CN113930433B

CN113930433B - Plant coding sequence, amplification primer and application of amplification primer in optimizing plant height

Info

Publication number: CN113930433B
Application number: CN202111357971.8A
Authority: CN
Inventors: 白戈; 谢贺; 杨大海; 费明亮; 袁诚; 童治军; 肖炳光; 李永平; 陈学军
Original assignee: Yunnan Academy of Tobacco Agricultural Sciences
Current assignee: Yunnan Academy of Tobacco Agricultural Sciences
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2023-04-11
Anticipated expiration: 2041-11-16
Also published as: CN113930433A

Abstract

The invention relates to a plant coding sequence, an amplification primer and application thereof in optimizing plant height, wherein the coding gene comprises the following components: the nucleotide sequence of the tobacco NtbHLH137 gene is shown as SEQ ID No:1 is shown in the specification; the amino acid sequence of the encoded protein is shown as SEQ ID No:2 is shown in the specification; the over-expression of the gene can increase the plant height of tobacco plants, and the NtbHLH137 has wide application prospect in the field of tobacco plant type breeding. The protein NtbHLH137 is a plant MYC transcription factor gene, the expression of the NtbHLH137 is regulated, the plant height of a tobacco plant can be regulated, and the over-expression of the gene in tobacco can obviously increase the plant height of the tobacco. Therefore, the gene NtbHLH137 for regulating the plant type has a wide application prospect in the field of plant type breeding, and has great economic benefit potential.

Description

Plant coding sequence, amplification primer and application of amplification primer in optimizing plant height

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a plant coding sequence, an amplification primer and application thereof in optimizing plant height.

Background

Plant types are generally classified into leaf types, stem types, ear types, root types, and the like. The plant height obviously affects the structural condition and microclimate of the population in the field, and the research on the plant height has important significance for utilizing and improving the microclimate in the field, and improving the light energy utilization rate and the crop yield.

At present, research on plant type breeding is mainly focused on corn and rice. In corn, plant types can be divided into two main types, namely flat type and compact type. The division is careful, and the corn plant type can be mainly divided into leaf type, stem type, root type, spike type and the like. Tobacco is currently less studied for plant height improvement as an important commercial crop. The plant height of tobacco has a correlative relation with the yield, the quality and the like, and is one of important agronomic traits. Importantly, the high yield of the tobacco needs to be achieved by taking reasonable close planting as a center to establish a reasonable group structure, wherein one important index is the plant height, the plant height can be increased to improve the closing in the field, enhance the ventilation and light transmission conditions, and improve the photosynthetic utilization efficiency of the lower leaves of the plants, so that the photosynthetic utilization rate of the whole plants is improved, and the effect of increasing the yield is achieved.

The tobacco leaves are large, so that the light transmittance of the lower leaves is not good, and meanwhile, the ventilation condition of the tobacco field is also influenced due to the fact that the tobacco leaves are large in sheet opening. However, currently, the research on breeding of tobacco plant types, particularly plant height traits, is relatively few, and how to improve the light transmittance and ventilation of tobacco by improving the plant height of tobacco and finally improve the photosynthetic utilization rate of tobacco and the total yield of tobacco is an important research direction of tobacco breeding at present.

Disclosure of Invention

The invention aims to solve the defects of the problems and provides a plant coding sequence, an amplification primer and application thereof in optimizing plant height.

The invention is realized by adopting the following technical scheme.

The plant coding sequence of the invention has an amino acid residue sequence shown in SEQ ID No:2, respectively.

The nucleotide sequence of the plant coding sequence is shown as SEQ ID No:1 is shown.

The present invention relates to an expression vector comprising the plant coding sequence of

claim

1 or 2.

The present invention relates to a cell line comprising a plant coding sequence according to

claim

1 or 2.

The present invention relates to host bacteria comprising the plant coding sequences of

claim

1 or 2.

The primer for amplifying the plant coding sequence is a forward primer:

5 'ATGGCTGCTTTTTCAGACCAATTAC-3'; reverse primer:

5’-TTAATGGAAAGAACAAAAGTTGTTG-3’。

the method for obtaining PCR amplification products by using the primers comprises the following steps:

the PCR reaction system is as follows: 50ul system: template DNA 1. Mu.l, primer-F (10 uM) 1. Mu.l, primer-R (10 uM) 1. Mu.l, 5 XBuffer 10. Mu.l, dNTP mix (10 mM) 1. Mu.l, phusionDNA Polymerase 0.5. Mu.l, ddH ₂ O is added to 50ul;

the PCR amplification conditions are as follows: 98 ℃ for 5min,35 PCR cycles (98 ℃ 30s, 58 ℃ 30s; extension at 72 ℃ for 5 minutes and storage at 4 ℃.

