CN111072762A

CN111072762A - Mao bamboo senescence-associated NAP transcription factor, and coding gene and application thereof

Info

Publication number: CN111072762A
Application number: CN202010033270.8A
Authority: CN
Inventors: 高健; 谢丽华
Original assignee: International Center for Bamboo and Rattan
Current assignee: International Center for Bamboo and Rattan
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2020-04-28
Anticipated expiration: 2040-01-13
Also published as: CN111072762B

Abstract

The invention discloses a transcription factor PheNAP8 related to moso bamboo aging, a coding gene and application thereof, wherein the nucleotide sequence is shown as SEQ ID NO.1, and the protein sequence is shown as SEQ ID NO. 2. Through cloning and expression of the Phyllostachys pubescens PheNAP8 gene and heterologous transformation of Arabidopsis thaliana, the remarkable phenotype of growth retardation and early senescence of a transgenic Arabidopsis thaliana plant over-expressing PheNAP8 is found, and the gene plays an important role in the regulation and breeding of the senescence of the Phyllostachys pubescens, can be used for screening and identifying the varieties of the bamboo with easy senescence, and is suitable for industrial application.

Description

Mao bamboo senescence-associated NAP transcription factor, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a moso bamboo senescence-associated NAP transcription factor, and a coding gene and application thereof.

Background

The flowering and senescence of moso bamboos, the aging of bamboo shoots and the rapid senescence of picked bamboo shoots can cause serious economic loss and ecological problems, at present, the research on the senescence phenomenon and senescence mechanism of moso bamboos is few, and the senescence regulation mechanism is not clear. The moso bamboo belongs to perennial lignified herbaceous plants, the aging types of the moso bamboo are common individual aging, organ aging and gradual aging, group aging, forest aging and bamboo shoot aging removing, and various aging processes of the moso bamboo are mutually overlapped. The specificity and complexity of the biological characteristics and aging types of moso bamboos increases the difficulty of studying the aging mechanism of moso bamboos.

The NAC transcription factor family is one of the largest plant-specific transcription factor families. The NAC domain was first reported by Aida et al 1997 (Aida et a1.,1997), taking the initials of petunia NAM gene, Arabidopsis ATAF1/2 and CUC2 genes and naming NAC. NAP transcription factor is found in 1998 as a target gene of AP3/PI for controlling flower development by Sablowski and Meyerowitz, and further analyzed for its sequence to determine that it belongs to NAC family, which is finally named NAP (NAC-Like, Activated by AP3/PI), which has a crucial role in the growth and development of plants, whole plant senescence and organ senescence regulation. NAP, an important senescence-associated gene, has been discovered and identified in different species as involved in senescence regulation, such as: AtNAP (Arabidopsis), OsNAP (rice), HvNAC005 (barley), BeNAP (Pseudobulbus Cremastrae seu pleiones), SiNAC1 (millet), GhNAP (cotton), SINAP2 (tomato), Csat NAP (saffron), and TaNAM-B1 (wheat) (Uauy et al, 2006; Kalivas et al, 2010; Chen et al, 2011; Christiansen et al, 2014; Fan et al, 2015; Ren et al, 2017; Balazadeh et al, 2018).

However, many studies on senescence regulation by the NAP subfamily genes have focused on annual model plants or crops. The moso bamboo belongs to perennial lignified herbaceous plants, the aging types of the moso bamboo are common individual aging, organ aging and gradual aging, group aging, forest stand aging and bamboo shoot aging removing, and various aging processes of the moso bamboo are mutually overlapped and are obviously different from other annual plants or crops. In addition, the specificity and complexity of the biological properties and senescence type of bamboo increases the difficulty of studying the senescence mechanism of bamboo. Therefore, the aging phenomenon and mechanism of moso bamboo are rarely studied at present, the mechanism of aging regulation is less clear, and the research on the aging of the moso bamboo by NAP members is limited to the evolutionary analysis and the cloning of the gene PeNAC1(NAP subfamily member), and the analysis of the regulation function is lacked. Therefore, NAC (PhyNAP) gene resources related to the bamboo senescence are excavated, the PhyNAP gene is researched to regulate the senescence mechanism of the bamboo, the establishment of a method for regulating senescence is facilitated, and a foundation is laid for the adversity-resistant genetic improvement and molecular breeding of the bamboo.

The invention is provided in view of the above.

