vector is transformed into agrobacterium and then injected into tobacco leaves for fluorescent microscope observation. The results are shown in FIG. 1 (B). It can be seen that AHL9 and AHL11 are further proved to be positioned on the nucleus at the subcellular level, which lays a foundation for the specific function of the nucleus.

Example 2

On the basis of example 1, the inventors further constructedAHL9AndAHL11overexpression of the genes for further study of the function of these two genes. The construction of the specific overexpression lines is briefly described below.

(I) construction of 35S promoter-driven overexpression vector35S::AHL9/AHL11-GFP(namely:35S::AHL9-GFPand35S::AHL11-GFP）

with reference to the schematic diagram 2 (B) and the following steps (the specific operation is performed with reference to the prior art):

firstly, using arabidopsis cDNA as a template, designing a primer according to a gene sequence, and carrying out PCR amplification on a target gene by using high-fidelity DNA polymerase;

subsequently, the Gateway vector construction technology is used for constructing the vector, namely:

carrying out BP reaction, carrying out PCR amplification by using a designed target gene amplification primer containing attB1 and attB2 site sequences and high-fidelity polymerase, carrying out electrophoresis detection, and recovering a purified product; then, carrying out escherichia coli competence transformation by using protease K;

and the second step of LR reaction, adding an expression vector pGWB405 on the basis of ensuring the correct construction of the BP fusion vector pENTR containing the target gene segment, reacting for 1 h at 25 ℃, and then carrying out escherichia coli competence transformation and screening.

(II) transformation of Agrobacterium

Carrying out plasmid PCR identification, enzyme digestion identification and sequencing verification on the overexpression vector plasmid vector constructed in the step (I) to ensure that the overexpression vector plasmid vector is transformed to agrobacterium GV3101 after the overexpression vector plasmid vector is correctly recombined and constructed; specifically, the method comprises the following steps:

adding 1-2 μ L of successfully sequenced recombinant Escherichia coli plasmid into 100 μ L of Agrobacterium-infected GV3101, standing on ice for 5 min, then liquid nitrogen for 5 min, reacting at 37 deg.C for 5 min, then standing on ice for 5 min, culturing at 220 rpm on 28 deg.C shaking table for 2-3 hr, spreading the cultured Agrobacterium on corresponding resistant plate, and culturing in 28 deg.C incubator for 36-48 hr.

(III) transformation

Immersing the arabidopsis inflorescence in the agrobacterium liquid obtained in the step (II) by adopting an inflorescence infection transformation method, and further carrying out resistance screening to obtain the arabidopsis inflorescenceAHL9/AHL11The specific transformation operation of the overexpression transgenic plant is as follows:

the agrobacterium cultured overnight is centrifuged at 4000 rpm for 5 min to collect the bacteria after the bacteria are preserved, the supernatant is poured out, then a resuspension (0.023 g MS and 5 g sucrose are added into 100 ml of ultrapure water) is added to adjust the OD value to about 1.0, a proper amount of surfactant is added to shake evenly, and then the arabidopsis inflorescence is immersed for a certain time and treated in the dark for 24 h.

Is homozygous forahl9The single mutant plants are bred and observed with phenotype, and compared with wild type plants,ahl9there were no significant phenotypic differences in the single mutations (see FIG. 2A).

For the positive plant of resistance screening (part of screening results are shown in fig. 2 (C)), when the plant leaves are slightly bigger, the seedling leaves are cut to extract genome DNA respectively, and PCR identification is performed to ensure that the obtained overexpression material is successful in transgenosis. The partial identification results are shown in FIGS. 2 (D) and 2 (E). It can be seen that the overexpression transgenic plants obtained by the resistance screening are all positive target plants.

Further, qRT-PCR detection was performed on the transgenic positive homozygote material for evaluationAHL9、AHL11The level of gene expression.

