CN111206039A - Bambusa multiplex transcription factor BmMYB83 gene and application thereof - Google Patents

Abstract

The invention discloses a filial bamboo transcription factor BmMYB83 gene and an application thereof, belonging to the technical field of plant genetic engineering. The transcription factor BmMYB83 provided by the present invention belongs to the MYB transcription factor family and is derived from Bamboo filial piety. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The invention reveals the phylogenetic status of the gene encoding the BmMYB83 transcription factor of Bamboo filial piety through gene cloning, expression and comparative analysis, and provides a method for applying the gene. Overexpression of the gene can change the plant type of Arabidopsis thaliana, including the Dwarfing, leaf curling, increase in the number of flower stems, etc.; it can also shorten the root length of rice. The genes of the present invention will play an important role in the improvement of plant traits.

Description

Bambusa multiplex transcription factor BmMYB83 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a transcription factor BmMYB83 gene of Bambusa multiplex (Bambus amultiplex) and application thereof.

Background

MYB transcription factors are one of the largest class of transcription factors in plants and are involved in a variety of activities during plant growth and development, including stress response (Baldoni, E. et al, International Journal of molecular sciences, 16, 15811-15851, 2015), hormone synthesis (Wang et al, BMC Genomics, 16: 17, 2015), signal transduction. In the process of secondary growth of plant cell walls, MYB transcription factors present a regulation and control mode of a hierarchical network and participate in synthesis of components such as cell wall cellulose, hemicellulose, lignin and the like. MYB46 and MYB83 are main switches of network regulation and are directly regulated by two types of NAC transcription switch factors (Zhang et al, Frontiers in Plant Science, 9: 1535, 2018).

Genetic studies have shown that the Arabidopsis double mutants of MYB46 and MYB83 produce severe secondary wall synthesis defects, whereas overexpression of both genes can activate up-regulation of the expression of synthetic genes for cellulose, lignin and xylan (Zhong et al, Plant Cell, 19(9), 2776-; whereas overexpression of OsMYB46 or ZmMYB46 in the double mutant of Arabidopsis myb46myb83 restored the phenotypic defect to some extent, moreover, overexpression of OsMYB46 and ZmMYB46 in Arabidopsis, respectively, resulted in a very severe leaf curl phenotype (Zhong et al, Plant cellphysiology, 52(10), 1856-one 1871, 2011).

At present, the study on MYB transcription factors in bamboo plants is only embodied in moso bamboos. Researchers have classified the PheR2R3MYBs transcription factors in Phyllostachys pubescens into 17 subgroups by a systematic analysis method, and verified that the gene can increase the cold tolerance of plants and improve the sensitivity of the plants to drought and salt stress by a technology of ectopically expressing PhemYB4-1 in Arabidopsis thaliana (Hou et al, Frontiers in Plant Science, 9, 738, 2018). In addition, the study of other MYB transcription factors in bamboo plants is nearly blank.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a transcription factor BmMYB83 gene of Bambusa multiplex. The invention aims to solve another technical problem of providing the application of the transcription factor BmMYB83 gene of the Bambusa multiplex.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a transcription factor BmMYB83 gene of Bambusa multiplex has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the transcription factor BmMYB83 gene of the Bambusa multiplex is shown in SEQ ID NO. 2.

The Bambusa multiplex transcription factor BmMYB83 gene vector.

The Bambusa multiplex transcription factor BmMYB83 gene is applied to the change of plant dwarfing, leaf curling, increased number of flower stems or root length shortening of plant phenotype.

The application comprises the following steps:

1) constructing a vector of the transcription factor BmMYB83 gene of the Bambusa multiplex;

2) transforming the constructed vector of the transcription factor BmMYB83 gene into a plant or a plant cell;

3) and cultivating and screening to obtain the plants with dwarfing plants, curled leaves, increased flower stem number or shortened root length.

In the application, the plant is arabidopsis thaliana or rice.

Compared with the prior art, the invention has the beneficial effects that:

the transcription factor BmMYB83 provided by excavating MYB transcription factor resources in the Bambusa multiplex with the help of model plants Arabidopsis thaliana and rice has a nucleotide sequence shown as SEQ ID NO.1, and an amino acid sequence of encoded protein is shown as SEQ ID NO. 2. Through gene clone expression and comparative analysis, the phylogenetic position of the encoding gene of the transcription factor BmMYB83 of the Bambusa multiplex is revealed, and a method for applying the gene is provided, wherein the plant type of an arabidopsis plant can be changed by over-expressing the gene, and the plant type comprises plant dwarfing, blade curling, increased flower stem number and the like; can also shorten the root length of rice. The curling of leaves and the increase of the number of flower stems are useful characters in agriculture, the shortening of root length and the dwarfing of plants are also useful character changes in crop breeding, and the gene plays an important role in the improvement of plant characters.