Disclosure of Invention

The first purpose of the invention is to provide gene analysis and identification for obviously up-regulating expression in different aging processes of moso bamboo;

the second purpose of the invention is to provide a moso bamboo aging related NAP transcription factor, a coding gene and application thereof;

the third purpose of the present invention is to provide a transgenic production method based on the bamboo senescence-associated NAP transcription factor;

it is a fourth object of the present invention to provide a method for detecting or identifying a senescence-susceptible variety based on the bamboo senescence-associated NAP transcription factor.

In order to achieve the purpose of the invention, the invention provides a novel moso bamboo transcription factor PheNAP8, which is related to moso bamboo senescence, and surprisingly shows extremely obvious expression quantity difference in different moso bamboo senescence degrees, is characterized in senescence of different organs, can be used for screening and identifying moso bamboo senescence-prone varieties, and can also regulate plant leaf development and plant senescence, preferably promote plant senescence. The transcription factor is derived from bamboo (Phyllostachys edulis) and named PhyNAP 8, the nucleotide sequence of the transcription factor consists of 1077 basic groups, as shown in SEQ ID NO.1, and the expressed protein sequence consists of 358 amino acids, as shown in SEQ ID NO. 2. Specifically, the method comprises the following steps:

the invention provides a protein-transcription factor PheNAP8, which is characterized in that the amino acid sequence is shown in SEQ ID NO. 2;

in some embodiments, the protein sequence is (1) an amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2; (2) an amino acid sequence of a protein which has at least 90 percent of homology with the amino acid sequence shown as SEQ ID No.2 and has the same function; preferably, the homology is at least 95%; more preferably at least 99%.

The invention provides a coding gene of a coding protein PheNAP 8;

in some embodiments, the coding gene sequence is shown in SEQ ID NO. 1;

in some embodiments, the sequence is at least 90% homologous to SEQ ID No. 1; preferably, the homology is at least 95%; more preferably at least 99%.

The invention provides an expression vector, a recombinant plasmid, an expression cassette, a recombinant bacterium or a recombinant cell containing the PheNAP8 encoding gene;

the invention provides a transgenic plant cell, transgenic plant part or transgenic plant variety comprising the gene encoding PheNAP8, and corresponding hybrid varieties;

the invention provides a construction method of a transgenic plant, which is characterized in that the coding gene of PheNAP8 is introduced into a target plant to obtain the transgenic plant, and the leaf development of the transgenic plant is faster than that of the target plant;

in some embodiments, the plant is a monocot or dicot, preferably, the plant is phyllostachys pubescens or arabidopsis thaliana;

in some embodiments, the construction method specifically comprises:

constructing a plant expression vector of a PheNAP8 gene;

2. carrying out enzyme digestion detection on the recombinant plasmid PheNAP8-pCAMBIA 2300-35S;

3. preparation, transformation and identification of agrobacterium GV3101 competent cells;

4. agrobacterium-mediated arabidopsis transformation;

5. screening positive transformants of Arabidopsis;

6. PCR and RT-PCR detection of over-expressed plants.

The invention provides application of the protein PheNAP8 and PheNAP8 coding genes, corresponding expression vectors, recombinant plasmids, expression cassettes, recombinant bacteria or recombinant cells in regulation and control of plant leaf development and plant senescence;

the invention also provides the application of the coding gene of the transcription factor PheNAP8 related to the bamboo senescence in the molecular mechanism for regulating leaf senescence;

in some embodiments, the plant is a monocot or dicot, more preferably, the plant is phyllostachys pubescens or arabidopsis thaliana;

the invention provides application of the protein PheNAP8 and the coding gene in identification of the bamboo varieties easy to age;

preferably, the method is applied to identification of the varieties of the moso bamboos which are easy to age in the seedling stage;

the invention provides a method for screening/identifying a moso bamboo easy-aging variety, which is characterized by detecting whether the protein PheNAP8 or a coding gene thereof exists in the moso bamboo variety; or detecting the content of the protein PheNAP8 or the coding gene thereof in the bamboo varieties, and determining whether the bamboo varieties are easy-to-senesce varieties or not by evaluating and comparing the content of the protein or the gene. It is understood in the art that conventional known detection means for genes or proteins are within the scope of the present invention;

the invention provides a kit for screening/identifying plant varieties easy to senesce, which is characterized by comprising components for detecting the protein PheNAP8 or coding genes thereof in the claim;

in some embodiments, the components include, but are not limited to, components in the form of primers, probes, or sequencing reagents;

in some embodiments, the primer sequences are shown as

SEQ ID NOs

3 and 4.