When qRT-PCR detection is carried out, the primer sequence is designed as follows:

AHL9-1-qRT-F：5’-TCCGTAACCGAACTGGAAAC-3’，

AHL9-1-qRT-R：5’-TGAACAGGACTTGCAGCTATTA-3’；

AHL9-2-qRT-F：5’-GATCACCACGTTCTGGACAA-3’，

AHL9-2-qRT-R：5’-GCATGAGCATGACGCATATC-3’；

AHL11-1-qRT-F： 5’-GGTCAAACCCAAACCCAAATC-3’，

AHL11-1-qRT-R：5’-GGAGAAGAGTCAACGGGTAAAG-3’；

AHL11-2-qRT-F：5’-GGTGGTTCTGTTTCGTTGGC-3’，

AHL11-2-qRT-R：5’-TCGAAAACACGCAGAAATTCCTAA-3’；

ACTIN-qRT-F：5’-GTAACATTGTGCTCAGTGGTGGTA-3’，

ACTIN-qRT-R：5’-GATAGAACCACCAATCCAGACACT-3’。

it should be noted that, in the detection and analysis, two pairs of different primers (AHL 9-1-qRT-F, AHL9-1-qRT-R, AHL9-2-qRT-F, AHL 9-2-qRT-R; AHL11-1-qRT-F, AHL11-1-qRT-R, AHL11-2-qRT-F, AHL 11-2-qRT-R) are suitable for useAHL9、AHL11The qRT-PCR detection of the gene adopts different primers, mainly for ensuring the accuracy of the detection result, thereby being convenient for more accurate evaluationAHL9、AHL11Gene expression level profile. For quantitative analysis, Arabidopsis thaliana was usedACTINThe gene is used as an internal reference.

The partial detection results are shown in fig. 3 (a) and 3 (B). As can be seen,AHL9/AHL11higher levels of expression were exhibited in multiple overexpression lines. And only choose to follow up observation and studyAHL9OE-10 (which was discarded because the OE9 was less stable) and OE-11, which were expressed in higher amounts,AHL11Two strains of OE-5 and OE-6 with increased expression levels were subsequently investigated.

Example 3

On the basis of example 2, the inventors further carried out phenotypic observations and other experiments on seeds of homozygotes of the T3 generation overexpression lines obtained, to determineAHL9 and AHL11The relationship between gene and plant aging regulation and control, correlationThe experimental summary is as follows.

(1) Influence on leaf senescence

And performing phenotype analysis on the obtained over-expression plants, planting completely dried T3 generation over-expression homozygote seeds and wild seeds harvested at the same period in nutrient soil, culturing in a long-day plant material room, and periodically observing the phenotype.

A summary of some of the phenotypic cases is shown in FIG. 4. The comparison can find that:

AHL9the overexpressed plant leaves were elongated and curled outward (FIG. 4 (A)), andAHL11the leaves of the over-expressed plants were significantly wrinkled and curled (FIG. 4 (C)), whereas compared with the wild type,AHL9andAHL11the over-expression plants showed obvious leaf yellowing after 32 days of growth (FIG. 4 (A), FIG. 4 (C)).

Further, the inventors performed leaf yellowing analysis (fig. 4 (B), fig. 4 (D)) on leaves of 5-week-old wild-type plants and over-expressed plants to determine specific senescence phenotype differences. It can be seen that: compared with the wild type plants, the method has the advantages that,AHL9/AHL11the tip of the leaf of the overexpressed plant turned yellow first andAHL9the aging degree of the over-expression plant leaves is lower than that of the over-expression plant leavesAHL11And (5) over-expressing the plant.

By combining these phenotypic results, an initial determination can be madeAHL9、AHL11The gene is associated with the plant leaf morphology (curled phenotype), in particular with the early leaf senescence phenotype, i.e., by regulationAHL9、AHL11The change of the gene expression level can regulate and control the growth cycle of the plant (or the life cycle of the plant can be shortened by the over-expression of the two genes).

(2) Influence on expression of specific senescence genes

In the process of observing the senescence phenotype of the over-expression strain, the inventor further detects the senescence-associated gene expression conditions of different growth periods of leaves to preliminarily discussAHL9、AHL11The regulation mechanism of genes for the aging process. The specific experimental summary is as follows.

