CN111172172B - Regulatory gene PdeMIXTA02 for initial development of populus deltoides and application thereof - Google Patents

Abstract

The invention discloses a regulatory gene PdeMIXTA02 for the initial development of populus deltoids and application thereof, belonging to the technical field of plant genetic engineering. The invention discloses a gene PdeMIXTA02 for regulating and controlling poplar initial development for the first time by gene sequence evolution analysis, gene expression analysis of different time points before and after poplar initial development and genetic transformation functional verification of arabidopsis thaliana. The gene belongs to a MIXTA transcription factor family, sequence evolution analysis shows that key genes GhMML3 and GhMML4 which are used for regulating and controlling the initial development of cotton wool are the same MIXTA subclass branch, and are only specifically expressed within 5 days after pollination of female cotton wool of poplar, and the overexpression in arabidopsis thaliana can greatly increase the number of epidermal hairs and promote the epidermal cells of arabidopsis thaliana to be convexly differentiated into the epidermal hairs. The result proves that the PdEMIXTA02 gene regulates the initial development of poplar catkins and can be used for cultivating new varieties of non-flying poplar.

Description

Regulatory gene PdeMIXTA02 for initial development of populus deltoides and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a regulatory gene PdeMIXTA02 for the initial development of Populus deltoids (Poplus delides) poplar catkins and application thereof.

Background

The poplar (Populusspp.) is the most widely distributed and most adaptable tree species in the world. The cultivation of poplar in China is mainly concentrated in the areas of North China, northeast China, northwest China and the like. The poplar is not only an important fast-growing high-yield timber forest tree species, but also an important tree species for environmental greening; it is also a suitable feedstock for second generation biofuel production due to its extremely fast growth rate and good cell wall chemistry; it has strong habitat adaptability, and can be widely used in ecological protection forest, three-north protection forest and agriculture and forestry protection forest. The economic value, the ecological value and the social value of the tree self reflect the superiority, and other tree species cannot possess and replace the superiority.

The problem of seasonal environmental pollution caused by poplar catkins becomes an important factor restricting the development of poplar industry at present. The poplar is a male and female heterostrain in reproductive characteristics, the female strain of the poplar has good quality and the growth performance of the female strain is generally superior to that of the male strain, so the excellent clone of the poplar popularized by breeding is mainly the female strain, however, after the capsule of the female strain of the poplar grows to be mature, the fruit cracks to generate a plurality of poplar catkins which fly around, and the duration of the catkins is long, and usually can reach half a month. The spread of poplar catkins can not only cause environmental pollution, but also cause unsmooth respiratory tract of people, and is easy to cause fire because of flammability. Therefore, the shade tree is forbidden to plant female poplar, male poplar is selected to replace female poplar, but the male poplar is generally weaker than female poplar in growth character, a great amount of pollen is generated after the male poplar is mature, and serious environmental pollution is also brought. Therefore, how to exert the strong ecological benefit and the higher economic benefit of the poplar and control the seasonal floatage pollution of the poplar is a problem to be solved urgently in the current society.