The invention also provides a primer pair, and the sequences of the primer pair are respectively shown as SEQ ID NO 3 and SEQ ID NO 4.

The invention also provides a composition which comprises the primer pair.

The invention has the beneficial technical effects that:

1) the invention clones and extracts the protein PheNAP8 and the gene thereof from the phyllostachys pubescens for the first time, and proves that the protein can regulate the development of plant leaves and promote the senescence of plants for the first time.

2) Compared with other known senescence genes PheNAP2 and PheNAC3 in the phyllostachys pubescens, the PheNAP8 gene has extremely obvious up-regulation expression in the senescence phyllostachys pubescens, the up-regulation multiple is more than 120 times, and meanwhile, the gene is obviously up-regulated in multiple tissue parts such as leaves, leaves and the like, so that the gene has unexpected effect; can be used for screening and identifying moso bamboo varieties which are easy to age, has very important significance for breeding the moso bamboo varieties, and is suitable for industrialized application.

3) The overexpression of the PheNAP8 gene in Arabidopsis can obviously promote the senescence process of Arabidopsis, and the effect is obvious.

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 some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the expression pattern analysis of the Phyllostachys pubescens PheNAP8 gene in different senescence process of leaves in Phyllostachys pubescens.

1A is the relative expression quantity of the PheNAP8 gene in the leaves of the three-year-old seedling of the phyllostachys pubescens; young: young leaf, match: mature leaf, Old: senescent leaves; 1B is the relative expression quantity of the gene PheNAP8 in the leaves of the phyllostachys pubescens; 4.26: flower bud differentiation period; 5.26 and 6.7: elongation period of inflorescence; 6.16: full bloom period; 6.26: a young embryo forming stage;

FIG. 2 shows the homology analysis of the Phyllostachys pubescens PheNAP8 transcription factor with NAP transcription factors of other species;

FIG. 3 is the electrophoresis picture of Phyllostachys pubescens PheNAP8 gene amplification; wherein, M is D2000 DNA Marker,

lanes

1 and 2 are PCR amplified fragments of PheNAP8 gene;

FIG. 4 is the restriction enzyme digestion verification diagram of the overexpression vector of Phyllostachys pubescens PheNAP8 gene, wherein M is D2000 DNA Marker, and Lane 1 is the restriction enzyme digestion of recombinant plasmid BamHI and SalI;

FIG. 5 shows qRT-PCR and RT-PCR analysis of PheNAP8 gene transgenic Arabidopsis plants. 6A: qRT-PCR analysis is carried out on transgenic arabidopsis plants with the gene PheNAP 8; WT: wild type Arabidopsis thaliana; 2. 4, 6, 8 and 12: transgenic lines 35S:PheNAP 8/WT-2, 35S:PheNAP 8/WT-4, 35S:PheNAP 8/WT-6, 35S:: PheNAP8/WT-8 and 35S:: PheNAP 8/WT-12. 6B: carrying out RT-PCR analysis on transgenic arabidopsis plants with the gene PheNAP 8; m: d2000 DNA Marker, WT: wild type DNA is negative control; lanes 1-5: the candidate PheNAP8 gene transferred different strains 35S:: PheNAP8/WT-2, 35S:: PheNAP8/WT-4, 35S:: PheNAP8/WT-6, 35S:: PheNAP8/WT-8 and 35S:: PheNAP8/WT-12, lanes 6-10 are the same as lanes 1-5; lane 7, vector plasmid DNA as positive control; lane 8: ddH₂O；

FIG. 6 shows the RT-PCR detection of transgenic Arabidopsis plant with PhyNAP 8 gene. Wherein, M is D2000 DNAmarker, CK < - > is wild type DNA as negative control; water: blank control; CK + (CK + is positive control) by using vector plasmid DNA; lanes 1-6, different strains of the candidate transgenic PheNAP8 gene;

FIG. 7 phenotype of a wild-type, over-expressing transgenic line of the PheNAP8 gene when grown normally for 20 days;

FIG. 8 is a graph showing the expression of a senescence-associated gene AtSAG12 in leaves of a transgenic line which is a wild type and overexpresses the PheNAP8 gene when the wild type grows normally for 20 days. WT: wild type Arabidopsis thaliana; 2. 4, 6, 8 and 12: transgenic lines 35S:PheNAP 8/WT-2, 35S:PheNAP 8/WT-4, 35S:PheNAP 8/WT-6, 35S:: PheNAP8/WT-8 and 35S:: PheNAP 8/WT-12.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. 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.

