CN109735550B - Nucleotide sequence and application thereof in improving density of plant secretory glandular hairs - Google Patents

Abstract

The invention discloses a nucleotide sequence and application thereof in improving the density of secretory glandular hairs of plants, and relates to the technical field of plant genetic engineering, wherein the nucleotide sequence is selected from the following sequences: 1) The amino acid sequence of SEQ ID NO: 1. 3, 5 or a nucleotide sequence shown in any one of the above; 2) SEQ ID NO: 1. 3 or 5 by substitution, deletion or addition of one or more nucleotides; 3) A nucleotide sequence having at least 80% homology with any one of SEQ ID NOs 1, 3, 5. The invention transfers any one of the nucleotide sequences into plants by means of genetic engineering, can obviously improve the density of secretory glandular hairs of plants, fully exerts the potential of the secretory glandular hairs of plants in pest resistance and production of specific metabolites and has extremely high practical production and application values.

Description

Nucleotide sequence and application thereof in improving secretory glandular hair density of plants

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a nucleotide sequence and application thereof in improving secretory glandular hair density of plants.

Background

Glandular hairs are protuberant structures derived from plant epidermal cells, and are classified into non-secretory glandular hairs and secretory glandular hairs according to whether they can secrete and store secondary metabolites. Secretory glandular hairs are distributed on the epidermis of about 30% of the aerial organs of vascular plants in nature, such as peppermint and basil of the lamiaceae family, southernwood and sunflower of the Compositae family, tobacco and tomato of the Solanaceae family, alfalfa and alfalfa of the Leguminosae family, and hop of the Moraceae family. The secretory glandular hairs can help plants to resist plant diseases and insect pests and ultraviolet injury, and can secrete a large amount of chemical substances, such as terpenes, methyl benzenealkanes, flavonoids, methyl ketones, acyl saccharides and the like, and the chemical substances can be used for preparing medicines, producing pesticides, essential oil and the like and have high commercial value. Secretory glandular hairs are also called plant factories. Among them, artemisia annua (Artemisia annua), also known as Artemisia annua, is a traditional Chinese herbal plant of Artemisia of Compositae, and has secretory glandular hair (gland trichomes) and T-shape non-secretory glandular hair (non-gland trichomes); artemisinin is a sesquiterpene lactone compound containing a peroxy-bridge structure, artemisinin and its derivatives are mainly used for treating malaria, and artemisinin-based combination therapy is the most effective method recommended by the world health organization for treating malignant malaria. While artemisinin is synthesized and stored only in the secretory glandular hairs of Artemisia annua.

The secretory glandular hairs are distributed in different numbers in plants, and in some crops with important economic value, the quantity and density of the secretory glandular hairs are insufficient to limit the yield of secondary metabolites.

Therefore, those skilled in the art have been devoted to developing a method for increasing secretory glandular hair density in plants by means of transgenic technology.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to increase the density of secretory glandular hairs of plants.

To achieve the above object, the present invention provides a nucleotide sequence selected from the following sequences:

1) SEQ ID NO: 1. 3, 5 or a nucleotide sequence shown in any one of the above;

2) SEQ ID NO: 1. 3 or 5 by substitution, deletion or addition of one or more nucleotides;

3) A nucleotide sequence having at least 80% homology with any one of SEQ ID NOs 1, 3, 5.

In another aspect, the present invention also provides an amino acid sequence selected from the group consisting of:

1) The amino acid sequence of SEQ ID NO: 2. 4, 6;

2) SEQ ID NO: 2. 4 or 6 by substitution, deletion or addition of one or more amino acids;

3) An amino acid sequence having at least 80% homology with any of SEQ ID NOs 2, 4, 6.

On the other hand, the invention also provides the application of the nucleotide sequence or the amino acid sequence in improving the density of the secretory glandular hairs of the plant.

Further, the plant is one or more of Labiatae, leguminosae, and Compositae.

Further, the above plants are one or more of Mentha arvensis (Mentha haplocalyx Briq), mentha spicata (Mentha spicata Linn), artichoke (Cynara cardunculus var. Scolymus), sunflower (Helianthus annuus), tomato (Solanum lycopersicum), wild species Solanum pennellii (Solanum pennellii) and potato (Solanum tuberosum).