Drawings

FIG. 1 is a gel electrophoresis chart of BmMYB83 transcription factor amplification product;

FIG. 2 is a BmMYB83 transcription factor system occurrence tree diagram;

FIG. 3 is an alignment chart of the amino acid sequence of the BmMYB83 transcription factor;

FIG. 4 is a graph of the root length phenotype of BmMYB83 transgenic rice and wild type rice "Nipponbare" plants, WT represents wild type plants, and Line 4, 10, 14, 17 represent different lines of transgenic rice, respectively;

FIG. 5 is a leaf phenotype diagram of BmMYB83 transgenic Arabidopsis and wild type Arabidopsis plants, wherein col-represents the Arabidopsis Columbia wild type plant, and Line1, 2, 4, 5, 6, 8 represent different strains of the transgenic plant, respectively;

FIG. 6 is a graph of the whole plant type, plant size and flower stem number of BmMYB83 transgenic Arabidopsis and wild Arabidopsis, and lines 1, 4 and 8 respectively represent different strains of the transgenic Arabidopsis.

Detailed Description

The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1: obtaining of BmMYB83 transcription factor coding gene of Bambusa multiplex

RNA extraction

The extraction material is bamboo shoot of Bambusa multiplex, RNA extraction is performed by using a rapid extraction kit of protoporphyrin RNAPure, extraction is performed according to an operation instruction, and the obtained product is stored at-80 ℃ for later use

First Strand cDNA Synthesis and reverse transcription PCR

Total RNA was reverse transcribed into cDNA using a Whole gold (Beijing) one-stepc DNA synthesis kit according to the instructions. Reaction System and reaction conditionsRespectively, the following steps: mu.g of prepared total RNA, Anchored Oligo (dT)₁₈Primell. mu.L, 10. mu.L of 2 XTS Reaction Mix, 1. mu.L of LTrans script RT/RI Enzyme Mix, 1. mu.L of gDNAremover supplemented with RNase-free Water to 20. mu.L, gently mixed and centrifuged. Reverse transcription is carried out for 30min at 42 ℃ by a PCR instrument and 5s at 85 ℃; cooling on ice; centrifuging and storing at-20 ℃ for later use.

PCR amplification of the CDS region of BmMYB83

According to the sequencing and gene annotation result of the Bambusa multiplex transcriptome, the full-length gene sequence of MYB83 is obtained, and the sequence ID is BmuUn 039904. Two primers MYB83-S (5'-AGCCCTTTTTCCATCCTTG-3') and MYB83-A (5'-GTACTCCTTGGCAGCAGCTA-3') were designed upstream and downstream of the CDS region as primers for PCR.

The PCR enzyme is TOYOBO high fidelity enzyme, and the reaction system is 50 mu L: ddH₂O，34μL；10×Buffer，5μL；dNTP，5μL；Mg²⁺2 μ L; forward primer, 1 μ L; reverse primer, 1 μ L; cDNA, 1 uL; KOD-Plus enzyme, 1. mu.L. The PCR procedure was: 5min at 94 ℃; circulating for 38 times at 94 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 55 s; 10min at 72 ℃. The PCR product was separated by 1.2% agarose gel electrophoresis to obtain a fragment of about 1152bp, as shown in FIG. 1. After purification and recovery, the product was sequenced by Soxhlet Biotech, Inc. (Nanjing). The sequencing result is shown in SEQ ID NO.1, and the length of the nucleotide sequence is 1152 bp; the expression sequence is shown in SEQ ID NO.2, and the protein consists of 383 amino acid residues.

Example 2: functional analysis of BmMYB83 transcription factor of Bambusa multiplex

1. Comparative analysis of sequences

In order to analyze the genetic relationship between BmMYB83 of the Bambusa multiplex and MYB transcription factors of other species, a phylogenetic tree of BmMYB83 and MYB transcription factors of other species is established by MEGA 7. The results (FIG. 2) show that BmMYB83 has the closest relationship with OsMYB83 and OsMYB46 in the model plant rice and belongs to the same cluster.