Example 1 analysis and identification of Phyllostachys pubescens senescence-associated Gene PheNAP8

1) According to the Li (2019) bamboo shoot postharvest senescence transcriptome sequencing results, the number of upregulated expression was the greatest in NAC family members, of which 11 phernac genes were not substantially expressed at 0h postharvest (RPKM <0.3), and were significantly upregulated during postharvest storage (table 1). Wherein the PheNAP8 has similar expression patterns, is quickly up-regulated in 12-24h after the harvest, has higher up-regulation multiple (more than 120) than other PheNAC genes, and has extremely obvious and even unexpected up-regulation effect.

TABLE 1.11 expression analysis (transcriptome sequencing) of Phyllanthus PhyNAC genes after Phyllostachys Pubescens harvesting

2) Expression analysis of PheNAP8 in seedling leaves and flowering bamboo leaves

A. According to the judgment of the development stage of the leaves by the Rey army (2018), young leaves, mature leaves and aged leaves in the natural growth state are taken. The leaves of the flowering bamboos are taken according to the judgment of the flowering process of the bamboos (zhang et al, 2014).

B. Extraction of total RNA from leaves: the total RNA of the leaves is extracted according to the instruction of a TaKaRa RNAioso Plus kit, and the specific operation is as follows: a. grinding 0.2g of sample in liquid nitrogen to powder, and collecting the powder into a 2mL centrifuge tube; b. adding 1mLRNAiso Plus, reversing, mixing, and standing at room temperature for 5 min; c.12,000rpm, 4 ℃ and 5min, centrifuging, and taking the supernatant into an RNase-free centrifuge tube; d. adding 200 μ L chloroform into each tube, reversing, mixing for 5min, and standing at room temperature for 5 min; e.12,000rpm, 4 ℃, 15min, centrifuging, taking the supernatant to a new RNase-free centrifuge tube; f. adding equal volume of isopropanol into each tube, reversing, mixing, and standing at room temperature for 10 min; g.12,000rpm, 4 ℃, 10min, centrifuging and then discarding the supernatant; h. adding 1mL of 75% ethanol, washing the precipitate at 7,500rpm at 4 ℃ for 5min, centrifuging, and then removing the supernatant; i. adding 30 μ L of RNase-free H₂Dissolving the precipitate with O, and storing at-80 deg.C for use.

C. Reverse transcription

According to PrimeScript^TMThe RT reagent Kit with gDNA Eraser (TaKaRa) Kit carries out the first strand reverse transcription of cDNA. The reaction system and the reaction conditions are respectively as follows: mu.g total RNA, 2. mu.L gDNA Eraser Buffer, 1. mu.L gDNA Eraser, water to 10. mu.L, at 42 ℃ for 2min, quickly placed on ice. Then 1. mu.L of RT Primer Mix, 4. mu.L of LPrimeScript Buffer2, 1. mu.L of RNase Free dH were added in sequence₂O, 1. mu.L PrimeScript RT Enzyme Mix, 15min at 37 ℃, 5sec at 85 ℃, 85 ℃ for storage.

D. The expression patterns of the Phyllostachys pubescens PheNAP8 gene in different aging processes are detected by qRT-PCR.

The qRT-PCR reaction system was mixed according to the instructions of SYBR Green I Master (Roche).

The quantitative primers of the gene PheNAP8 are RT-PheNAP8-F: 5'-AGAGAGCCAGCTGTACGGGAC-3' (SEQ ID NO.3) and RT-PheNAP8-R:5 '-GTCGGGCCGTGCCATCT-5' (SEQ ID NO. 4). The expression pattern of the target gene was analyzed with TIP41 as an internal reference (Fanet al, 2013). The quantitative primers of TIP41 are RT-TIP41-F: 5'-AGAGAGCCAGCTGTACGGGAC-3' (SEQ ID NO.5) and RT-TIP41-R: 5'-GTCGGGCCGTGCCATCT-3' (SEQ ID NO. 6). And (3) PCR reaction conditions: reaction procedure: 5min at 95 ℃; 95 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 20s, 45 cycles.

The results show that the Phyllostachys Pubescens PheNAP8 gene is up-regulated and expressed with the increase of the aging degree of the leaf blade in the leaf blade of 3-year-old seedling or the leaf blade of flowering Phyllostachys Pubescens, has strong significance, is superior to the existing aging gene, and proves that the gene can be used as a senescence marker gene of the Phyllostachys Pubescens (figure 1).