In another aspect, the present invention also provides a method for increasing secretory glandular hair density of a plant by using a transgene, comprising the following steps:

(1) Obtaining a target gene by adopting a gene cloning method, wherein the target gene is any one of the nucleotide sequences;

(2) Constructing a plant expression vector with the target gene;

(3) Transforming the plant expression vector with the target gene into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing the plant expression vector with the target gene;

(4) Transforming the agrobacterium tumefaciens strain containing the target gene plant expression vector into a plant, and carrying out PCR (polymerase chain reaction) detection to obtain a transgenic plant integrated with the target gene;

(5) And counting the glandular hair density on the transgenic plant leaves integrated with the target gene to obtain the plant with the enhanced glandular hair density.

Further, in the step (1), the gene cloning method comprises the following steps: extracting total RNA of plant genome, reverse transcribing to synthesize cDNA, designing primer based on the target gene sequence to perform PCR amplification and sequencing to obtain the target gene.

Further, in the step (2), constructing a plant expression vector having the gene of interest comprises the steps of: amplifying a target gene sequence by high-fidelity enzyme, respectively introducing BamH I and Xba I enzyme cutting sites in front of and behind the target gene, connecting the target gene with a vector by using ligase, transforming a host cell, selecting a monoclonal, and extracting a plasmid for PCR detection and enzyme cutting verification.

Further, in the step (3), transforming Agrobacterium tumefaciens comprises the steps of: the plant expression vector with the target gene is transferred into agrobacterium tumefaciens by a freeze thawing method, and PCR verification is carried out.

Further, in step (4), the conversion comprises the steps of: pre-culturing explants; co-culturing agrobacterium and explants; and (4) screening the regenerated plants containing the target genes by using the resistance culture medium.

Further, in a specific embodiment, the plant is artemisia apiacea.

Furthermore, the target gene is any one of a sweet wormwood AaWRKY75b gene, a wild mint MhWRKY75b gene and a spearmint MsWRKY75b gene.

Furthermore, the sequence of the southernwood AaWRKY75b gene is shown as SEQ ID NO. 1; the wild mint MhWRKY75b gene sequence is shown in SEQ ID NO. 3; the sequence of the Mentha spicata MsWRKY75b gene is shown in SEQ ID NO. 5.

Furthermore, the amino acid sequence coded by the southernwood AaWRKY75b gene is shown in SEQ ID NO. 2; the amino acid sequence coded by the wild mint MhWRKY75b gene is shown in SEQ ID NO. 4; the amino acid sequence of the spearmint MsWRKY75b gene code is shown in SEQ ID No. 6.

Further, in the step (1), the primer sequence is shown as SEQ ID NO:7 and 8.

Further, cDNA was synthesized by reverse transcriptase PowerScript.

Further, the pre-culture of the explant comprises the following steps: soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, drying surface water with sterile absorbent paper, inoculating in hormone-free MS solid culture medium, culturing at 25 deg.C under 16 hr illumination/8 hr dark condition to obtain herba Artemisiae Annuae sterile seedling, and cutting sterile seedling leaf explant for transformation after the seedling grows to about 5 cm;

further, co-cultivation of Agrobacterium with the explant comprises the following steps: transferring the leaf explant to a co-culture medium added with Acetosyringone (AS), dropwise adding activated 1/2MS bacterial liquid of agrobacterium tumefaciens containing a target gene plant expression vector, fully contacting the leaf explant with the bacterial liquid, and performing dark culture at 28 ℃ for 3 days.

Further, the screening of resistant regenerated plants comprises the following steps: transferring leaf explants co-cultured for 3 days into germination selection medium containing 6-benzylaminopurine (6-BA), naphthylacetic acid (NAA), hygromycin (Hyg) and carbenicillin (Cb); culturing at 25 ℃ under 16-hour illumination/8-hour dark conditions, subculturing once every two weeks, obtaining Hyg resistant clustered buds after 2-3 subcultures, shearing the well-grown resistant clustered buds, transferring the clustered buds to a rooting culture medium for culturing until rooting, and obtaining Hyg resistant regenerated sweet wormwood plants.

Further, the method also comprises the step (6) of carrying out HPLC-ELSD measurement on the artemisinin content in the transgenic southernwood, and screening to obtain transgenic southernwood plants with increased artemisinin content.