Recovery and purification of the PCR amplification product as described above: after gel electrophoresis, cutting off the gel with the target fragment by using a clean blade, and putting the gel into a centrifugal tube, wherein the gel is not required to be cut too large so as to avoid that a DNA fragment solution contains a large amount of impurities during recovery;

adding a QG solution (volume/colloid amount) with the volume of 3 times into the centrifuge tube, and then carrying out warm bath for 10min at the temperature of 50 ℃ until the colloid is completely melted; transferring the solution in the centrifugal tube to a 2ml adsorption column, centrifuging for 1min, and discarding the liquid phase; adding 0.5ml of QG solution into the adsorption column again, centrifuging for 1min, and discarding the liquid phase; adding 0.75ml of PE solution into the adsorption column, centrifuging for 1min, and discarding the liquid phase; and after centrifuging for 1min again, placing the adsorption column on a new centrifuge tube, adding 50 mu l of dissolving solution, standing for 1min, and finally centrifuging for 1min to obtain a liquid phase, namely the recovered DNA solution.

The plant coding sequence is applied to optimizing the height of the tobacco plant.

The application method of the invention is to excessively express the coding sequence of the plant to increase the plant height of the tobacco.

The method for over-expressing the plant type related protein coding gene in the plant comprises the methods such as a plant virus vector mediated gene over-expression method, agrobacterium-mediated transformation over-expression vector, optimization and modification of a gene coding frame, optimization of a gene promoter to achieve an over-expression effect and the like.

The invention has the beneficial effects that the plant height related protein and the coding gene thereof provide gene and technical support for crop, especially tobacco plant type breeding. The protein NtbHLH137 is a plant transcription factor. The over-expression of the gene in tobacco can increase the plant height of the tobacco and improve the plant type of the tobacco plant, so that the plant type regulatory gene NtbHLH137 has wide application prospect in the field of plant type breeding and has huge economic benefit potential.

The invention is further explained below with reference to the drawings and the detailed description.

Drawings

FIG. 1 electrophoretogram of PCR product of NtbHLH137;

FIG. 2 is a pdonr-zeo vector map;

FIG. 3 is a diagram of a plant expression vector PB2GW7 of the NtbHLH137 gene;

FIG. 4 is a histogram showing the expression level of the NtbHLH137 gene in a tobacco strain transformed with the NtbHLH137 gene;

FIG. 5 is a picture of the phenotype of increased plant height of tobacco plants with over-expression of the NtbHLH137 gene.

Detailed Description

The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.

The first purpose of the invention is to provide a gene NtbHLH137 for improving the plant type of tobacco by improving the plant height of the tobacco; the second aim is to provide a cloning method of the tobacco improving plant type gene NtbHLH137, and the third aim is to provide application of the tobacco improving plant type gene NtbHLH 137.

The first purpose of the invention is realized by that the nucleotide sequence of the gene NtbHLH137 for increasing the plant height of tobacco is shown as SEQ ID No:1 is shown.

The second object of the present invention is achieved by comprising the steps of:

A. tobacco leaf cDNA Synthesis: extracting total RNA of tobacco leaves, and performing reverse transcription to obtain first-strand cDNA;

B. PCR amplification of the NtbHLH137 gene: and (3) designing a primer according to the sequence of the NtbHLH137 gene by taking the tobacco leaf cDNA as a template, carrying out PCR amplification, recovering and purifying a PCR amplification product, and sequencing.

The third purpose of the invention is realized by the application of the gene NtbHLH137 for increasing the plant height of tobacco in optimizing the plant type of the tobacco.

The present invention provides a novel protein determining plant strain height and a coding gene thereof.

The plant height related protein provided by the invention is named as NtbHLH137, is derived from Yunyan 87 cultivated tobacco, and is a protein with one of the following amino acid residue sequences:

1) SEQ ID No:2;

2) And (3) mixing the sequence table SEQ ID No:2 through substitution and/or deletion and/or addition of one or more amino acid residues and is related to the plant spacing length.

The sequence 2 in the sequence table consists of 366 amino acids.