DNAMAN was used to analyze the homology of BmMYB83 with closely related MYB transcription factors in other species. The results (FIG. 3) show that BmMYB83 has higher homology with OsMYB83 in rice, and the sequence similarity is 58.84%. And the sequence similarity with rice OsMYB46 and Arabidopsis AtMYB83 is 39.44% and 38.39%, respectively.

2. Analysis of Rice transgenes

2.1 vector construction

The correctly sequenced PCR product was ligated into the pEASY-Blunt vector of the full-scale gold (Beijing) using the ligation system: 4.5. mu.L DNA, 0.5. mu.L Vector. After gently mixing, the mixture was left at room temperature for 5min and then placed on ice.

Melting competent cells Trans1-T1 on ice, adding the connected carrier, flicking, mixing, and ice-cooling for 30 min; heat shock in 42 deg.C water bath for 30s, immediately placing on ice for 2 min; adding 1mL of LB culture medium without antibiotic at room temperature, and culturing at 200rpm and 37 ℃ for 1 h; the cells were centrifuged at 8000rpm for 1min to obtain the cells, most of the supernatant was aspirated, and 200. mu.L of the culture medium was left to suspend the cells. Spread to LB plates containing ampicillin; the cells were incubated overnight in an incubator at 37 ℃. 10 single clones were picked and shaken separately in tubes containing 4mLLB medium, after 18 hours at 37 ℃ and 200rpm, plasmids were extracted and sent to Kinsley for sequencing. The plasmid with the correct sequencing was designated pEASY-Blunt-BmMYB 83.

2.2 expression vector construction

This section is operated by Wuhan Boehfar Biometrics.

Designing primers with enzyme cutting sites to carry out PCR reaction, wherein the primer sequences are respectively as follows:

BmMYB83(+)：5′-cagtCGTCTCacaacatgaggaagccggagttccc-3′；

BmMYB83(-)：5′-cgatCGTCTCattcaacttggaaatcaagga-3′；

fusion of GFP (+): 5'-cagtGGTCTCatccctgtatcgtgaagggcgagga-3', respectively;

fusion to GFP (-): 5'-cagtGGTCTCatacatcagttgtagagctcgtcca-3' are provided.

2 50 μ L reaction systems: buffer, 5 μ L; dNTPs, 2. mu.L; forward primer, 2 μ L; reverse primer, 2 μ L; cDNA, 1 uL; ddH₂O, 34 μ L; taq enzyme, 2U; mg (magnesium)²⁺4 μ L. The PCR procedure was: 5min at 94 ℃; 30 times of circulation at 94 ℃, 45s at 50 ℃ and 56s at 72 ℃; 10min at 72 ℃ and 30min at 16 ℃. After 1% agarose gel electrophoresis, the electrophoresis bands of 1149bp and 717bp are respectively cut off,putting the mixture into the same system for sol recovery (the recovered product is marked as rDNAmG2), sequencing without errors, and then performing enzyme digestion and connection.

The vector enzyme cutting system is 20 mu L: buffer: 2 μ L, BsaI/Eco 31I: 1 μ L, pBWA (V) HU-ccdB: 4 μ L, H₂O: 13 μ L. 1h at 37 ℃. The enzyme cutting system of rDNAmG2 is 20 mu L: buffer: 2 μ L, BsaI/Eco 31I: 1 μ L, rDNAmG 2: 4 μ L, H₂O：13μL。37℃1h。

Combining and recovering the carrier enzyme cutting product and the recovered fragment enzyme cutting product, and connecting the carrier enzyme cutting product and the recovered fragment enzyme cutting product after the purification of a PCR purification kit, wherein the connecting system is 10 mu L: buffer: 1 μ L, T4-1 igase: 1 μ L, H₂O: 5.5. mu.L, product recovered: 2.5. mu.L. 20 ℃ for 1 h.

5-10. mu.L of the ligation product was transformed into E.coli competent cells, plated on kanamycin-resistant plates, incubated at 37 ℃ for 12 hours, and 10 plaques were picked while 1.5mL of EP inocula and PCR identification were performed. The primers used for identification are pBWA (V) HU-ccdB identification primers: pubiseq +, NOSseq-R, Pbw2+, Pbw 2-.