In recent years, the research on the shape of poplar seed flocks has been advanced, and the poplar seed flocks are flocculent fibers attached to the end parts of seeds and play a role in assisting the diffusion of the seeds, and the fiber length is about 9.5-15.9 mm, and the diameter is about 8-11 μm and exceeds the length of the seeds. Paraffin section found the placenta at the base of the ovary as the origin of poplar fiber generation, and observed the enlargement of nucleus when the fiber cell bulges, and the replication of nucleus occurs, but the cell does not divide, which indicates that the replication cycle bypasses normal mitosis and is a potential mechanism for maintaining the single cell structure of poplar fiber. Genes associated with cellulose synthesis and cell wall biosynthesis were found to be abundant in this bioprocess in differential expression analysis, suggesting that this component of poplar fiber structure is consistent with that found in cotton fibers from epidermal cells of the seed coat. The observation of a scanning electron microscope shows that the poplar seed is formed by the bulge development of the placenta epidermal cells 1 day after pollination, and the process is similar to the process of the cotton seed which is formed by the bulge development of ovule epidermal cells 1 day after pollination. Therefore, the research on the initial development regulation of the poplar catkins can use the initial development regulation mechanism of the cotton catkins as reference. At present, researches find that after GhMML3 gene of cotton is silenced, ovule epidermal cells do not bulge and develop any more, and then a non-flocculent phenotype appears; after GhMML4 silencing of cotton, the number of protrusions of ovule epidermal cells is reduced remarkably, and a little flocculent phenotype appears, and both belong to the members of the MIXTA gene family. In addition, in the cotton boll-free mutant, the expression level of 10 MIXTA genes of cotton is greatly reduced by 6 gene family members, including GhMML3, GhMML4, GhMML7, GhMML8, GhMML9 and GhMML 10. Therefore, according to the regulation effect of the MIXTA gene on the initial development of cotton wool, it is speculated that the MIXTA gene in the poplar also has the regulation effect on the initial development of the cotton wool.

The MIXTA gene belongs to the ninth subclass of the R2R3-MYB gene family, and was first found in a snapdragon (Antirrhinum maju) mutant to regulate the development of cone cell shapes in the petal epidermis. In Arabidopsis, members of this gene family include the three genes AtMYB16, AtMYB17, and AtMYB 106. The MIXTA gene comprises, in addition to the R2R3-MYB domain, a motif which is AQWESAR AE RL RES. In terms of biological functions, the MIXTA firstly regulates the differentiation of cone cells of petals and the formation of trichomes, such as AmMIXTA, AmMYBML2, AmMYBML3 of snapdragon (antirhinum maju) and GhMYBML10 of upland cotton (Gossypium hirsuta) and the like; and secondly, to the density of the epidermal hairs of the leaves, such as AtMYB16 of Arabidopsis thaliana (Arabidopsis thaliana), MgMYBML8 of Scrophulariaceae Hericium erinaceus (Mimulus guttatus), and DcMYBML1 of Dendrobium pigeon (Dendrobium crutum); finally, it is also possible to regulate the differentiation of ovule epidermal cells, such as GhMYB25, GhMML3 and GhMML4 of cotton (Gossypium hirsuta). The relevant function reports of the MIXTA gene in different plants are all related to the regulation and control of the differentiation of epidermal hair of different parts of the plants, and are mainly concentrated on ovules, petals and leaves, so that the function of the MIXTA gene in different plants is conserved. Populus catkin is formed by differentiation of epidermal cells of placenta and has biological characteristics similar to those of fibers formed by differentiation of ovule epidermis of cotton. Therefore, it was concluded that the MIXTA gene plays a key regulatory role in the initial development of poplar catkin.

With the large-area planting of poplar, the pollution of poplar catkins to the environment and the harm to the society are increasingly serious. But the molecular mechanism of poplar catkin generation is not well understood at present. Therefore, by researching the initial development regulation mechanism of the poplar catkin, the key gene for regulating the initial development of the poplar catkin is discovered, and the method has important significance for obtaining new varieties of poplar trees without floating catkins.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a regulatory gene PdeMIXTA02 for the initial development of populus deltoides. The invention aims to solve another technical problem of providing the application of a regulatory gene PdeMIXTA02 for the initial development of populus deltoids in breeding new varieties of non-flying populus trees.

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

a regulatory gene PdeMIXTA02 for the initial development of Populus deltoides catkin has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the regulatory gene PdeMIXTA02 for the initial development of the populus deltoids is shown as SEQ ID NO. 2.

The vector, the recombinant bacterium or the host cell contains the regulatory gene PdeMIXTA02 for the initial development of the populus deltoids.

Further, the vector of the regulatory gene PdeMIXTA02 for the initial development of populus deltoids is pCAMBIA1301-PdeMIXTA 02.

The application of the regulatory gene PdeMIXTA02 for the initial development of populus deltoids in breeding new varieties of non-flying populus deltoids.