The substitution and/or deletion and/or addition of one or more amino acid residues refers to the substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The gene encoding the NtbHLH137 (NtbHLH 137) also falls within the scope of the present invention.

The NtbHLH137 coding gene can have one of the following nucleotide sequences: 1) SEQ ID No: 1; 2) Encoding SEQ ID No:2 a polynucleotide of protein sequence; 3) Can be combined with the sequence shown in SEQ ID No:1 to a DNA sequence defined in the specification; 4) And SEQ ID No:1, and the DNA sequence has over 70 percent of homology and codes the same functional protein.

Expression vectors, cell lines and host bacteria comprising the NtbHLH137 of the invention are within the scope of the invention. Primer pairs for amplifying any one of the NtbHLH137 fragments also belong to the protection scope of the invention.

The invention also provides a method for improving and increasing the plant height of tobacco by using the gene, which is to over-express the plant-related protein coding gene in plants.

The overexpression of the plant growth height-increasing related protein coding gene NtbHLH137 can be realized by various methods, such as a method for plant virus vector mediated gene overexpression, an agrobacterium-mediated transformation overexpression vector, optimization modification of a gene coding frame, optimization of a gene promoter to achieve an overexpression effect and the like. The method of overexpressing a gene in the present invention is not limited to the above-described methods as long as the NtbHLH137 can be overexpressed.

The NtbHLH137 enables the plant to show an increase in plant height by using any gene overexpression or gene modification method; any vector capable of guiding the expression of the exogenous gene in the plant is utilized, the NtbHLH137 provided by the invention is transferred into the plant, and the plant shows that the plant height is high.

When the NtbHLH137 gene is constructed into a plant expression vector, any one of an enhanced promoter and an inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, vectors to be used may be processed, for example, by adding a plant selectable marker (GUS gene, luciferase gene, etc.) or an antibiotic marker having resistance (gentamicin, kanamycin, etc.). The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: tobacco, rice, wheat, corn, cucumber, tomato, poplar, turf grass or alfalfa and the like. From the safety of transgenic plants, the transformed plants can be directly screened according to the plant height and length without adding any selective marker gene. The expression vector carrying the NtbHLH137 gene of the invention can be used for transforming plant cells or tissues by using Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and other conventional biological methods, and culturing the transformed plants into plants through tissues.

Specifically, the nucleotide sequence of the tobacco plant type related gene NtbHLH137 is shown as SEQ ID No:1 is shown.

The amino acid sequence coded by the tobacco plant height regulating gene NtbHLH137 is shown as SEQ ID No:2, respectively.

The cloning method of the tobacco plant height related gene NtbHLH137 comprises the following steps:

The primer in the step B is as follows:

a forward primer: 5' ATGGCTGCTTTTTCAGACCAATTAC-3

Reverse primer: 5' TTAATGGAAAGAACAAAGTTGTTG-3

The PCR reaction system and the amplification conditions in the step B are as follows:

TABLE 1 PCR reaction System and conditions

The specific operation of sequencing in step B is to deliver invitrogen for sequencing.

The specific operations for recovering and purifying PCR amplification products described in step B are as follows:

after gel electrophoresis, the gel with the target fragment is cut off by a clean blade and placed in a centrifuge tube, and the gel does not need to be cut too large so as to avoid that a DNA fragment solution contains a large amount of impurities during recovery.

The invention relates to an application of a tobacco plant height regulating gene NtbHLH137, and an application of the tobacco plant height related gene NtbHLH137 in optimizing the plant type of tobacco.

The method for obtaining the tobacco plant with the optimized plant type comprises the following steps:

A. constructing a vector:

designing a primer according to a screened NtbHLH137 gene sequence in a tobacco genome, adding a sequence of 5 'GGGGACAAGTTTGTTACAAAAAGCAGGCTGC3' before a forward primer and adding a sequence of 5 'GGGGACCATTTGTTACAAAGCTGGGTC3' before a reverse primer according to the BP reaction requirement in a Gateway system. PCR cloning is carried out by Phusion high-fidelity polymerase in all PCR reactions. These fragments were cloned into pDONR-Zeocin vectors by BP reaction, and then cloned into respective target vectors by LR reaction.