Detection primer set 1:

pubiseq+：5′-CCTGCCTTCATACGCTATTTATTTGCTTGG-3′

NOSseq-R：5′-CAAGACCGGCAACAGGATTCAATC-3′

detection primer set 2:

Pbw2+：5′-GCAACGCTCTGTCATCGTTACAAT-3′

Pbw2-：5′-GCGATTAAGTTGGGTAACGCCAGGG-3′

and (3) PCR reaction identification, wherein the reaction system is 25 mu L, and the components are as follows: h₂O：16.5μL，buffer：2.5μL，Mg²⁺: 2 μ L, dNTP: 1 μ L, pubiseq +/Pbw2 +: 1 μ L, NOSseq-R/Pbw 2-: 1 μ L, taq enzyme: 1U, Template: 1 μ L. The reaction procedure is as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 50 ℃ for 45s, 72 ℃ for 56 s; 10min at 72 ℃; 30min at 16 ℃. The target band is about 1456 bp.

And (3) taking 100 mu L of bacterial liquid corresponding to 1-3 positive bands, carrying out sample feeding sequencing, inoculating the rest 400 mu L of bacterial liquid into 5-10mL LB containing kanamycin resistance, shaking the bacteria in a test tube, carrying out bacterial liquid sequencing, and extracting the agrobacterium tumefaciens to be transformed from the plasmid from the bacterial liquid with the sequence consistent with that of BmMYB 83.

2.3 Agrobacterium transformation

1)200ng of plasmid, 40. mu.L of Agrobacterium-infected competent cells were added and mixed, and then electrotransformation (U, 1.8 KV; r, 200 Ω; c, 25. mu.F).

2) Adding 800 μ L SOC liquid culture medium after electric shock, and culturing at 28 deg.C for 1 h; the SOC liquid culture medium comprises the following components: 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl₂20mM glucose.

3) The cells were collected by centrifugation at 4000rpm for 10 minutes, resuspended at 200. mu.L SOC, plated on LB solid medium containing 100. mu.g/mL spectinomycin, 40. mu.g/mL rifampicin, and 100. mu.g/mL streptomycin, and subjected to inverted culture at 28 ℃ for 48 to 72 hours.

2.4 Agrobacterium-mediated transformation of Rice calli

(1) Induction of Rice calli

Taking mature seeds of japonica rice variety Nipponbare, mechanically shelling, selecting plump sterile spot high-quality seeds, and sterilizing; inoculating the sterilized seeds to a callus induction culture medium for induction, and inducing the callus by taking the mature embryos of the rice as materials.

Callus induction medium (NB): macroelement N6, trace element B5, organic matter B5, Fe salt, sucrose (30 g/L), glutamine (500 mg/L), proline (500 mg/L), casein hydrolysate (300 mg/L), 2mg/L2, 4-D and 3.0g/L plant gel, and adjusting the pH to 5.8.

N6 macroelements include: 1650mg/L NH₄NO₃，1900mg/L KNO₃，440mg/L CaCl₂·2H₂O，370mg/L MgSO₄·7H₂O，170mg/L KH₂PO₄。

Trace elements B5 include: 0.83mg/L KI, 6.2mg/L H₂BO₃，22.3mg/L MnSO₄.4H₂O，8.6mg/LZnSO₄.7H₂O，0.25mg/L Na₂MoO₄·2H₂O，0.025mg/L CuSO₄·5H₂O，0.025mg/L CoCl₂·6H₂O。

B5 organics included: 100mg/L of 0 inositol, 0.5mg/L of nicotinic acid, 0.5mg/L of pyridoxine hydrochloride, 0.1mg/L of thiamine hydrochloride and 2mg/L of glycine.

(2) Agrobacterium transformation

Following the EHA105 competent instructions. Melting Agrobacterium tumefaciens strain at-80 deg.C under normal temperature, and inserting into ice when it is in ice-water mixed state; adding 0.01-1 mu g of constructed plasmid of the target gene into every 100 mu L of competence, lightly stirring the mixture at the bottom of a centrifuge tube, uniformly mixing, and standing on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min in sequence; adding 700 mu L of nonreactive LB liquid medium, and performing shake culture at 20 ℃ for 2-3 h; centrifuging at 6000rpm for 1min, removing most of supernatant, leaving about 100 mu L of supernatant, gently blowing and beating the resuspended thallus, coating an LB plate containing kanamycin and rifampicin, and performing inverted culture for 2-3 d.