Further, the application of the regulatory gene PdeMIXTA02 for the initiation development of the populus deltoids in breeding new varieties of the populus deltoids comprises the following steps:

1) preparing an antisense vector, an RNAi vector or a CRISPR vector of the PdeMIXTA02 gene;

2) gene expression silencing or gene editing is carried out on PdeMIXTA02 in populus tremuloides by utilizing a genetic transformation system of the populus tremuloides, so that the function of the PdeMIXTA02 gene is lost;

3) breeding and screening the transgenic material with the PdEMIXTA02 gene inactivated to obtain a new transgenic poplar variety with female plants without flying.

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

the invention determines the initial development time point of poplar catkin to be 1 day after pollination through a scanning electron microscope, the poplar catkin is formed by the initial development of placenta epidermal cells, and the MIXTA gene is deduced to be a candidate gene for regulating the initial development of the poplar catkin by combining the regulation mechanism of the initial development of cotton fibers. Eight MIXTA genes are identified in populus tremuloides genome, and the PdEMIXTA02 gene is determined to be a key gene for controlling the initial development of populus tremuloides through gene sequence evolution analysis, expression pattern analysis of different periods of the initial development of populus tremuloides and genetic transformation verification in Arabidopsis thaliana. The result shows that when the gene sequence is analyzed by evolution, the gene and the gene for regulating and controlling the initial development of cotton wool are in the same evolutionary branch; the analysis of the gene expression pattern shows that the gene is specifically expressed in the initial development period of the poplar catkin and is not expressed in other tissues; the genetic transformation of Arabidopsis shows that the number of the epidermal hair of the transgenic material of the gene is remarkably higher than that of the wild material, and the gene can promote the epidermal cell of Arabidopsis to bulge and differentiate into the epidermal hair. In conclusion, the PdEMIXTA02 gene can be verified to be a key gene for regulating and controlling the initial development of populus tremula, and genetic operations such as gene silencing or gene editing are carried out on the gene, so that a new variety of populus tremules without flying catkins can be obtained.

Drawings

FIG. 1 is a scanning electron micrograph of the Populus catkin initial development site and the initial time point; the results show that poplar catkin cells initiate from placenta epidermal cells and start to bulge 1 day after pollination (1DPA), and rapidly elongate 3-5 days after pollination (3DPA, 5 DPA);

FIG. 2 is a graph of the results of cluster evolution analysis of the MIXTA gene sequence; the results showed that the gene PdEMIXTA02 of Populus deltoides is clustered in a clade with genes GhmML3 and GhmML4, which regulate the initial development of cotton boll, and the genes in the clade belong to the genes MIXTA, 8 of which are derived from Populus deltoides (Pde: Populus deltoides), 10 are derived from Gossypium hirsutum (Gh: Gossypium hirsutum), 3 are derived from Arabidopsis thaliana (At: Arabidopsis thaliana), 7 are derived from maize (Zm: Zeamays), 5 are derived from rice (Os: Oryzasativ), 8 are derived from trees (Spu: Salixwillowaria), 3 are derived from Eucalyptus globulus (Eg: Eucalyptus grandis), and 11 are derived from tobacco (Nt: Nicotiana uvarum L);

FIG. 3 is a graph showing the results of analysis of qPCR expression pattern of PdEMIXTA02 gene; selecting 7 different tissues of a female inflorescence (-3DPA) before pollination, a female inflorescence (0DPA) before pollination, a female inflorescence (3DPA) after pollination, a female inflorescence (5DPA) after pollination, a leaf (leaf), a leaf bud (bud) and a bark (bark) for gene expression pattern analysis, wherein the number of a histogram represents the average value of the three biological repetitions, and an error line is the standard error result of the three biological repetitions;

FIG. 4 is a diagram showing the cloning of PdEMIXTA02 gene and the construction result of overexpression vector; FIG. 4A is an electrophoresis gel of a gene full-length clone using cDNA of a female inflorescence 1 day after pollination as a template; FIG. 4B is the electrophoresis gel diagram of the constructed over-expression vector; FIG. 4C is a schematic diagram of the construction of an overexpression vector;