By using

The vector is constructed by the technology, and the principle can be briefly described as follows:

BP reaction

(1) Prepare 8. Mu.l of the reaction in a 200. Mu.l centrifuge tube, comprising: 1-7 mul attB-PCR product (about 15-150 ng, concentration is more than or equal to 10 ng/mul), 1 mul pDONR carrier (150 ng/mul) and a proper amount of TE buffer solution (pH8.0), and evenly mixing at room temperature;

(2) BP close ^TM II standing the enzyme mixture on ice for 2min to melt and lightlyLightly shaking for 2 times, and mixing uniformly for later use;

(3) To the sample prepared in (1), 2. Mu.l of BP clone was added ^TM II, mixing the enzyme mixture gently;

(4) BP close ^TM II, putting the enzyme mixture back to-20 ℃ or-80 ℃ for storage;

(5) The reaction system is put into a warm bath at 25 ℃ for 1h;

(6) Adding 1 mu l of proteinase K solution into the reaction system, shaking gently, and then placing the sample in a warm bath at 37 ℃ for 10min so as to terminate the BP reaction;

(7) And (3) after the mixed solution is transformed into escherichia coli, coating the transformed bacterium solution on an LB (lysogeny broth) plate containing Zeacin resistance, picking a bacterial colony to a culture medium solution containing corresponding antibiotics for shake culture, and extracting positive cloned plasmids for later use after confirmation.

LR reaction

(1) Prepare 8 μ l of the reaction in a 200 μ l centrifuge tube, including: 1-7 mul of pDONR-Zeocin plasmid (50-150 ng) obtained by 2.2.10.1, 1 mul of target vector (150 ng/mul) and a proper amount of TE buffer solution (pH8.0) are mixed evenly at room temperature;

(2) LRClonase is added ^TM II, standing the enzyme mixture on ice for 2min to melt, and lightly shaking for 2 times to mix uniformly;

(3) Add 2. Mu.l of LRClonase ^TM II, mixing the enzyme mixture by gentle shaking;

(4) LRClonase ^TM II, storing the enzyme mixture in a refrigerator at-20 ℃ or-80 ℃;

(5) Placing the reaction system at 25 ℃ for warm bath reaction for 1h;

(6) Adding 1 mul of proteinase K solution into the reaction system to stop the LR reaction, after gently shaking, placing the sample at 37 ℃ and standing for 10min;

(7) And transforming the LR reaction product into escherichia coli, coating a plate, screening positive clones, extracting plasmids, and performing experiments such as yeast double-hybrid and agrobacterium transformation.

B. And (3) agrobacterium transformation:

adding 1 mu g (200 ng/mu l) of target plasmid into 100 mu l of competent agrobacterium, uniformly mixing, standing on ice for 5min, freezing in liquid nitrogen for 5min, taking out from the liquid nitrogen, putting into a water bath kettle at 37 ℃ for water bath for 5min, standing on ice for 5min, adding 500 mu l of LB solution, recovering and culturing for 4h under the condition of 28 ℃ and full shaking, finally uniformly coating the bacterial solution on a selective plate culture medium, and culturing for 48h at 28 ℃.

C. Culturing a transgenic plant:

(1) Under the aseptic condition, putting the tobacco seeds into an EP tube, and washing the tobacco seeds for 2 to 3 times by using sterile water;

(2) Soaking in 75% ethanol for 30-60sec;

(3) Treating with 0.1% mercuric chloride for 5min, and washing with sterile water for 5 times;

(4) Sowing on MS culture medium, culturing in tissue culture room of Yunnan tobacco agricultural science research institute, and dark culturing for 4 days. Culturing at 25 deg.C under illumination for 20-30 days.

(5) When the tobacco seedling grows to 3-5cm (20-30 days), taking the terminal bud, placing on MS + BA0.2mg/L (strong bud for rapid growth) culture medium, and subculturing.

(6) After 14 days of subculture (only small leaves are needed), the leaves are taken, the size is 1cmX1cm, petioles are cut off, the surfaces and the edges of the leaves are scratched, the leaves are placed on a preculture medium with MS and BA1.0mg/L and pH of 6.0-6.5, the front side of the preculture medium is downward and is tightly attached to the culture medium, and the preculture is carried out for 2-3 days under dark conditions.