Example 2 PheNAP8 gene evolution analysis.

The reported amino acid sequences of the members of the NAP subfamily are downloaded separately from the NCBI database. The protein sequence encoded by the Phyllostachys pubescens PhyNop 8 gene was subjected to multiple sequence alignment by ClustalX1.83(http:// www.clustal.org /) with the NAP protein sequences of other species (Thompson et al, 1997), and a rootless phylogenetic tree was constructed by the neighbor-joining method (bootstrap value set to 1000) by MEGA 7.0. The result shows that Phyllostachys Pubescens PheAP 8 has higher homology with other reported NAP transcription factors, PheAP 8 has the highest sequence similarity with BeNAC1 of pleione bamboo (figure 2), but no 100% homologous gene exists, and the PheNAP8 is proved to be a brand new gene which is not reported in the prior art and is an NAC transcription factor. Members of the NAP subfamily are mainly from Arabidopsis thaliana (ANAC018, NP 175696; ANAC025, EFH 64368; NP 564966; AtNAP, ANAC047, NP 187057, ANAC056, NP 188170), alfalfa (Medicago truncatula) (MtNAC969, XP 003607731), kidney bean (PvNAP, AAK84884), grape (VvNAP, ACX47024), soybean (GnNAC 1, AAY46121), cotton (GhNAP, XP 017640689), tomato (SINAP1, Solyc05g007770.1; SINAP2, Solyc04g005610.2.1), rice (ONAC010, LOC _ Os07g37920, OsNAC/ONAC 058, NP 913; OsOsNAC 3624234, NACtXP 015620532), golden silk bamboo (BealNAP 58NAP 686), Setarian wheat (wheat straw patchouli 39 1, wheat straw patchouli) (DNAsn-B39768), wheat straw patchouli-wheat straw patchouli) (PvNAP 39 1, NAC 6326, NAC 638, wheat straw patchouli). Candidate PheNAC protein sequences were subjected to multiple sequence alignment by ClustalX1.83(http:// www.clustal.org /) (Thompson et al, 1997) and an unrooted phylogenetic tree (bootstrap value set to 1000) was constructed by the neighbor-joining method by MEGA 7.0.

Example 3 obtaining of Gene encoding Phyllostachys Pubescens PheNAP8 transcription factor

The material adopted by the embodiment is moso bamboo leaves, and the moso bamboo leaves are stored at the temperature of minus 80 ℃ for standby after being quickly frozen by liquid nitrogen.

1) Extraction of total RNA from moso bamboo leaves

Extraction of Total RNA and first Strand cDNA Synthesis according to the method of example 1

3) PheNAP8 gene sequence amplified by RT-PCR

Designing full-length primers according to a moso bamboo genome database, wherein the full-length primers are respectively as follows: NAP1-F: 5'-ATGATGATGTCGAACCCGG-3' (SEQ ID NO.7) and NAP1-R: 5'-TCAGTTCATCCCCATGTTTGAAT-3' (SEQ ID NO. 8). PCR amplification was performed using the reverse transcription product as a template and NAP1-F and NAP1-R as primers. The PCR product was subjected to agarose gel electrophoresis (FIG. 3), and the objective product was recovered, ligated with pGEMT-easy, transformed into E.coli, and subjected to sequencing analysis by Jinweizhi corporation.

Example 4 functional analysis of the PheNAP8 Gene

1) Construction of plant expression vector of PheNAP8 gene

The CDS sequence of PheNAP8 gene is amplified by primers OE-PheNAP8-F and OE-PheNAP8-R (enzyme cutting sites KpnI and XbaI are respectively introduced at two ends), and is connected with pGEM-T easy, and after the sequencing is carried out, the plasmid is extracted and stored for later use. The target fragment is recovered by using KpnI and XbaI double enzyme digestion, and is connected with a constructed super expression vector pCAMBIA2300-35S, and the name is PheNAP8-pCAMBIA 2300-35S. The sequences of the primers OE-PheNAP8-F and OE-PheNAP8-R are 5'-CGGGGTACCATGATGATGTCGAACCCGG-3' (SEQ ID NO.9) and 5'-GCTCTAGATCAGTTCATCCCCATGTTTGAAT-3' (SEQ ID NO. 10).

2) Enzyme digestion detection of recombinant plasmid PheNAP8-pCAMBIA2300-35S

BamHI and SalI were chosen for double restriction detection of recombinant plasmids, and FIG. 4 shows that bands of the same size as the target fragment were obtained after restriction. The recombinant plasmid is shown to contain a target gene sequence and can be used for subsequent infection transformation experiments.