FIG. 5 is a functional verification result chart of the transformed poplar PdeMIXTA02 gene in Arabidopsis; wherein FIG. 5A shows an Arabidopsis seedling grown in soil for 10 days; FIG. 5B shows the leaves of 5A Arabidopsis thaliana, in growth order, with the gray circle being the statistical area for the number of coat hairs, 3mm in diameter, and Bar 1.5 mm; fig. 5C shows that overexpression of PdeMIXTA02 gene in arabidopsis significantly increased epidermal hair density, Bar 0.5 mm; fig. 5D shows statistics of the number of epidermal hairs in the same leaf area at the same site in the leaves of transgenic arabidopsis thaliana and WT, the histogram value represents the average of the number of epidermal hairs in the same area of the first true leaf of 50 arabidopsis thaliana, the error bars are standard errors, and the x represents the very significant difference from wild type arabidopsis thaliana (P < 0.005); FIG. 5E shows the electrophoresis gel of the semi-quantitative RT-PCR analysis of different transgenic lines of PdMEMIXTA 02 gene.

Detailed Description

The invention is further described with reference to specific examples.

Example 1:

(1) determination of Populus initial development site and time point

In the campus of Nanjing forestry university (32 degrees in North latitude and 118 degrees in Western longitude), female populus tremuloides of the major push variety Nanlin 895 fast growing populus (NL895) with the growth time of more than 15 years is selected.

Collecting female inflorescence flowers of NL895 poplar 1 day (1DPA), 3 days (3DPA) and 5 days (5DPA) after pollination in 3 months in 2018, placing in FAA stationary liquid, and vacuumizing for storage. Longitudinally planing the female florets stored in the stationary liquid, dehydrating by using gradient alcohol concentration, drying, and observing by using a scanning electron microscope. The results show that poplar catkins started at time point 1DPA, began to bulge from epidermal cells of the placenta, and poplar catkins cells elongated rapidly after time point 3DPA (fig. 1). This physiological process is similar to the initial development of cotton wool. At present, known research results show that the genes for regulating the initial development of cotton wool are mainly MIXTA genes, including GhMML3, GhMML4 and the like. The function of the MIXTA gene in different plants is mainly combined to regulate the initial development of epidermal hair of different tissue parts, and the function is highly conserved among plants, so that the MIXTA gene is inferred to play a regulating role in the initial development process of poplar catkin.

(2) Identification of MIXTA gene and sequence evolution analysis

The MIXTA class of genes belongs to the ninth subclass of R2R3-MYB, and its sequence features include two MYB conserved domain units, as well as the Motif sequence of AQWESAR AE RL RES. Based on the sequence characteristics of MIXTA, 8 MIXTA genes were identified in Populus tremuloides (Populus deltoids), and members of MIXTA gene families were also identified in the following species, including 10 cotton (Gossypium herbaceum), 3 Arabidopsis thaliana (Arabidopsis thaliana), 7 maize (Zea mays), 5 rice (Oryza sativa), 8 willow (Salix purpurea), 3 Eucalyptus (Eucalyptus grandis) and 11 tobacco (Nicotiana tabacum L), respectively. A total of 55 protein sequences, and an evolutionary Tree was constructed according to the maxim likehood Tree method using MEGA5.0 software. The results show that the PdeMIXTA02 gene is clustered in the same branch as a key gene that regulates the initial development of cotton wool (fig. 2). Presumably, it may have a regulatory effect on the initial development of poplar catkins.