(7) Then taking out the pre-cultured leaf or stem section, and putting the leaf or stem section into the invasion dye solution for infection. And (5) shaking the agrobacterium tumefaciens bottle 2 at night one day before infection. 2ml centrifuge tube full of bacteria liquid, 4000rpm centrifugation for 5min, using suspended bacteria liquid to wash twice. Placing the suspension liquid according to the proportion of 1;

(8) Putting the leaves and the stem segments back on a pre-culture medium, and culturing for 2-3 days at 28 ℃ in the dark until the periphery of the cut of the leaves is provided with the bacterial plaque;

(9) Washing bacteria, taking out co-cultured tobacco leaves and stem segments, washing with sterile water added with 500mg/LCef for 5 times, shaking in a shaking table for 30min for the first time, and then 5min for each time to wash away agrobacterium on the surface of the explant;

(10) Taking out, sucking dry with filter paper, transferring to tobacco bud inducing culture medium (MS + BA1.0mg/L + Bar25mg/L + Cef500mg/L, pH5.8); after 2 weeks, if no growth was observed, the Cef concentration was reduced. If the bacteria grow, the concentration of Cef is continuously maintained.

(11) The medium was changed every two weeks until adventitious shoots were established (typically 2 weeks). Cutting off regenerated plantlet (about 1 cm), transferring into subculture medium MS + BA0.2-0.1mg/L + Bar25mg/L + Cef500mg/L, and pH5.8;

(12) When the plantlet grows to 2cm long (only a small bud is available), the plantlet is transferred to a rooting culture medium MS and NAA for 0.2-0.1mg/L, and the plantlet is cultured at 24 days and 1 ℃, 12h of illumination and 1500lx for about three weeks to grow a thick and strong root system.

(13) Carrying out PCR preliminary detection on the rooted plants, and transplanting the plants with positive results to peat after seedling hardening: vermiculite =7, 1, placed in a climatic chamber for culturing, and the growth of the medium is observed and recorded.

Preparation of agrobacterium infection liquid

(1) Taking agrobacterium which is preserved in a refrigerator at the temperature of minus 80 ℃ and contains an expression vector, performing plate-cutting culture, and adding 50mg/LKan and 50mg/LRif into an LB solid plate;

(2) Picking single bacterial plaque to 5mLLB liquid culture medium containing 50mg/LKAn and 50mg/LRif, putting into a shaking table, culturing overnight (12 h-16 h) at 28 ℃ and 200 rpm;

(3) Storing the strain, adding 750ul of the strain liquid into 250ul of sterilized glycerol, and storing in a refrigerator at-80 ℃ for later use.

(4) Shaking, adding 10ul Kan (required concentration 50 mg/L) and 10ul Rif (required concentration 50 mg/L) into 10ml LB liquid culture medium, and culturing at 10ul bacteria liquid at 28 deg.C and 200rpm overnight (12 h-16 h).

(5) When the concentration of the bacterial liquid reaches about OD600=1.5, adding 2mL of bacterial liquid into a centrifugal tube, and centrifuging at 4000rpm for 5min;

(6) The supernatant was decanted, 1mL of fresh MS liquid medium was aspirated, the Agrobacterium was resuspended, and centrifuged at 4000rpm for 5min.

(7) Repeating the step (6) once;

(8) After the suspension of the bacteria in 1mL of MS liquid culture medium, the suspension was added to 40mL of MS liquid culture medium (containing 40ul25mg/L of As), thus obtaining the infection solution. Standing for more than 2h, and infecting again.

200ml of suspension liquid

20X Large volume 10ml

200 Xorganic 1ml

200 Xiron salt 1ml

200 XTrace 1ml

Sucrose 5.6g

The invention is further illustrated by the following specific examples:

example 1

1. Tobacco leaf cDNA Synthesis

The total RNA of tobacco leaves was extracted with TRIZOL (Invitrogen, USA) reagent, and 1. Mu.g of the total RNA was quantified in a 1.5ml centrifuge tube and reverse-transcribed according to the instructions of First Strand cDNA Synthesis Kit of Invitrogen to obtain tobacco leaf cDNA.

PCR amplification of NtbHLH137 Gene

And (3) designing a primer according to tobacco genomic database information by taking the tobacco leaf cDNA as a template, and carrying out PCR amplification on the NtbHLH137 gene to obtain a PCR amplification product.

The primers are as follows:

a forward primer: 5' ATGGCTGCTTTTTCAGACCAATTAC-3

Reverse primer: 5' TTAATGGAAAGAACAAAGTTGTTG-3

The PCR product obtained by amplification was electrophoresed on 0.8% agarose gel, and as a result of gel electrophoresis (as shown in FIG. 1), a fragment of 1104bp in size was obtained.