Firstly, the fourth and fifth rosette leaves are aged in different growth stagesThe phenotype was summarized, as shown in FIGS. 5 (A) and 6 (A), and it was found that, as leaf age increased,AHL9/AHL11the tips of the leaves of the overexpressing plants turned yellow first, which is consistent with the phenotypic observations described previously.

Further, the inventors analyzed physiological parameters (mainly chlorophyll content) related to aging of mature leaves at different growth stages, and analyzed the aging-related genesSAGs(comprising:SAG12、SAG13、SAG113) And photosynthesis-related genes: (CAB1AndRBCS1) qRT-PCR measurements were performed at the transcript levels of different growth stages of the leaf.

For qRT-PCR analysis, the relevant primers were designed as follows:

SAG12-qRT-F ：5’-ATCCAAAAGCAACTTCTATTACAGG-3’，

SAG12-qRT-R：5’-CCACTGCCTTCATCAGTGC-3’；

SAG13-qRT-F ：5’-AGGAAAACTCAACATCCTCGTC-3’，

SAG13-qRT-R：5’-GCTGACTCGAGATTTGTAGCC-3’；

SAG113-qRT-F：5’-CCATGGCTGTTCCCATGTA-3’，

SAG113-qRT-R：5’-AAGCTACGCGCCATTGAC-3’；

CAB1-qRT-F：5’-GCAAGGAACCGTGAACTAGAA-3’，

CAB1-qRT-R：5’-TCCGAACTTGACTCCGTTTC-3’；

RBCS-qRT-F：5’-CGCTCCTTTCAACGGACTTA-3’，

RBCS-qRT-R：5’-AGTAATGTCGTTGTTAGCCTTGC-3’；

ACTIN-qRT-F：5’-GTAACATTGTGCTCAGTGGTGGTA-3’，

ACTIN-qRT-R：5’-GATAGAACCACCAATCCAGACACT-3’。

when the chlorophyll content in the mature leaf of arabidopsis is determined, the specific determination method refers to the following steps:

selecting the fourth and fifth rosette leaves of arabidopsis, weighing the fresh leaves, placing the leaves into an EP tube with the specification of 2 mL, adding 1 mL of chlorophyll extracting solution (95% ethanol), and incubating for 24h at room temperature in the dark (during the incubation, uniformly mixing the leaves by blowing for 4-5 times to ensure the incubation effect);

centrifuging, adding 200 μ L of supernatant into enzyme labeling plate, measuring absorbance values of the solution at 665 nm and 649 nm, respectively, and using chlorophyll extractive solution as negative control;

the calculation formula of the chlorophyll content is as follows:

ChlorophyⅡ a（μg/mL） = 13.95 × A665 - 6.88 × A649；

ChlorophyⅡ b（μg/mL） = 24.96 × A649 - 7.32 × A665；

Total Chlorophyll（mg/g FW）= Ca + Cb）× V/W；

in the formula: v, volume of extract, 1 mL in this method; w: weight of fresh leaves.

The detection results are shown in fig. 5 and 6. Specifically, the method comprises the following steps:AHL9、AHL11the gene expression level is obviously higher than that of the wild type (figure 5 (C) and figure 6 (C)), namely, the genetic stability of the transgenic plant is more stable; further phenotypic observation showed that the chlorophyll content in the leaves of the overexpressed plants was significantly lower than that of the wild type (fig. 5 (a), 5 (B), 6 (a), 6 (B)) compared to the wild type at the same growth period, i.e., the chlorophyll-lowering rate of the new transgenic variety of the overexpressed plants was significantly higher than that of the wild type; and photosynthesis-related genesCAB1AndRBCS1the transcription level detection result shows that the transcription level of the photosynthesis related gene in the transgenic plant is obviously lower than that of the wild type (figure 5 (G), figure 5 (H), figure 6 (G) and figure 6 (H)); genes associated with further senescenceSAG12、SAG13AndSAG113the results of the transcription level detection of (A) show that the transcription level of senescence-associated genes in the transgenic lines was significantly higher than that of the wild type (FIG. 5 (D), FIG. 5 (E), FIG. 5 (F), FIG. 6 (D), FIG. 6 (E), FIG. 6 (F)).