(3) Analysis of expression patterns of PdMEMIXTA 02 gene at different time points of poplar catkin initial development

In 2018 spring, collecting female inflorescences of different time points of the initial development of poplar seed, wherein the female inflorescences comprise 4 time nodes of 3 days (-3DPA) before pollination, 0DPA (on the day of pollination), 3 days (3DPA) after pollination and 5 days (5DPA) after pollination. In addition, 3 vegetative organs in total were selected as controls for leaf (leaf), bud (bud) and bark (bark). And extracting RNA of the collected sample to perform reverse transcription of cDNA. Quantitative primers for the PdeMIXTA02 gene were designed as follows:

N00954F：5′-GTTTGCCGGCGATCATAACGC-3′；

N00954R：5′-GAGAAGCATCGACCAGATCATTGAGTAAGCTAT-3′。

the PtUBQ gene is taken as an internal reference gene, and the primer sequence of the internal reference gene is as follows:

PtUBQF：5′-GTTGATTTTTGCTGGGAAGC-3′；

PtUBQR：5′-GATCTTGGCCTTCACGTTGT-3′。

a20. mu.L reaction system was prepared, including 100ng of cDNA, 4pmol of upstream and downstream primers, 10. mu.L of AceQqPCR SYBR Green Master Mix, and sterile ultrapure water to 20. mu.L.

Real-time quantitative PCR experimental procedure: first 95 ℃ denaturation for 3 min, then 40 reaction cycles including 95 ℃ denaturation for 15 sec, 60 ℃ annealing for 15 sec, and 72 ℃ extension for 30 sec were performed.

Calculation of Gene expression Using 2-^ΔCTMethod, i.e. Δ CT ═ CT_{Target gene}-CT_{Internal reference gene}. The relative expression pattern of PdeMIXTA02 gene in different tissues of populus deltoids was analyzed using the T-test method. The results show that PdeMIXTA02 is a gene specifically expressed within 5 days after pollination and is not expressed in other tissues (fig. 3). The specific expression time node of the gene is completely coincided with the time node of the initial development of the poplar catkin. Combining the results of the previous sequence analysis, the PdMEXTA 02 gene is deduced to be a poplar catkin initial development regulating gene.

(4) PdeMIXTA02 gene homologous cloning and overexpression vector construction

The primer MIXTA02F was designed based on the full-length sequence of the PdEMIXTA02 gene identified in the Populus deltoides genome: 5'-ATGGTTAAGTCTCAATGCTTTGAGAAGGT-3', MIXTA 02R: 5'-TCAAAATACAGATGACCCAGTTGGAGAAGC-3', using cDNA of 3 days inflorescence after NL895 pollination as template, cloning PdeMIXTA02 (figure 4A) in a homologous way, connecting the target fragment to Blunt vector for sequencing, and determining that the full length of the gene PdeMIXTA02 is 1149bp, the specific sequence is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein is shown as SEQ ID NO. 2.

The pCambia1301 vector is used as a backbone vector, the full length of the PdEMIXTA02 gene is recombined between two restriction enzymes KpnI and XbaI of a multiple cloning site by a homologous recombination method, and CaMV35S is used as a promoter of a target gene. Double restriction validation using KpnI and XbaI yielded clear restriction gel electrophoresis bands (FIG. 4B) confirming the completion of the vector construction, which was named pCAMBIA1301-PdEMIXTA02 (FIG. 4C).

(5) Functional verification of PdeMIXTA02 gene transferred arabidopsis thaliana

Columbia wild type Arabidopsis thaliana (col-0) used for plant transformation was from laboratory storage. Arabidopsis seeds were surface-sterilized in 75% ethanol for 30 seconds, then washed in sterile water 3 times, then surface-sterilized in 10% NaClO (v/v) for 10 minutes, then washed again with sterile water 6 times, and then uniformly sown in 1/2MS medium containing 3% sucrose and 0.8% agar, pH 5.8. Seeds were first cultured in the dark at 4 ℃ for 3 days for vernalization, then transferred to a growth chamber (22-23 ℃, 16h light/8 h dark) for germination, and arabidopsis seedlings grown for about two weeks were transplanted into a nutrition pot, nutrient soil: black soil: perlite: vermiculite 3: 3: 1: 1, grown under the same conditions (22-23 ℃, 16h light/8 h dark).