Selecting an agrobacterium tumefaciens single colony, placing the agrobacterium tumefaciens single colony in a YEB culture medium containing a proper amount of antibiotics, performing shake culture at 28 ℃, and rotating at 220rpm until the OD value is about 1-1.2; centrifuging at 5000rmp for 10min, removing supernatant, resuspending the thallus with sucrose suspension (5% sucrose and 0.03% surfactant silwet), and adjusting OD₆₀₀The value is 0.8 to 1.0, and the activation is carried out for 2 to 3 hours under light.

YEB culture medium is: beef extract 5g/L, yeast extract 1g/L, peptone: 5g/L, sucrose: 5g/L, MgSO₄·7H₂O: 0.5g/L, pH 7.0, Kana: 50mg/L, Rif: 50 mg/L.

(3) Screening and differentiation of rice callus

Selecting light yellow, granular and hard and compact callus, placing the callus in agrobacterium suspension carrying target genes for infection, and then placing the callus on a co-culture medium for culture. The co-culture medium is as follows: macroelement N6, trace element B5, organic matter B5, Fe salt, maltose 30g/L, casein hydrolysate 500mg/L, inositol 2g/L, inositol 100 mu mol/LAS 3g/L, plant gel 2mg/L2, 4-D, and pH is adjusted to 5.5.

Taking out the well-grown callus, airing, transferring the well-grown callus to a screening culture medium for primary screening, and transferring the initial callus with the resistant callus to a new culture medium for secondary screening. And finally, selecting the resistant callus, transferring the resistant callus into a differentiation culture medium for induced differentiation to form seedlings, and transferring the seedlings to a rooting culture medium after the seedlings grow to about 1 cm. And simultaneously, inducing and differentiating callus tissues which do not infect agrobacterium to be used as wild type control seedlings.

The screening culture medium is as follows: NB +50mg/L hygromycin +200mg/L cefazolin sodium +200mg/L amoxicillin, pH was adjusted to 5.8.

The callus presorting culture medium comprises: NB +20g/L sorbitol +5mg/L ABA +3mg/LCuSO₄+3mg/L BA +1mg/L LNAA +50mg/L hygromycin +100mg/L cefazolin sodium +100mg/L amoxicillin, pH was adjusted to 5.8.

The callus differentiation culture medium is: macroelement N6, trace element B5, organic matter B5, sucrose, casein hydrolysate, BA, NAA, hygromycin, plant gel, penicillin 100mg/L, and cefazolin sodium 100mg/L, wherein the total content of the macroelement N6, the trace element B5, the organic matter B5, the sucrose, the sorbitol and the casein hydrolysate is 20g/L, the total content of the casein hydrolysate is 2mg/L, the total content of the BA, the NAA and the hygromycin is 50mg/L, the plant gel is 3.0g/L, the amox.

The rooting culture medium comprises: 1/2MS +20g/L sucrose +3.0g/L plant gel +100mg/L amoxicillin +100mg/L cefazolin sodium, pH adjusted to 5.8.

(4) Identification of Positive cloned seedlings of Rice

The rice test-tube plantlet grows to about 10cm, DNA is extracted by a CTAB method, and PCR identification is carried out on the test-tube plantlet. The method comprises the following specific steps:

grinding: taking transgenic seedlings, adding CTAB, grinding, and adding a proper amount of CTAB; dissolving in water: shaking every 10min for 30 min; cooling the sample to normal temperature, adding chloroisopentyl alcohol, oscillating for 20min, wherein the volume of the chloroisopentyl alcohol is the same as that of CTAB; centrifuging for 10min, and transferring the supernatant to an EP tube; adding 0.7 times of isopropanol (pre-cooling in a refrigerator at-20 deg.C), shaking to observe filament, and standing in the refrigerator for 30 min; centrifuging for 5min, and discarding the supernatant; cleaning with 70% ethanol, blowing with a pipette to suspend, and adding ddH into each tube₂O and RNase; detection on 1% agarose gel.

And (3) placing the rice seedlings detected as positive clones in a rice incubator for culturing, and opening a bottle cap for hardening the seedlings under the conditions of illumination of 700UM, temperature of 28 ℃, humidity RH 70%, illumination for 14h and darkness for 10 h. And taking out the plantlets after three days, washing off the root culture medium, and culturing by using a potting method, wherein the used soil is field soil.

After harvesting the BmMYB83 transgenic plant, accelerating germination under the same conditions with wild type seeds, after budding, culturing the plant by using a rice nutrient solution for one week, and observing the phenotype of the plant, wherein the root length of the obtained transgenic rice plant is shorter than that of the wild type plant under the condition that the seedling heights of the two plants are not obviously different (figure 4).