3) Preparation, transformation and identification of Agrobacterium GV3101 competent cells

A Single colony of Agrobacterium tumefaciens GV3101 was picked and inoculated into 10ml of YEP medium (containing rifampicin 50 mg. L)^-1) Culturing in liquid culture medium at 28 deg.C and 220rpm overnight;

b2 mL of the above-mentioned bacterial suspension was added to 50mL of YEP medium (containing rifampicin 50 mg. multidot.L)^-1) Centrifuging at 28 deg.C and 220rpm for 3-4 hr until OD600 is about 0.6, 5000rpm for 5min, and discarding supernatant;

c, adding 40mL of precooled 10% glycerol to resuspend the thalli, carrying out ice bath for 30min, carrying out 4 ℃, carrying out 5000rpm for 5min, and discarding the supernatant;

d, adding 30mL of precooled 10% glycerol to resuspend the thalli, carrying out treatment at 4 ℃ and 5000rpm for 5min, discarding the supernatant, and repeating the step D;

e, adding 2mL of 10% glycerol for suspension, subpackaging in a centrifuge tube with 1.5mL (100 mu L of each tube), quickly freezing by using liquid nitrogen, and storing at-80 ℃ for later use.

F, respectively adding 1 μm L of PheNAP8-pCAMBIA2300-35S recombinant plasmid into the Agrobacterium infected cells, electrically stimulating at 2500V, adding 300 μ L of LB liquid culture medium, 28 deg.C, 180rpm, 3 hr;

g, coating 100 mu L of culture on an LB plate (containing rifampicin 50 mg. L-1; kanamycin 50 mg. L-1), and culturing at 28 ℃ for 36 h;

h, picking a single clone, carrying out PCR detection on a bacterial liquid by using a universal primer 35S-F and an NOS-R on a carrier (the PCR system is the same as the above), and storing a positive transformant for later use.

4) Agrobacterium-mediated transformation of Arabidopsis thaliana

Seeds infecting Arabidopsis plants were harvested by transformation of Arabidopsis by inflorescence dip-dyeing (Clough et al, 1998) (T₀Seed generation), air drying and storing for later use.

5) And (4) screening positive transformants of Arabidopsis thaliana.

A T to be obtained₀Soaking the seeds in 75% ethanol for 10min, reversing for several times every 5min, washing with anhydrous ethanol for 3 times, placing the seeds on sterilized filter paper, and blow-drying in a super clean bench (about 30 min);

b the air-dried Arabidopsis seeds are evenly sown on 1/2MS solid culture medium (containing Kan 50 mg. L)^-1) At 4 ℃, dark culture is carried out for 2 d;

c, transferring to an arabidopsis room for long-day culture, and culturing at 23 ℃ for 16h in light/8 h in dark for 8 d;

d, transplanting the screened Kan resistant Arabidopsis plants into nutrient soil for long-day culture (23 ℃, 16h of light/8 h of dark).

5) PCR and RT-PCR detection of over-expressed plants.

PCR detection of transgenic plants A: taking a proper amount of leaves of wild arabidopsis thaliana and transgenic plants, and extracting genome DNA of the leaves of arabidopsis thaliana according to a CTAB method. The recombinant vector plasmid PheNAP8-pCAMBIA2300-35S is used as a positive control, the total DNA of wild arabidopsis thaliana is used as a negative control, and primers RT-PheNAP8-F and 2300-R (designed from the vector pCAMBIA 2300-35S) are used for carrying out PCR identification on the target gene. The results show that the candidate transgenic line 35S, PheNAP8/WT-1 is false positive, and the rest are transgenic plants (figure 5).

And B, RT-PCR detection of transgenic plants: total RNA of wild type Arabidopsis thaliana and transgenic Arabidopsis thaliana was extracted according to the method of example 1, and reverse transcription was performed to obtain first strand cDNA, and Actin2-F and Actin2-R and RT-PheNAP8-F and RT-PheNAP8-R were used to amplify Actin and the desired gene. The results show that the PheNAP8 gene is overexpressed in arabidopsis thaliana in the transgenic lines (fig. 6).