In the specific implementation mode of the invention, the Artemisia apiacea AaWRKY75b gene or the gene with 80% or more homology with the Artemisia apiacea AaWRKY75b gene (such as the Mentha arvensis MhWRKY75b gene or the Mentha spearmint MsWRKY75b gene) is over-expressed in the plant with secretory glandular hairs by a transgenic means, so that the density of the secretory glandular hairs in the transgenic plant is obviously improved, the content of secondary metabolites such as artemisinin in the transgenic plant is improved, and the invention has extremely high practical production and application values. For plant artemisia apiacea, the density of secretory glandular hairs on the surface of the obtained leaves is obviously improved to obtain transgenic plants, so that the secretory glandular hairs of the artemisia apiacea are converted into biochemical factories with high yield, and HPLC-ELSD measurement also shows that the artemisinin content of the plants with the improved secretory glandular hairs is also obviously improved, the potential of the plants in insect resistance and specific metabolite production is fully exerted, and the transgenic plants have extremely high practical production and application values.

The conception, the specific implementation steps and the technical effects generated by the present invention will be further described in conjunction with the accompanying drawings so as to fully understand the objects, the characteristics and the effects of the present invention.

Drawings

FIG. 1 is a comparison graph of the density of glandular hairs of leaf of Artemisia annua transgenic for AaWRKY75b and wild Artemisia annua in one embodiment of the invention;

FIG. 2 is a graph showing the statistical results of the glandular hair density of leaf blades of Artemisia apiacea and wild type Artemisia apiacea with transferred AaWRKY75b gene in one embodiment of the invention;

FIG. 3 is a graph showing the results of the detection of the contents of AaWRKY75b transgenic Artemisia annua and wild type Artemisinin in one embodiment of the present invention;

FIG. 4 is a comparison graph of leaf glandular hair density of Artemisia annua and wild type Artemisia annua transferred MhWRKY75b gene or MsWRKY75b gene in one embodiment of the invention;

FIG. 5 is a graph showing the statistics of leaf glandular hair density of Artemisia annua transgenic for MhWRKY75b and wild type Artemisia annua in one embodiment of the present invention;

FIG. 6 is a graph showing the statistical results of leaf glandular hair density of transgenic Artemisia annua L-75 b gene and wild type Artemisia annua L in one embodiment of the invention.

Detailed Description

The technical contents of the present invention will be further described with reference to the accompanying drawings and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, such as molecular cloning in Sambrook, etc.: the Laboratory Manual is described in New York, 1989 edition of Cold Spring Harbor Laboratory Press, or as recommended by the manufacturer.

The agrobacterium tumefaciens EHA105 related to the invention has been found in Huangya Lian, jianghuang, panglong, guo Ping and Zhu Chang Xiong; study of agrobacterium tumefaciens mediated genetic transformation of trichoderma harzianum, journal of chinese bioengineering, 2008, 28 (3): 38-43, respectively. Agrobacterium tumefaciens EHA105 is commercially available from publicly available sources, such as the CAMBIA corporation, australia, under the strain number Gambar1.

Example 1

Extraction of total RNA of sweet wormwood

The Artemisia apiacea leaves are quickly ground into powder in liquid nitrogen, and the total Artemisia apiacea RNA is extracted by using an RNA kit of TIANGEN company according to the experimental procedures of the kit. And (3) taking a part of the obtained RNA solution, carrying out agarose gel electrophoresis detection to identify the total RNA quality and measuring the RNA content by using the concentration of a spectrophotometer, and storing the rest part at-80 ℃.

Example 2

Cloning of Artemisia apiacea AaWRKY75b gene

Synthesizing cDNA by PowerScript reverse transcriptase by using the total RNA extracted in the example 1 as a template; a gene specific primer is designed according to the sequence of the Artemisia apiacea AaWRKY75b gene (GenBank accession number: KX 465129.1), the sequence of the primer is shown in table 1, the AaWRKY75b gene of the Artemisia apiacea is obtained by PCR amplification from total cDNA, a PCR reaction system is shown in table 2, and DNA sequencing is carried out after gel electrophoresis recovery of an amplification product. The full-length coding sequence of the gene is obtained after sequencing, and is shown as SEQ ID NO.1, wherein, the initiation codon is ATG, the termination codon is TAG, and the protein coding sequence is deduced, and is shown as SEQ ID NO. 2.