After electrophoresis is finished, a PCR product purification kit of Qiagen company is adopted, the PCR product is recovered and purified according to the product instruction, invitrogen is sent for sequencing, and the sequence result is verified, so that the sequence is completely the same as the data in the genome database.

Example 2

1. Construction of plant expression vectors

Using the full-length NtbHLH137 fragment of example 2 as a template, PCR amplification was performed using a primer containing a gateway linker sequence, and the amplification product was purified by PCR and inserted into pdonr-zeo vector of invitrogen through BP reaction. The constructed BP reaction vector is used for replacing the NtbHLH137 segment into a PB2GW7 vector through an LR reaction.

The gateway reaction primer sequences are as follows:

NtbHLH137_F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGCTGCTTTTTCAGACCAATTAC-3’

NtbHLH137_R：

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATGGAAAGAACAAAAGTTGTTG-3’。

2. agrobacterium-mediated tobacco transformation and identification of transgenic plants

The agrobacterium LBA4404 is transformed by the recombinant plasmid which is identified to be correct through PCR reaction and sequencing through a freeze-thaw method, a positive agrobacterium strain is determined through colony PCR, and a tobacco variety Yunyan 87 is transformed by an agrobacterium-mediated leaf disc method.

The specific method comprises the following steps:

(1) Under the aseptic condition, putting the tobacco seeds into an EP tube, and washing the tobacco seeds with sterile water for 2-3 times;

(2) Soaking in 75% ethanol for 30-60sec;

(7) Then taking out the pre-cultured leaf or stem section, and putting the leaf or stem section into the invasion dye solution for infection. Shake the agrobacterium 2 bottles the night before infection. 2ml centrifuge tube full of bacteria liquid, 4000rpm centrifugation for 5min, using suspended bacteria liquid to wash twice. Placing the suspension liquid according to the proportion of 1;

(8) Putting the leaves and the stem sections back on a pre-culture medium, and co-culturing for 2-3 days at 28 ℃ under a dark condition until the periphery of the cut of the leaves is provided with the bacterial plaque;

(10) Taking out, sucking dry with filter paper, transferring to tobacco bud inducing culture medium (MS + BA1.0mg/L + Bar25mg/L + Cef500mg/L, pH5.8); after 2 weeks, if no growth was observed, the Cef concentration was reduced. If the strain grows, the Cef concentration is continuously maintained.

(11) The medium was changed every two weeks until adventitious buds grew (typically 2 weeks). Cutting off regenerated plantlet (about 1 cm), transferring into subculture medium MS + BA0.2-0.1mg/L + Bar25mg/L + Cef500mg/L, and pH5.8;

(13) And (5) growing the roots to 2-3cm. When the height of the seedling is about 7-10cm, moving out the triangular flask to wash away the root culture medium, transplanting the seedling into a flowerpot, and culturing in a greenhouse.

Extracting genome DNA of the transgenic tobacco seedlings by adopting a DNA extraction kit of Qiagen company, designing a Basta resistance gene primer for PCR amplification, screening positive plants, and detecting 25 positive plants.

Total RNA of wild-type plants and 10 plants transformed with the T0 generation of the NtbHLH137 gene were extracted as described in example 1, and Real time-PCR analysis was performed, with the reference gene being 26s, to analyze the expression of different lines. Two plants with the highest expression level are selected for photographing.

NtbHLH137qRT primer

NtbHLH137_qRT_F：5’-AAGGCCCTCATGTTGGATGA-3’

NtbHLH137_qRT_R：5’-AGTAGGGCTAGTTTGCTGCA-3’

26s reference gene primer

26s_F：5’-GAAGAAGGTCCCAAGGGTTC-3’

26s_R：5’-TCTCCCTTTAACACCAACGG-3’

Example 3

Uniformly sowing the wild type Yunyan 87 plants and 2 transgenic lines (OE-1 and OE _ 2) of transgenic tobacco seeds into small pots containing nutrient soil, culturing in a light culture chamber, and observing the plant height phenotype. The heights of the transgenic OE-1 and OE-2 tobacco plants of the NtbHLH137 are obviously superior to those of wild tobacco plants (figure 5), and statistical data of the heights of other transgenic plant materials show that the heights of different transgenic plants are also higher than those of non-transgenic plants. The transgenic tobacco over-expressed by OE-1 and OE-2 is shown to have important significance for the breeding of the tobacco plant type in the future.