6) Phenotypic observation of over-expressed plants

Harvested T₁The generation transgenic seeds are indicated to be disinfected and then are broadcast on 1/2MS culture medium containing kanamycin, and dark culture is carried out at 4 ℃ for 2 d; then, the culture is switched to long-day culture (16h light/8 h dark) at 23 ℃ for 8 d; kan-resistant Arabidopsis plants were transplanted into nutrient soil for long-day culture (23 ℃, 16h light/8 h dark). The phenotype of the transgenic arabidopsis is observed under natural conditions, and the result shows that after the transgenic arabidopsis is transferred to nutrient soil for 10 days, the transgenic plant is delayed in development and blooms in advance, and after 20 days, the leaves of the transgenic plant begin to yellow and age, but the leaves of the wild arabidopsis do not have the senescence phenotype (figure 7), which shows that the gene can effectively regulate and control the plant senescence.

7) Observation of overexpression plant genotype

Taking the quantitative primers RT-PheNAP8-F and RT-PheNAP8-R of the PheNAP8 gene as examples, the RT-PCR kit for screening/identifying the bamboo senescence varieties is constructed, and comprises other conventional reagent components. Extracting leaf RNA of transgenic arabidopsis and wild arabidopsis, carrying out reverse transcription to obtain first-strand cDNA, detecting the expression of a senescence-associated gene PheNAP8 by using the first-strand cDNA as a template and an actin gene as an internal reference through RT-PCR, and displaying that the senescence-associated gene PheNAP8 in a transgenic strain is up-regulated (figure 8).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> International bamboo rattan center

<120> A Mao bamboo aging related NAP transcription factor, its coding gene and application

<130>2020

<160>10

<170>PatentIn version 3.5

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atcgccgacg tcgacatcta caagttcgac ccatgggacc tcccatccaa ggctgcgtac 180

ggggacaagg aatggtactt cttcagcccg agggaacgca agtacccgaa tgggatccgg 240

ccgaaccgcg cggcggggtc tggctactgg aaggccaccg gcaccgacaa gcccattcac 300

agcagcgcca ccggcgagag cgtcggcgtc aagaaggccc tcgtcttcta caagggccgt 360

ccgcccaagg gcaccaagac caactggatc atgcacgagt accggctcga ctccgccgac 420

gctaaggccg gcaacaccta ccgccccacg aagttccgaa acgcctccat gaggttggac 480

gactgggtgc tgtgccggat ctacaagaag agcagtcacg tgtcgccgat ggcggtgccg 540

ccgctgtccg accacgagca ggacgagccg tgcgccttcg gagagagcca gctgtacggg 600

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ctggcgcagc tcctcgacga cggcgccccg gccgagatgg cacggcccga ccagcacgcc 780