Research shows that leaf senescence can be induced by the stress of dark environment, and the research aims to discussAHL9AndAHL11the inventor analyzes the senescence phenotype of wild type and over-expression plants under the induction of darkness, and prepares WT,AHL9AndAHL11the first to overexpress plantsFour or five rosette leaves were dark-treated in a MES buffer.

In the experimental process, for dark treatment of the isolated leaf of Arabidopsis thaliana, the following operations are referred to:

selecting mature leaves (usually the fourth rosette leaf) which grow at the same position for about three weeks, photographing and recording before dark treatment, and measuring the chlorophyll content of the mature leaves;

putting the leaves into an MES buffer for incubation under a continuous dark environment at 22 ℃, and observing and photographing records every day;

and after dark treatment for 3 days, observing the yellowing phenomenon of the leaves, taking a picture for recording, cleaning the leaves for 2-3 times by using double distilled water, and measuring the chlorophyll content of the leaves.

The results of the detection are shown in FIG. 7. It can be seen that: after 3 days of dark induction, it was found that the yellowing of the leaves of the over-expressed plants under dark induction was more pronounced and the leaves senesced faster than the wild type by treating the color change of the leaves (FIGS. 7 (A), (C)). Besides, we also detected the chlorophyll content of the dark-treated leaves, and the analysis found that the chlorophyll content of the over-expressed plant leaves also decreased significantly faster than that of the wild-type (fig. 7 (B), (D)). This result indicates thatAHL9AndAHL11can regulate and control leaf senescence induced by darkness.

The combination of the above results shows that: the reduction of chlorophyll content, the reduction of photosynthesis-related gene transcription level and the increase of senescence-related gene expression level in the process of leaf senescence of the over-expressed plants are consistent with the senescence phenotype that leaves turn yellow in advance, which shows thatAHL9AndAHL11the gene plays an important role in regulating leaf senescence, and particularly can obviously promote the leaf senescence process when the gene is over-expressed.

SEQUENCE LISTING

<110> university of Henan

<120> application of AHL9 and AHL11 genes in regulation and control of plant leaf senescence