SEQ ID No：1:

ATGGCTGCTTTTTCAGACCAATTACAGCACACAAACCCTTTCCTTCTTGACTCAGTTTTTTTGCCAAGTTCTCCTATTAAGATGTCTGGTTTTTTAGAGGAACAAAACAATTCTATAGTGCAGAATTGTTTTACTCAATTTTACCAACCAGAATCTTTTCAGCAGCTCCCAACTGCCAATGTGATTGTTCATGAAAGTAGCTATTGCCTTGACCAAAGTACAAATGTTACACTTAGCCAAAATGAGCTTAATTCTATGACCAACAACAGTAGCAGCAGTGTTAGCTTGGATATGGATTCTTCCTCTGTTACTGATAAAATAGAAAGTGGGAATAAGCCTAATTTTATTCCTATGGACAAGAAAAGAAAATCCAGAGAAGGGTCTTCCTCAATGAGTTCTGCTCATTCTAAGAATGTAAAACAGGTTGATAATGGGAAAAAGAAGAAAAGCAATAGCCAATCAGTAGGCAAAGATGAGAAAAAGGGAAAAGATGACAACAAAAAAGAGGAAAAGAAAGCTAATGAAGAGGCTCCAACAGGCTACATTCATGTTAGAGCAAGAAGGGGTCAAGCAACAGACAGCCATAGTCTTGCTGAAAGGGTGAGGAGAGAGAAAATAAGTGAAAGGATGAAGATACTGCAATCTCTTGTTCCTGGTTGTGACAAGGTGAATAACAAAGTAACTGGGAAGGCCCTCATGTTGGATGAGATAATCAATTATGTCCAATCTTTGCAAAACCAAGTTGAGTTTCTCTCCATGAAACTTGCTTCTTCGAATCCAATGTACTATGACTTTGGCATGGACTTAGATGCACTCATGGTCAGACCTGACCAGAGTTTGAGTGGATTGGGAACACCACTGCCAAACATGCAGCAAACTAGCCCTACTAACATTACATCACAGGCAGCTGAAGTTATTCCTAACATTAATAATAGTGGCTATCCTTTCTTGGATAATTCAGCTTCACTCATGTTTCAACAAGTCCATTTTCCTAATTCCATTTCTCAGGGTAATGGACAGCTCTTATGGGGTGCAGATGACCAAAGACAAAAATTAATTAATCAGTCAGGACTCAGCAACAACTTTTGTTCTTTCCATTAA

SEQ ID No：2:

MAAFSDQLQHTNPFLLDSVFLPSSPIKMSGFLEEQNNSIVQNCFTQFYQPESFQQLPTANVIVHESSYCLDQSTNVTLSQNELNSMTNNSSSSVSLDMDSSSVTDKIESGNKPNFIPMDKKRKSREGSSSMSSAHSKNVKQVDNGKKKKSNSQSVGKDEKKGKDDNKKEEKKANEEAPTGYIHVRARRGQATDSHSLAERVRREKISERMKILQSLVPGCDKVNNKVTGKALMLDEIINYVQSLQNQVEFLSMKLASSNPMYYDFGMDLDALMVRPDQSLSGLGTPLPNMQQTSPTNITSQAAEVIPNINNSGYPFLDNSASLMFQQVHFPNSISQGNGQLLWGADDQRQKLINQSGLSNNFCSFH

The foregoing is only a part of the specific embodiments of the present invention and specific details or common general knowledge in the schemes have not been described herein in more detail. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and it is obvious for those skilled in the art that all the technical solutions obtained by using the equivalent substitution or the equivalent change fall within the protection scope of the present invention. The scope of the claims of the present application shall be defined by the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

<110> research institute of tobacco agricultural science in Yunnan province

<120> plant coding sequence, amplification primer and application thereof in optimizing plant height

<160>12

<210>1

<211>1101

<212>DNA

<213> Artificial sequence

<400>1

ATGGCTGCTTTTTCAGACCAATTACAGCACACAAACCCTTTCCTTCTTGACTCAGTTTTTTTGCCAAGTTCTCCTATTAAGATGTCTGGTTTTTTAGAGGAACAAAACAATTCTATAGTGCAGAATTGTTTTACTCAATTTTACCAACCAGAATCTTTTCAGCAGCTCCCAACTGCCAATGTGATTGTTCATGAAAGTAGCTATTGCCTTGACCAAAGTACAAATGTTACACTTAGCCAA