TABLE 1 AaWRKY75b Gene clone PCR primers

TABLE 2 reaction System for PCR

Example 3

Construction of plant overexpression vector containing AaWRKY75b gene

AaWRKY75b gene sequence is amplified through high fidelity enzyme, the sequence of an amplification primer is shown in Table 3, wherein a BamHI restriction site is introduced into a forward primer, an XbaI restriction site is introduced into a reverse primer, and the forward primer and the reverse primer are connected to a pCAMbia 1305.1 vector (purchased from Youbao biology) through ligase to carry out sequencing to confirm the correctness of the gene.

TABLE 3 PCR primers for AaWRKY75b-pCAMbia 1305.1 vector construction

Example 4

Obtaining of AaWRKY75b overexpression-containing agrobacterium tumefaciens engineering bacteria

The plant overexpression vector containing the AaWRKY75b gene in the example 3 is transferred into agrobacterium tumefaciens by a freeze-thaw method, and PCR verification is carried out, wherein verification primers are shown as SEQ ID NO. 11 and SEQ ID NO. 12. Obtaining the agrobacterium tumefaciens strain of the plant over-expression vector containing the AaWRKY75b gene.

Example 5: artemisia apiacea transformed by agrobacterium tumefaciens mediated AaWRKY75b gene

(1) Pre-culture of explants

Soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, blotting surface water with sterile absorbent paper, inoculating in hormone-free MS (Murashige and Skoog, 1962) solid culture medium, and culturing at 25 deg.C for 16h/8h (light/dark) for 5-7 days to obtain herba Artemisiae Annuae sterile seedling. After the seedling grows to about 5cm, shearing a sterile seedling leaf explant for transformation.

(2) Co-culture of Agrobacterium and explant

Transferring the leaf explant to a co-culture medium (1/2MS + AS100 mu mol/L) added with Acetosyringone (AS), dropwise adding 1/2MS suspension of the activated Agrobacterium tumefaciens engineering bacteria containing the AaWRKY75b plant overexpression vector, fully contacting the explant with a bacterial solution, and performing dark culture at 28 ℃ for 3 days. The control was leaf explants to which 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the desired gene was added.

(3) Selection of resistant regenerated plants

Transferring the sweet wormwood herb explants cultured for 3 days in a co-culture manner into a germination screening culture medium (MS +6-BA 0.5mg/L + NAA 0.05mg/L + Hyg (overexpression) 50mg/L + Cb 500 mg/L) added with 6-benzylaminopurine (6-BA), naphthylacetic acid (NAA), hygromycin (Hyg) and carbenicillin (Cb) for carrying out light culture at 25 ℃ for 16h/8h (light/dark), subculturing once every two weeks, and obtaining Hyg resistant cluster buds after 2-3 subcultures. Shearing off the well-grown resistant cluster buds, and transferring the cluster buds to a rooting culture medium (1/2MS + Cb125mg/L) for culturing until the cluster buds grow to root, thereby obtaining a Hyg resistant regenerated sweet wormwood plant.

Example 6

PCR detection of transgenic southernwood plant

A forward primer (35SF. The result shows that the designed PCR specific primer can be used for amplifying a specific DNA fragment. When non-transformed genomic DNA of Artemisia annua is used as a template, no fragment is amplified.

In the embodiment, the plant expression vector is used for transforming agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing the AaWRKY75b plant over-expression vector for transforming artemisia apiacea, and the constructed agrobacterium tumefaciens strain is used for transforming artemisia apiacea to obtain a transgenic artemisia apiacea plant detected by PCR. The transgenic southernwood plant can be directly used for screening southernwood plants with increased secretory glandular hair density and increased artemisinin content.

Example 7:

statistics of transgenic southernwood epidermal glandular hair density and total glandular hair number

The leaves of non-transgenic Artemisia annua and AaWRKY75b over-expressing transgenic Artemisia annua were observed under excitation light with a BX51 type microscope from Olympus under a wavelength of 450nm to 480 nm. Taking Artemisia apiacea leaves with the same size, randomly sampling at different 5 positions, measuring the total area of the Artemisia apiacea leaves by using ImageJ software, and counting the glandular hair density, wherein CK represents a wild type control group, the leaf glandular hair density of the Artemisia apiacea plant with the AaWRKY75 b-transformed gene is obviously higher than that of the wild type Artemisia apiacea, and the maximum leaf glandular hair density is 41.59/mm ² The wild type sweet wormwood is 20.65 times of the wild type sweet wormwood.