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1047

<212> DNA

<213> Arabidopsis thaliana

<400> 1

atggatcgaa gagatgcaat gggattatcc gggtcaggtt cttactatat ccatagagga 60

ttatccgggt cgggtcctcc aacgtttcat ggatcaccac agcaacagca aggtcttcgt 120

cacttaccta atcaaaactc tccattcggg tcaggctcca ctggtttcgg atctccttct 180

ttacacggtg atccttctct ggcaacagca gccggaggag ccggagctct tcctcatcat 240

atcggcgtta atatgattgc tcctcctcca cctcccagtg aaactccgat gaaacgaaag 300

agaggacggc ctagaaaata cggtcaagac ggctctgttt ctttggctct gtcgtcttcc 360

tctgtttcga ccattactcc caacaactct aacaaacgcg gccgtggtcg acctccgggc 420

tccggcaaga aacagagaat ggcttccgtt ggtgaactga tgccttcatc ttctggaatg 480

agcttcacgc cacatgttat cgcggtttca ataggagaag atattgcatc aaaggttata 540

gctttctctc aacaaggtcc gagagccatt tgcgttttat ctgcaagtgg tgcagtctct 600

actgcaacac ttattcaacc atcagcatct cccggagcca ttaaatacga gggccggttt 660

gaaatcctag cgttatcaac atcttatata gtggcaactg atggaagctt ccgtaaccga 720

actggaaact tatcggtttc gcttgctagc cccgatgggc gtgtgattgg cggtgccatt 780

ggtgggcctt taatagctgc aagtcctgtt caggttattg tagggagctt tatatgggca 840

gctccaaaga tcaagagcaa gaaacgagaa gaagaagctt ctgaagttgt tcaagaaact 900

gatgatcacc acgttctgga caataataac aacacgattt cgcctgtccc tcagcagcag 960

ccaaaccaaa acctgatttg gtcaacaggt tcaaggcaaa tggatatgcg tcatgctcat 1020

gctgatattg atttaatgcg cggttga 1047

<210> 2

<211> 1065

<212> DNA

<213> Arabidopsis thaliana

<400> 2

atggatcgaa gagacgcaat ggcgttatcc gggtcgggtt cttactatat ccaaagagga 60

atccccggtt ctggtcctcc tcctcctcaa actcaaccaa cgtttcacgg atcacaagga 120

tttcatcatt tcaccaattc catctctcct tttgggtcaa acccaaaccc aaatccaaac 180

cctggaggtg tctctactgg attcgtgtct cctcctttac ccgttgactc ttctccggct 240

gattcgtcag cggcggcggc gggagctttg gttgctcctc cttcaggtga cacgtctgtg 300

aagcggaaga gaggacggcc tagaaaatat ggacaagatg gtggttctgt ttcgttggca 360

ttgtctcctt ctatctccaa cgtttccccg aactctaaca aacgtggccg tggaagacct 420

cctggctccg gcaagaagca acggctatct tccattggtg aaatgatgcc ttcatcaact 480

gggatgagct tcacaccgca tgtaatcgta gtttccattg gtgaagacat tgcttcaaag 540

gttatatcgt tctcgcatca aggtccacga gcgatatgtg tcttatccgc aagtggtgct 600

gtctctactg caactcttct tcagccagca ccttctcatg gaactattat atacgagggt 660

ctattcgagc tcatatctct ctcaacttct tatctgaaca caactgacaa tgactaccca 720

aaccgcactg gaagtctagc ggtctcactt gctagccccg atggtcgtgt cattggtggt 780

ggaattggag gtcctctaat agcagcaagc caagtccagg tcattgttgg cagcttcatt 840

tgggcaattc cgaaagggaa gattaaaaaa cgtgaagaaa cttctgaaga tgtccaagat 900

actgatgctt tggaaaacaa caacgataac acagcagcaa cgtcacctcc tgttcctcag 960

caaagtcaga acattgttca gactcctgta ggcatttggt caactggttc aaggtcaatg 1020

gatatgcatc acccccatat ggacattgat ctaatgcgtg gatga 1065

Claims (3)

1. The application of the Arabidopsis AT hook family gene in the aspect of regulating and controlling the senescence of plant leaves is characterized in that the Arabidopsis AT hook family gene refers to ArabidopsisAHL9A gene;

the Arabidopsis thalianaAHL9The gene is related to the senescence of plant leaves, and the coded protein serving as a transcription factor can influence the expression of senescence-related genes and photosynthesis-related genes, so that the senescence process of the leaves is regulated by regulating ethylene accumulation and senescence signal pathways;

the senescence-associated genes SAGs include:SAG12、SAG13、SAG113；

the photosynthesis-related genes include:CAB1、RBCS1；

the regulation is toAHL9After the gene is over-expressed, compared with the wild type, the transcription level of the genes related to photosynthesis in the leaves of an over-expression strain is obviously lower than that of the wild type, the transcription level of the genes related to senescence is obviously higher than that of the wild type, and the leaves are senescent in advance in phenotype;

the aging shows the following specific phenotypic aspects: compared with the wild type, the chlorophyll content in the leaves of the over-expression plants is obviously lower and the etiolation area is obviously larger than that of the wild type in the process of entering the senescence stage of the growth cycle from the late stage of the growth of the plants.

2. Use of the Arabidopsis thaliana AT hook family gene of claim 1 for regulating senescence in leaves of a plant, characterized in that it is prepared during overexpressionAHL9When the gene is used, the gene is prepared by taking arabidopsis cDNA as a template and adopting a PCR amplification method, and during PCR amplification, a primer sequence is designed as follows:

AT2G45850-F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGATCGAAGAGATGCAATGG-3’，

AT2G45850-R：

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTGACCGCGCATTAAATCAATATCA-3’。

3. the use of the Arabidopsis thaliana AT hook family gene of claim 1 for regulating leaf senescence in plants, wherein genetic engineering techniques are used to control leaf senescence in plantsAHL9After the gene is over-expressed, a new plant variety with a short growth cycle is prepared; the new plant variety is specifically a new arabidopsis variety.

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