AATGAGCTTAATTCTATGACCAACAACAGTAGCAGCAGTGTTAGCTTGGATATGGATTCTTCCTCTGTTACTGATAAAATAGAAAGTGGGAATAAGCCTAATTTTATTCCTATGGACAAGAAAAGAAAATCCAGAGAAGGGTCTTCCTCAATGAGTTCTGCTCATTCTAAGAATGTAAAACAGGTTGATAATGGGAAAAAGAAGAAAAGCAATAGCCAATCAGTAGGCAAAGATGAGAAA

AAGGGAAAAGATGACAACAAAAAAGAGGAAAAGAAAGCTAATGAAGAGGCTCCAACAGGCTACATTCATGTTAGAGCAAGAAGGGGTCAAGCAACAGACAGCCATAGTCTTGCTGAAAGGGTGAGGAGAGAGAAAATAAGTGAAAGGATGAAGATACTGCAATCTCTTGTTCCTGGTTGTGACAAGGTGAATAACAAAGTAACTGGGAAGGCCCTCATGTTGGATGAGATAATCAATTAT

GTCCAATCTTTGCAAAACCAAGTTGAGTTTCTCTCCATGAAACTTGCTTCTTCGAATCCAATGTACTATGACTTTGGCATGGACTTAGATGCACTCATGGTCAGACCTGACCAGAGTTTGAGTGGATTGGGAACACCACTGCCAAACATGCAGCAAACTAGCCCTACTAACATTACATCACAGGCAGCTGAAGTTATTCCTAACATTAATAATAGTGGCTATCCTTTCTTGGATAATTCA

GCTTCACTCATGTTTCAACAAGTCCATTTTCCTAATTCCATTTCTCAGGGTAATGGACAGCTCTTATGGGGTGCAGATGACCAAAGACAAAAATTAATTAATCAGTCAGGACTCAGCAACAACTTTTGTTCTTTCCATTAA

<210>2

<211>366

<212>PRT

<213> Artificial sequence

<400>2

MAAFSDQLQHTNPFLLDSVFLPSSPIKMSGFLEEQNNSIVQNCFTQFYQPESFQQLPTANVIVHESSYCLDQSTNVTLSQNELNSMTNNSSSSVSLDMDSSSVTDKIESGNKPNFIPMDKKRKSREGSSSMSSAHSKNVKQVDNGKKKKSNSQSVGKDEKKGKDDNKKEEKKANEEAPTGYIHVRARRGQATDSHSLAERVRREKISERMKILQSLVPGCDKVNNKVTGKALMLDEIINY

VQSLQNQVEFLSMKLASSNPMYYDFGMDLDALMVRPDQSLSGLGTPLPNMQQTSPTNITSQAAEVIPNINNSGYPFLDNSASLMFQQVHFPNSISQGNGQLLWGADDQRQKLINQSGLSNNFCSFH

<210>3

<211>25

<212>DNA

<213> Artificial sequence

<400>3

ATGGCTGCTTTTTCAGACCAATTAC

<210>4

<211>25

<212>DNA

<213> Artificial sequence

<400>4

TTAATGGAAAGAACAAAAGTTGTTG

<210>5

<211>31

<212>DNA

<213> Artificial sequence

<400>5

GGGGACAAGTTTGTACAAAAAAGCAGGCTGC

<210>6

<211>30

<212>DNA

<213> Artificial sequence

<400>6

GGGGACCACTTTGTACAAGAAAGCTGGGTC

<210>7

<211>56

<212>DNA

<213> Artificial sequence

<400>7

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGCTGCTTTTTCAGACCAATTAC

<210>8

<211>55

<212>DNA

<213> Artificial sequence

<400>8

GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATGGAAAGAACAAAAGTTGTTG

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

AAGGCCCTCATGTTGGATGA

<210>10

<211>20

<212>DNA

<213> Artificial sequence

<400>10

AGTAGGGCTAGTTTGCTGCA

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<400>11

GAAGAAGGTCCCAAGGGTTC

<210>12

<211>20

<212>DNA

<213> Artificial sequence

<400>12

TCTCCCTTTAACACCAACGG

Claims

1. The application of the gene in optimizing the height of the tobacco plant is characterized in that the amino acid sequence of the protein coded by the gene is shown as SEQ ID No:2, respectively.

2. The use of claim 1, wherein the nucleotide sequence of said gene is as set forth in SEQ ID No:1 is shown.

3. The use according to claim 1, wherein overexpression of the gene increases the plant height of tobacco.