Example 8

Determination of artemisinin content in transgenic southernwood by HPLC-ELSD

(1) HPLC-ELSD conditions and system applicability and preparation of standard solution

HPLC: a water allelance 2695 system is adopted, a chromatographic column is a C-18 reverse phase silica gel column (SymmetyrShield TM C18,5 mu m,250x 4.6mm, waters), a mobile phase is methanol to water, the volume ratio of the methanol to the water is 70, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the sample injection amount is 10 mu L, the sensitivity (AUFS = 1.0), and the number of theoretical plates is not less than 2000 calculated according to an artemisinin peak.

ELSD: adopting a water alliance 2420 system, wherein the temperature of a drift tube of the evaporative light scattering detector is 40 ℃, the amplification factor (gain) is 7, and the carrier gas pressure is 5bar;

accurately weighing 2.0mg of artemisinin standard (Sigma company), dissolving completely with 1mL of methanol to obtain 2mg/mL of artemisinin standard solution, and storing at-20 deg.C for use.

The mobile phase in the invention is methanol (methanol): water in a proportion of 70%: at 30%, the retention time of artemisinin was 5.1min, and the peak pattern was good. The theoretical plate number is not less than 2000 calculated by artemisinin.

(2) Preparation of Standard Curve

And respectively injecting 2 mu L,4 mu L,6 mu L,8 mu L and 10 mu L of the reference substance solution under corresponding chromatographic conditions to record a chromatogram and chromatographic parameters, and respectively performing regression analysis on the contents (X and mu g) of the standard substance by using a peak area (Y). Through studies, artemisinin in this example exhibited a good log-log linear relationship in the 4-20g range. The log-log linear regression equation for the artemisinin control was: y =1.28e +000X +4.71e +000 ² ＝0.979546。

(3) Preparation of sample and determination of artemisinin content

2g of fresh leaves of Artemisia annua are taken from the upper part, the middle part and the lower part of the Artemisia annua plant, and are dried in a 45 ℃ oven until the weight is constant. And knocking off the leaves from the dried branches and grinding into powder. Weighing about 0.1g of dry powder into a 2mL Eppendorf tube, adding 2mL of ethanol, treating with 40W ultrasonic waves for 30min, centrifuging at 5000rpm for 10min, taking supernatant, and filtering with a 0.22 μm filter membrane to obtain the product for measuring the content of artemisinin by HPLC-ELSD.

And (3) measuring the content of artemisinin by adopting HPLC-ELSD, wherein the sample injection volume is 20 mu L, substituting the peak area into a linear regression equation to calculate the content (mg) of artemisinin in the sample, and dividing by the dry weight (g) of the artemisia apiacea leaves of the sample so as to calculate the content of artemisinin in the artemisia apiacea plants. The result is shown in figure 2, the content of artemisinin in the sweet wormwood plant with the AaWRKY75b gene is obviously related to the density of glandular hairs, the content of artemisinin is increased to 18.8mg/g along with the increase of the density of secretory glandular hairs, and the content of artemisinin in the wild sweet wormwood plant is 10.5mg/g which is about 1.8 times of that of the wild sweet plant.

Example 9

Artemisia apiacea AaWRKY75b gene sequence homology alignment

The results of the nucleic acid sequence homology comparison of the AaWRKY75b gene sequence shown in SEQ ID NO:1 by using NCBI database show that the sequence homology of the segment with artichoke (Cynara cardunculus var. Scolymus) capable WRKY transcription factor 75 is 89%; the sequence homology with sunflower (Helianthus annuus) capable WRKY transcription factor 75 is 86%; 86% homology with tomato (Solanum lycopersicum) WRKY transcription factor 75 sequence; the sequence homology with wild species tomato pennellii (Solanum pennellii) capable WRKY transcription factor 75 is 86%; has 85 percent of sequence homology with potato (Solanum tuberosum) WRKY1 gene; the sequence homology with the homologous gene MsWRKY75b (GenBank accession number: KT 372786.1) of spearmint (Mentha spicata Linn) is 80 percent; the sequence homology with the wild mint (Mentha haplocalyx Briq) homologous gene MhWRKY75b (shown as SEQ ID NO: 3) is 80%.

Example 10

Extraction of total RNA of spearmint mint and wild mint

The procedure was as in example 1.