3 Arabidopsis transformation

3.1 sowing

Mixing peat soil, vermiculite and perlite at a ratio of 1: 3: 0.5, and autoclaving at 121 deg.C for 20 min. The sterilized soil is filled into a seedling pot, the bottom of the seedling pot is watered (flowers with the concentration of 1g/L can be added for the first watering), and the seedling can be sown when the soil in the pot absorbs water and becomes moist. Seeds of wild Columbia col-0 are placed in a refrigerator at 4 ℃ for about one week, taken out, uniformly scattered into soil, covered with a preservative film, placed in an artificial climate box for cultivation, and the cultivation temperature is 24 ℃ and the illumination is 120 UM. After the first true leaves of arabidopsis seedlings are unfolded (about 10 days), removing the preservative film, and culturing until the full-bloom stage for later use.

3.2 Agrobacterium transformation

The same as 2.4 (2) Agrobacterium transformation.

3.3 transformation of Arabidopsis thaliana by inflorescence Dipigmentation

Taking 3-4 pots of arabidopsis thaliana in the full-bloom stage, cutting out fruiting pods and bloomed flowers in advance, immersing inflorescences in the activated suspension for about 14s, culturing in a humid dark environment for 24h, taking out, and putting into an incubator for normal culture.

3.4 screening, Observation and identification

1) When the siliques of arabidopsis thaliana turn yellow or brown, bagging and stopping watering to ensure that the plants die slowly and then harvest uniformly to obtain transgenic T0 seeds;

the harvested T0 generation seeds are put into a refrigerator at 4 ℃ for low-temperature treatment for about 7 days (vernalization);

preparing an MS solid culture medium, placing the MS solid culture medium into a superclean bench for slow cooling after autoclaving, and adding hygromycin when the temperature of the culture medium is reduced to about 50 ℃, wherein the concentration of the hygromycin is 20 mg/L. Simultaneously preparing a hygromycin-free MS culture medium for later use;

taking a proper amount of vernalized transgenic T0 generation seeds, sterilizing with 75% ethanol for 5min, washing with sterile water for 5 times, uniformly spreading on an MS plate containing hygromycin, and meanwhile, spreading wild type col-0 seeds on an MS culture medium without antibiotics as a control;

and (3) putting the flat plate into an incubator for culturing, transplanting the seedlings into sterilized soil after the seedlings on the resistant MS culture medium take roots and the first pair of true leaves are unfolded (generally for 12-14 d), watering enough water to cover the preservative film until the preservative film survives, and removing the preservative film.

2) Taking 1-2 rosette leaves of transgenic positive seedlings of different strains to respectively extract RNA by using a TRIzol method, wherein the operation steps refer to an instruction book, and consumables such as centrifuge tubes, gun heads and the like are RNase-free.

Taking 1-2 leaves, grinding the sample with liquid nitrogen, adding 1mLTRIzol, blowing and uniformly mixing with a gun head, and standing for 5min at room temperature; adding 200mL of chloroform into 1mL of LTRIzol, shaking vigorously for 30s, and standing at room temperature for 3 min; centrifuging at 10000g and 4 deg.C for 15 min; after removal, the upper aqueous phase (about 600. mu.L supernatant per 1mL of LTRIzol) was transferred to a new centrifuge tube; adding 500 μ L isopropanol, reversing, mixing, and standing at room temperature for 10 min; centrifuging at 10000g and 4 ℃ for 10min to form colloidal precipitates at the bottom and the wall of the tube; removing supernatant, adding 1mL of 75% ethanol (prepared by DEPC water), and vigorously shaking by vortex; 7500g centrifuging at 4 deg.C for 5 min; removing supernatant, standing at room temperature for about 5min, and removing ethanol as much as possible; adding 55 mu L of RNA dissolving solution, and dissolving for 10min at the temperature of 55-60 ℃; 1.2% agarose gel electrophoresis checked the integrity of the extracted RNA.

The reverse transcription method was performed by first strand cDNA synthesis and reverse transcription PCR as described in example 1, and the cDNA was diluted 10-fold.

The qRT-PCR enzyme is ChamQ of Nanjing Novozam^TMUniversal SYBR in 20. mu.L: h₂O, 7.2 μ L; mix, 10 μ L; upstream/downstream primers: 0.4 mu L; cDNA template, 2. mu.L. Setting a program for pre-denaturation at 95 ℃ for 30 s; 10s at 95 ℃, 30s at 60 ℃ and 40 cycles; 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15 s. col-0 was used as control for the transgenic line and Actin as internal control.