The constructed over-expression vector pCAMBIA1301-PdeMIXTA02 was transformed into Agrobacterium tumefaciens GV3101(pMP 90). Agrobacterium was grown up, and after collecting the cells, the cells were adjusted to an OD of 0.8 with a suspension (1/2MS + 0.5% sucrose) and used for transformation experiments with arabidopsis thaliana. The wild arabidopsis transformation adopts a floral organ dip-dyeing method. In the full-bloom stage (about 4 weeks of growth) of wild type arabidopsis, the inflorescence is soaked in the prepared agrobacterium suspension for 30s, dark culture is carried out in a growth chamber for 24h, and then normal growth conditions are restored until the seeds are harvested by division after maturation (T1). Screening T1 generation transgenic positive plants by using an MS culture medium containing 30mg/L hygromycin, transplanting the screened positive plants into soil, and placing the soil in a growth room (22-23 ℃, 16h light/8 h dark) for normal management. When seedlings were grown in soil for 10 days (FIG. 5A), the number of epidermal hairs in a designated 1.5mm area of the first true leaf was observed under a microscope (FIG. 5B). The first true leaf of the PdeMIXTA02 transgenic arabidopsis 9# line was selected and the number of epidermal hairs in the indicated region was significantly increased compared to the wild type (fig. 5C). In order to verify the authenticity and reliability of the result, 5 lines (1#, 5#, 9#, 17# and 23#) of PdeMIXTA02 transgenic Arabidopsis are selected, 10 positive plants are selected in the T2 generation, and the number of epidermal hairs is counted in the same area of the first true leaf. The results show that PdeMIXTA02 gene can significantly increase the number of epidermal hairs in arabidopsis thaliana (fig. 5D). Meanwhile, for five positive plants of No.1, No. 5, No. 9, No. 17 and No. 23 of PdeMIXTA02 transgenic arabidopsis, the first true leaf is collected at the generation T2 for semi-quantitative RT-PCR. The results show that the target genes can be highly expressed in the positive plants (FIG. 5E). The transformation experiment of arabidopsis proves that the PdEMIXTA02 gene has the potential of promoting epidermal cells to be differentiated into bulge cells, and further proves the regulation and control effect of the gene on the initial development of poplar catkin. Therefore, the gene PdEMIXTA02 can be knocked out or silenced in the poplar by utilizing a genetic transformation technology, and the gene is used for cultivating a new species of the non-floating poplar.

Sequence listing

<110> Nanjing university of forestry

<120> Populus deltoides catkin initial development regulatory gene PdeMIXTA02 and application thereof

<130> 20200211

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1149

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gaagaccaga agcttttggc ttacgtcgaa gagcatggcc atggaagctg gcaagccttg 120

cctgccaaag ctggacttca gagatgcggg aagagctgta gactcaggtg gaccaactac 180

cttcggccag atatcaagag aggaaagttt aatttgcagg aagaacaatc aatcattcag 240

ctgcatgctc ttcttggaaa caggtggtca gccattgcta cacacttgcc gaaaagaacc 300

gataacgaga tcaagaatta ctggaacaca catcttaaga aaagattaga caaaatgggc 360

attgatcctg tgacccacaa gccaaaagct gattctttcg gctccggaag tggccattcc 420

aagggttctg ccaatttaag ccacatggct caatgggaga gtgctcgact agaggccgaa 480

gccagattgg tccgtgagtc aaagttaagt gtacctaacc ctcccaaaaa ccttctaggc 540

tttgcagttt cagctcaagt ctccgacaaa ggtagtgcag ctccaacaga aagaccacgg 600

tgtcttgatg tactcaaagc atggcaaggg gtcgttttca gcatgttctc ggttggcagc 660

agtgactctc tcgagtctcc aacgtccact ttgaatttct cagaaaatgc attagccatc 720

ccactcattg gagttcagaa aaatccaacc accacactag cttttgccac aaataatgct 780

acatgcaaca gagggactac ggccagtgag tttgataggg gaaaccagtt ggagtgctgt 840

gagaaactga aagaaccagc acaagtgaaa cggaatttgg acagttcaat ggcaattcat 900

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Trp Thr Pro Glu Glu Asp Gln Lys Leu Leu Ala Tyr Val Glu Glu His