gcgctcctcg gccaccccgt cgtgaaccaa tttcttgtgc acagcagagg caacaacatg 840

tggcagctcg cgcagatggg ctcgtcgacc tcgacgtcgg ccgcgggcga tggcgccact 900

ggaaaacgca agagatcgga agacggtggc aatactgggc taacgtgcca accgactgcg 960

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<400>2

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

1 5 10 15

Pro Thr Asp Glu Glu Leu Ile Leu His Tyr Leu Arg Asn Arg Ala Ala

20 25 30

Ser Ser Pro Cys Pro Val Asp Ile Ile Ala Asp Val Asp Ile Tyr Lys

35 40 45

Phe Asp Pro Trp Asp Leu Pro Ser Lys Ala Ala Tyr Gly Asp Lys Glu

50 55 60

Trp Tyr Phe Phe Ser Pro Arg Glu Arg Lys Tyr Pro Asn Gly Ile Arg

65 70 75 80

Pro Asn Arg Ala Ala Gly Ser Gly Tyr Trp Lys Ala Thr Gly Thr Asp

85 90 95

Lys Pro Ile His Ser Ser Ala Thr Gly Glu Ser Val Gly Val Lys Lys

100 105 110

Ala Leu Val Phe Tyr Lys Gly Arg Pro Pro Lys Gly Thr Lys Thr Asn

115 120 125

Trp Ile Met His Glu Tyr Arg Leu Asp Ser Ala Asp Ala Lys Ala Gly

130 135 140

Asn Thr Tyr Arg Pro Thr Lys Phe Arg Asn Ala Ser Met Arg Leu Asp

145 150 155 160

Asp Trp Val Leu Cys Arg Ile Tyr Lys Lys Ser Ser His Val Ser Pro

165 170 175

Met Ala Val Pro Pro Leu Ser Asp His Glu Gln Asp Glu Pro Cys Ala

180 185 190

Phe Gly Glu Ser Gln Leu Tyr Gly Thr Ser Ser Ala Gly Met Ile Gly

195 200 205

Gln Gly Gly Ala Asp Ala Phe Pro Leu Gln Ala Ala Ala Ala Thr Gln

210 215 220

Arg Met Pro Arg Thr Pro Ser Ile Ser Glu Leu Leu Asn Asp Tyr Ser

225 230 235 240

Leu Ala Gln Leu Leu Asp Asp Gly Ala Pro Ala Glu Met Ala Arg Pro

245 250 255

Asp Gln His Ala Ala Leu Leu Gly His Pro Val Val Asn Gln Phe Leu

260 265 270

Val His Ser Arg Gly Asn Asn Met Trp Gln Leu Ala Gln Met Gly Ser

275 280 285

Ser Thr Ser Thr Ser Ala Ala Gly Asp Gly Ala Thr Gly Lys Arg Lys

290 295 300

Arg Ser Glu Asp Gly Gly Asn Thr Gly Leu Thr Cys Gln Pro Thr Ala

305 310 315 320

Ala Gly Lys Lys Pro Asn Gly Ser Cys Phe Gly Ala Thr Phe Gln Ile

325 330 335

Gly Asn Gly Leu Gln Gly Ser Leu Gly Leu Gly His Gln Met Leu His

340 345 350

Ser Asn Met Gly Met Asn

355

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<400>3

agagagccag ctgtacggga c 21

<210>4

<211>17

<212>DNA

<213> Artificial sequence

<400>4

gtcgggccgt gccatct 17

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<400>5

agagagccag ctgtacggga c 21

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<400>6

gtcgggccgt gccatct 17

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<400>7

atgatgatgt cgaacccgg 19

<210>8

<211>23

<212>DNA

<213> Artificial sequence

<400>8

tcagttcatc cccatgtttg aat 23

<210>9

<211>28

<212>DNA

<213> Artificial sequence

<400>9

cggggtacca tgatgatgtc gaacccgg 28

<210>10

<211>31

<212>DNA

<213> Artificial sequence

<400>10

gctctagatc agttcatccc catgtttgaa t 31

Claims

1. A protein, wherein the protein sequence is any one of:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 2;

(3) an amino acid sequence of a protein which has at least 90 percent of homology with the amino acid sequence shown as SEQ ID NO.2 and has the same function; preferably, the homology is at least 95%; more preferably at least 99%.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein the gene sequence is any one of the following:

(1) as shown in SEQ ID NO. 1;

(2) a sequence having at least 90% homology to SEQ ID No. 1; preferably, the homology is at least 95%; more preferably at least 99%.

4. An expression vector, recombinant plasmid, expression cassette, recombinant bacterium or recombinant cell comprising the gene of claim 2 or 3.

5. A method for constructing a transgenic plant, comprising introducing the gene of claim 2 or 3 into a target plant to obtain a transgenic plant, wherein the leaves of the transgenic plant develop faster than the target plant;

preferably, the plant is a monocotyledon or dicotyledon, more preferably, the plant is a phyllostachys pubescens or arabidopsis thaliana.

6. Use of the protein of claim 1, the gene of claim 2 or 3, the expression vector, the recombinant plasmid, the expression cassette, the recombinant bacterium or the recombinant cell of the gene of claim 4 for regulating the development of plant leaves and senescence of plants; preferably, the plant is a monocotyledon or dicotyledon; more preferably, the plant is Phyllostachys pubescens or Arabidopsis thaliana.

7. Use of the protein of claim 1, the gene of claim 2 or 3 for screening/identifying an senescence-susceptible variety of Phyllostachys pubescens; preferably, the method is applied to identification of the bamboo varieties easy to age in the seedling stage.

8. A method for screening/identifying a species of phyllostachys pubescens which is susceptible to senescence, comprising detecting whether the protein of claim 1 or the gene of claim 2 or 3 is contained in the species of phyllostachys pubescens; or detecting the content of the protein of claim 1 or the gene of claim 2 or 3 in a moso bamboo variety.

9. A kit for screening/identifying a phyllostachys pubescens senescence-susceptible variety, comprising a component for detecting the protein of claim 1 or the encoding gene of claim 2; preferably, the components include, but are not limited to, components in the form of primers, probes, or sequencing reagents; more preferably, the kit comprises primer pairs as shown in SEQ ID NOs 3 and 4.

10. A primer pair is characterized in that the sequences of the primer pair are respectively shown as SEQ ID NO 3 and SEQ ID NO 4.