The primer sequences used for qRT-PCR experimental identification are as follows:

AtMYB83-S：5′-CCAACAGCCCATGAGCAGG-3′

AtMYB83-A：5′-ACGAGGATCGGCACCACG-3′

AtActin-S：5′-CTCTCCGCTTTGAATTGTCTCGTTG-3′

AtActin-A：5′-GGTACCATTGTCACACACGATTGGT-3′

the expression levels of the BmMYB83 of the obtained transgenic Arabidopsis thaliana strains 1, 2, 4, 5, 6 and 8 are 107.63, 142.43, 107.86, 27.68, 10.95 and 12.11 respectively. The phenotype showed mild (Line2, 5, 6) or severe (Line1, 4, 8) leaf curling phenomenon (fig. 5); dwarfing of the plants and an increase in the number of flower stems (FIG. 6).

Sequence listing

<110> Nanjing university of forestry

<120> Bambusa multiplex transcription factor BmMYB83 gene and application thereof

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ccggaggagg acgagaagct ggtggcgtac atgctgcgga gcgggcaggg gtcgtggagc 180

gacgtggcgc gcaacgccgg gctgcaaagg tgcggcaaga gctgccgcct ccggtggatc 240

aactacctcc gccccgacct caagcgcggc gccttctcgc cgcaggagga ggagctcatc 300

gtcaacctcc gcgccatcct tggcaacagg tggtctcaga tcgctgcccg gctgccgggg 360

cgcaccgaca acgagatcaa gaacttctgg aactccacca tcaagaagcg cccgaagaac 420

tcggcggcgt cctctccggc gacgaccgac tgcgcgtcgc cgccggagcc caagctcccg 480

gtcgacggcg gcgccagctg cctcgacctc gccggcgtcg aggacggcgc ccaccatgca 540

atgaaaagca tgtgggtgga ctcgtcctcc tcatcatcct cgtcgctgca gagccggccg 600

tccgcgatgg cggcggcggc tgccaggagc tacggcggcc tcctccagct gccagaccaa 660

gtctgcggcg tggcggccta cacgctggcg ccgttcttcc acgaccatgc atcgttcaaa 720

tttgctgcat tgcatggtgg tggttactac ggaagcactc accaagggat ggcaatggaa 780

ggaggtggaa gcttcatagg aggaggtgaa agcagcgtgc tctatagtgt gccccctctg 840

ctagagccca tagcagtaga agaagaccag accataatgg catcaagtaa caacaccact 900

accaaccctg aaaacaacag caacaacact actactgaga ctaccacaca gagtagcaac 960

aatggcagca gcatcacaga caacaacagt agcaacaaca agaacatcaa cattagccta 1020

ctgagcaacg atgtggtcta ctgggaggca ggtcaccaac agcccatgag caggaatgtc 1080

atgggggagt gggacctgga gttgatgaaa gatgtgtcat ccttaccttt ccttgatttc 1140

caagttgaat ga 1152

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Lys Leu Arg Lys Gly Leu Trp Ser Pro Glu Glu Asp Glu Lys Leu Val