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Gly His Gly Ser Trp Gln Ala Leu Pro Ala Lys Ala Gly Leu Gln Arg

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Cys Gly Lys Ser Cys Arg Leu Arg Trp Thr Asn Tyr Leu Arg Pro Asp

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Ile Lys Arg Gly Lys Phe Asn Leu Gln Glu Glu Gln Ser Ile Ile Gln

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Leu His Ala Leu Leu Gly Asn Arg Trp Ser Ala Ile Ala Thr His Leu

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Pro Lys Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu

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Lys Lys Arg Leu Asp Lys Met Gly Ile Asp Pro Val Thr His Lys Pro

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Lys Ala Asp Ser Phe Gly Ser Gly Ser Gly His Ser Lys Gly Ser Ala

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Asn Leu Ser His Met Ala Gln Trp Glu Ser Ala Arg Leu Glu Ala Glu

145 150 155 160

Ala Arg Leu Val Arg Glu Ser Lys Leu Ser Val Pro Asn Pro Pro Lys

165 170 175

Asn Leu Leu Gly Phe Ala Val Ser Ala Gln Val Ser Asp Lys Gly Ser

180 185 190

Ala Ala Pro Thr Glu Arg Pro Arg Cys Leu Asp Val Leu Lys Ala Trp

195 200 205

Gln Gly Val Val Phe Ser Met Phe Ser Val Gly Ser Ser Asp Ser Leu

210 215 220

Glu Ser Pro Thr Ser Thr Leu Asn Phe Ser Glu Asn Ala Leu Ala Ile

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Pro Leu Ile Gly Val Gln Lys Asn Pro Thr Thr Thr Leu Ala Phe Ala

245 250 255

Thr Asn Asn Ala Thr Cys Asn Arg Gly Thr Thr Ala Ser Glu Phe Asp

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Arg Gly Asn Gln Leu Glu Cys Cys Glu Lys Leu Lys Glu Pro Ala Gln

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Val Lys Arg Asn Leu Asp Ser Ser Met Ala Ile His Asp Thr Ser Pro

290 295 300

Phe Ala Gly Asp His Asn Ala Trp Phe Val Asp Ser Ser Ala Ile Glu

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

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Cys Asn Ser Leu Asp His Asn Pro Thr Cys Ser Gly Glu Asn Ile Asn

340 345 350

Asp Asn Tyr Ala Gly Asn Leu Glu Asp Asn Tyr Trp Asn Ser Leu Leu

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Asn Asp Leu Val Asp Ala Ser Pro Thr Gly Ser Ser Val Phe

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Claims (6)

1. A regulatory gene PdeMIXTA02 for the initial development of populus deltoids has the nucleotide sequence shown in SEQ ID NO. 1.

2. The expression protein of the regulatory gene PdexXTA 02 for the initiation of populus deltoids development of claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.

3. A vector or recombinant bacterium containing the regulatory gene PdeMIXTA02 for the initiation of the development of Populus deltoides catkin according to claim 1.

4. The vector of the gene PdexXTA 02 for controlling the initiation of the development of Populus deltoides catus floc according to claim 3, wherein the vector is pCAMBIA 1301-PdexXTA 02.

5. The use of the gene PdexXTA 02 for controlling the initiation of Populus deltoides catkin development according to claim 1 for breeding new varieties of Populus deltoides.

6. The use of the gene PdexXTA 02 for controlling the initiation of Populus deltoides floc development according to claim 5 in breeding new varieties of Populus deltoides, comprising the following steps:

1) preparing an antisense vector, an RNAi vector or a CRISPR vector of the PdeMIXTA02 gene;

2) gene expression silencing or gene editing is carried out on PdeMIXTA02 in populus tremuloides by utilizing a genetic transformation system of the populus tremuloides, so that the function of the PdeMIXTA02 gene is lost;

3) breeding and screening the transgenic material with the PdEMIXTA02 gene inactivated to obtain a new transgenic poplar variety with female plants without flying.

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