Example 11

Cloning of Mentha arvensis MhWRKY75b gene and Mentha spicata MsWRKY75b gene

The specific operation was performed according to the procedure of example 2, wherein the primers used are shown in table 4, the full-length coding sequence of the obtained spearmint MsWRKY75b gene is shown in SEQ ID No.3, and the protein coding sequence thereof is deduced to be shown in SEQ ID NO:4, obtaining the full-length coding sequence of the Mentha arvensis MhWRKY75b gene shown as SEQ ID NO:5, and deducing that the protein coding sequence is shown as SEQ ID NO: and 6.

TABLE 4 MhWRKY75b Gene and MsWRKY75b Gene cloning PCR primers

Example 12

Construction of plant overexpression vector containing MsWRKY75b gene or MhWRKY75b gene

The procedure was as in example 3, and the primer sequences used are shown in Table 5. The obtained plant overexpression vector is MhWRKY75b-pCAMbia 1305.1 vector and MsWRKY75b-pCAMbia 1305.1 vector.

TABLE 5 MhWRKY75b-pCAMbia 1305.1 vector and MsWRKY75b-pCAMbia 1305.1 vector construction PCR primers

Example 13

Obtaining of Agrobacterium tumefaciens engineering bacteria containing MhWRKY75b gene or MsWRKY75b gene over-expression

The detailed procedure was as in example 4. The verification primers are shown in SEQ ID NO. 11 and SEQ ID NO. 18.

Example 14

Agrobacterium tumefaciens mediated MsWRKY75b gene or MhWRKY75b gene transformed southernwood

The detailed procedure was carried out in accordance with the procedure of example 5.

Example 15

PCR detection of transgenic MsWRKY75b gene or MhWRKY75b gene sweet wormwood plant

The detailed procedure was carried out in accordance with the procedure of example 6. Wherein the primers used for identification are shown as SEQ ID NO. 11 and SEQ ID NO. 18.

Example 16

Statistics of epidermal glandular hair density and total glandular hair quantity of sweet wormwood herb with MsWRKY75b gene or MhWRKY75b gene transfer

The specific operation is carried out according to the steps in the example 7, and the results are shown in fig. 4, fig. 5 and fig. 6, wherein CK represents a wild type control group, the leaf glandular hair density of the transgenic southernwood plant with the MsWRKY75b gene is obviously higher than that of the wild type southernwood, and the maximum leaf glandular hair density is 38.65/mm ² The wild type is 20.34 pieces/mm ² 1.9 times of wild sweet wormwood; the density of glandular hairs of leaves of a sweet wormwood plant transformed with the MhWRKY75b gene is obviously higher than that of wild sweet wormwood, and the maximum density is 36.45 hairs/mm ² 20.10 pieces/mm ² About 1.8 times of wild type Artemisia annua.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

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<400> 5

atggataact attcccaacc ttcttcttca tcaagtctcg cacaaagctc tcatctatcc 60

atgctcaaca tgatgatgaa ctcccaacca cacgatcatc aactattcca acatcttgat 120

cagaataatg gacacgtggg cttcatccca tccgttgaaa ataatgatga tcataagtct 180

agttccgccg ccgttgaggt tgagggtggt ggtggtgcgg aggcggaaaa cgaggcggaa 240

ggcggcaaga gaaaggggga gaagaagtcc aagaaaccta ggtttgcctt ccaaacaaga 300

agccaagttg atatacttga tgatggttat aggtggagga aatatggtca aaaggctgtc 360

aagaacaata gatttcccag gagctactac agatgcacac atcaaggctg caatgtaaag 420

aaacaagtgc agaggctatc aaaagacgaa ggaatagtgg tgactactta tgaaggcgtc 480

cattctcatc ctatccaaaa atctaccgac aattttgacc acatccttag tcaaatgcag 540

atttatactg ccttctaa 558

<210> 6

<211> 185

<212> PRT

<213> spearmint (Mentha spicata Linn)