35 40 45

Ala Tyr Met Leu Arg Ser Gly Gln Gly Ser Trp Ser Asp Val Ala Arg

50 55 60

Asn Ala Gly Leu Gln Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile

65 70 75 80

Asn Tyr Leu Arg Pro Asp Leu Lys Arg Gly Ala Phe Ser Pro Gln Glu

85 90 95

Glu Glu Leu Ile Val Asn Leu Arg Ala Ile Leu Gly Asn Arg Trp Ser

100 105 110

Gln Ile Ala Ala Arg Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn

115 120 125

Phe Trp Asn Ser Thr Ile Lys Lys Arg Pro Lys Asn Ser Ala Ala Ser

130 135 140

Ser Pro Ala Thr Thr Asp Cys Ala Ser Pro Pro Glu Pro Lys Leu Pro

145 150 155 160

Val Asp Gly Gly Ala Ser Cys Leu Asp Leu Ala Gly Val Glu Asp Gly

165 170 175

Ala His His Ala Met Lys Ser Met Trp Val Asp Ser Ser Ser Ser Ser

180 185 190

Ser Ser Ser Leu Gln Ser Arg Pro Ser Ala Met Ala Ala Ala Ala Ala

195 200 205

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

210 215 220

Ala Ala Tyr Thr Leu Ala Pro Phe Phe His Asp His Ala Ser Phe Lys

225 230 235 240

Phe Ala Ala Leu His Gly Gly Gly Tyr Tyr Gly Ser Thr His Gln Gly

245 250 255

Met Ala Met Glu Gly Gly Gly Ser Phe Ile Gly Gly Gly Glu Ser Ser

260 265 270

Val Leu Tyr Ser Val Pro Pro Leu Leu Glu Pro Ile Ala Val Glu Glu

275 280 285

Asp Gln Thr Ile Met Ala Ser Ser Asn Asn Thr Thr Thr Asn Pro Glu

290 295 300

Asn Asn Ser Asn Asn Thr Thr Thr Glu Thr Thr Thr Gln Ser Ser Asn

305 310 315 320

Asn Gly Ser Ser Ile Thr Asp Asn Asn Ser Ser Asn Asn Lys Asn Ile

325 330 335

Asn Ile Ser Leu Leu Ser Asn Asp Val Val Tyr Trp Glu Ala Gly His

340 345 350

Gln Gln Pro Met Ser Arg Asn Val Met Gly Glu Trp Asp Leu Glu Leu

355 360 365

Met Lys Asp Val Ser Ser Leu Pro Phe Leu Asp Phe Gln Val Glu

370 375 380

<210>3

<211>19

<212>DNA

<213>MYB83-S(Artificial)

<400>3

agcccttttt ccatccttg 19

<210>4

<211>20

<212>DNA

<213>MYB83-A(Artificial)

<400>4

gtactccttg gcagcagcta 20

<210>5

<211>35

<212>DNA

<213>BmMYB83(+)(Artificial)

<400>5

cagtcgtctc acaacatgag gaagccggag ttccc 35

<210>6

<211>31

<212>DNA

<213>BmMYB83 (-)(Artificial)

<400>6

cgatcgtctc attcaacttg gaaatcaagg a 31

<210>7

<211>35

<212>DNA

<213> fusion GFP (+) (Artificial)

<400>7

cagtggtctc atccctgtat cgtgaagggc gagga 35

<210>8

<211>35

<212>DNA

<213> fusion GFP (-) (Artificial)

<400>8

cagtggtctc atacatcagt tgtagagctc gtcca 35

<210>9

<211>30

<212>DNA

<213>pubiseq+(Artificial)

<400>9

cctgccttca tacgctattt atttgcttgg 30

<210>10

<211>24

<212>DNA

<213>NOSseq-R(Artificial)

<400>10

caagaccggc aacaggattc aatc 24

<210>11

<211>24

<212>DNA

<213>Pbw2+(Artificial)

<400>11

gcaacgctct gtcatcgtta caat 24

<210>12

<211>25

<212>DNA

<213>Pbw2-(Artificial)

<400>12

gcgattaagt tgggtaacgc caggg 25

<210>13

<211>19

<212>DNA

<213>AtMYB83-S(Artificial)

<400>13

ccaacagccc atgagcagg 19

<210>14

<211>18

<212>DNA

<213>AtMYB83-A(Artificial)

<400>14

acgaggatcg gcaccacg 18

<210>15

<211>25

<212>DNA

<213>AtActin-S(Artificial)

<400>15

ctctccgctt tgaattgtct cgttg 25

<210>16

<211>25

<212>DNA

<213>AtActin-A(Artificial)

<400>16

ggtaccattg tcacacacga ttggt 25

Claims (6)

1. A transcription factor BmMYB83 gene of Bambusa multiplex has a nucleotide sequence shown in SEQ ID NO. 1.

2. The expressed protein of the Bambusa multiplex transcription factor BmMYB83 gene in claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.

3. A vector containing the transcription factor BmMYB83 gene of Bambusa multiplex as defined in claim 1.

4. The use of the Bambusa multiplex transcription factor BmMYB83 gene in claim 1 for dwarfing plant, curling leaves, increasing flower stem number or shortening root length.

5. Use according to claim 4, characterized in that it comprises the following steps:

1) constructing a vector of the transcription factor BmMYB83 gene of the Bambusa multiplex;

2) transforming the constructed vector of the transcription factor BmMYB83 gene into a plant or a plant cell;

3) and cultivating and screening to obtain the plants with dwarfing plants, curled leaves, increased flower stem number or shortened root length.

6. The use of claim 4 or 5, wherein the plant is Arabidopsis thaliana or rice.

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