<400> 6

Met Asp Asn Tyr Ser Gln Pro Ser Ser Ser Ser Ser Leu Ala Gln Ser

1 5 10 15

Ser His Leu Ser Met Leu Asn Met Met Met Asn Ser Gln Pro His Asp

20 25 30

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

35 40 45

Ile Pro Ser Val Glu Asn Asn Asp Asp His Lys Ser Ser Ser Ala Ala

50 55 60

Val Glu Val Glu Gly Gly Gly Gly Ala Glu Ala Glu Asn Glu Ala Glu

65 70 75 80

Gly Gly Lys Arg Lys Gly Glu Lys Lys Ser Lys Lys Pro Arg Phe Ala

85 90 95

Phe Gln Thr Arg Ser Gln Val Asp Ile Leu Asp Asp Gly Tyr Arg Trp

100 105 110

Arg Lys Tyr Gly Gln Lys Ala Val Lys Asn Asn Arg Phe Pro Arg Ser

115 120 125

Tyr Tyr Arg Cys Thr His Gln Gly Cys Asn Val Lys Lys Gln Val Gln

130 135 140

Arg Leu Ser Lys Asp Glu Gly Ile Val Val Thr Thr Tyr Glu Gly Val

145 150 155 160

His Ser His Pro Ile Gln Lys Ser Thr Asp Asn Phe Asp His Ile Leu

165 170 175

Ser Gln Met Gln Ile Tyr Thr Ala Phe

180 185

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggacaatt ttgtttctgt tttt 24

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctaaaacaaa ggtggatctt gta 23

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgggatccat ggacaatttt gtttctgttt tt 32

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gctctagact aaaacaaagg tggatcttgt a 31

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaagatgcct ctgccgacag tg 22

<210> 12

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctctagact aaaacaaagg tggatcttgt a 31

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggataact attcccaacc ttctt 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttagaaggca gtataaatct gcatt 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggataact attcccaacc ttctt 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttagaaggca gtataaatct gcatt 25

<210> 17

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgggatccat ggataactat tcccaacctt ctt 33

<210> 18

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gctctagatt agaaggcagt ataaatctgc att 33

<210> 19

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cgggatccat ggataactat tcccaacctt ctt 33

<210> 20

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gctctagatt agaaggcagt ataaatctgc att 33

Claims (6)

1. The application of a nucleotide sequence in improving the density of secreted glandular hairs of artemisia apiacea is characterized in that the nucleotide sequence is shown as SEQ ID NO:5, respectively.

2. A method for improving secretory glandular hair density of a plant by a transgene is characterized by comprising the following steps:

(1) Obtaining a target gene by adopting a gene cloning method, wherein the target gene is a gene sequence shown as SEQ ID NO: 5;

(2) Constructing a plant expression vector with the target gene;

(3) Transforming the plant expression vector with the target gene into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing the plant expression vector with the target gene;

(4) Transforming the agrobacterium tumefaciens strain containing the target gene plant expression vector into a plant, wherein the plant is sweet wormwood herb; PCR detection is carried out to obtain a transgenic plant integrated with the target gene;

(5) Counting the glandular hair density on the transgenic plant leaves integrated with the target gene to obtain a plant with improved glandular hair density;

(6) And (3) performing HPLC-ELSD (high performance liquid chromatography-evaporative light scattering) determination on the artemisinin content in the transgenic southernwood, and screening to obtain transgenic southernwood plants with increased artemisinin content.

3. The method for increasing secretory glandular hair density of a plant by using a transgene as in claim 2, wherein in the step (1), the gene cloning method comprises the following steps: extracting total RNA of plant genome, synthesizing cDNA through reverse transcription, and synthesizing the cDNA by using SEQ ID NO: and 7 and 8, performing PCR amplification and sequencing by taking the nucleotide sequences as primers to obtain the target gene.

4. The method for increasing secretory glandular hair density of a plant by using a transgene as in claim 2, wherein in the step (2), the construction of the plant expression vector with the target gene comprises the following steps: amplifying a target gene sequence by high-fidelity enzyme, respectively introducing BamH I restriction enzyme sites and Xba I restriction enzyme cleavage sites in front of and behind the target gene, connecting the target gene with a vector by using ligase, transforming a host cell, selecting a monoclonal, extracting a plasmid, and performing PCR detection and restriction enzyme digestion verification.

5. The method for increasing secretory glandular hair density of a plant by using a transgene as in claim 2, wherein in step (3), said transforming agrobacterium tumefaciens comprises the steps of: the plant expression vector with the target gene is transferred into agrobacterium tumefaciens by a freeze thawing method, and PCR verification is carried out.

6. The method for increasing secretory glandular hair density of a plant by using a transgene as in claim 2, wherein in step (4), said transformation comprises the following steps: pre-culturing explants; co-culturing agrobacterium and explants; and (4) screening the regenerated plants containing the target genes by using the resistance culture medium.

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