CN103436537B - Lily ester floral gene LoAAT1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of gene engineering, and in particular discloses a lily ester floral gene LoAAT1 and application thereof. A nucleotide sequence of the LoAAT1 gene is shown in SEQ ID NO: 1-2, and an amino acid sequence of protein coded by the gene is shown in SEQ ID NO: 3. The LoAAT1 gene is expressed in lily tissue with fragrance and is not expressed in lily varieties having no fragrance, and the expression s related to a development process of flower. The LoAAT1 gene can be linked to any one plant transformation vector and introduced into cells of lily or other plants, to obtain transgenic fragrance expressing the gene, thus being used for production; in addition, a molecular marker can be generated depending on the gene sequence information, and the molecular marker is used for identifying fragrance genotype of lily or other plants and for marker-assisted selective breeding, thus enhancing selecting efficiency of breeding.

Description

A lily ester floral fragrance gene LoAAT1 and its application

technical field

The invention relates to the technical field of genetic engineering, in particular to a lily ester floral fragrance gene LoAAT1 and its application.

Background technique

The floral fragrance of most plants is formed by a series of low-molecular-weight volatiles mixed together, and its chemical components are mainly terpenes, phenylpropanoic acid compounds/benzene ring compounds and fatty acid derivatives (Pichersky et al , 2006; Dudareva et al , 2004). Ethyl benzoate, a phenylpropane ester component formed through the shikimic acid pathway, is one of the main characteristic aroma components of lily (Fan Yanping, 2008).

Ethyl benzoate is a phenylpropionic acid compound/benzene ring compound, which is a precursor of L-phenylalanine. In plastids, phenylalanine is produced through the shikimate pathway, and cinnamic acid is catalyzed by phenylalanine lyase (PAL). Cinnamic acid is catalyzed by a series of enzymes to synthesize benzaldehyde and benzoic acid. Under the action of benzoic acid coenzyme A ligase (BZL), benzoic acid forms benzoyl CoA (Boatright J. et al , 2004), and finally in alcohol It is produced under the catalysis of acyltransferase (AAT), which is the terminal key enzyme that controls the formation of ethyl benzoate.

Nordstrom (1962) pointed out for the first time that ester synthesis in yeast mainly depends on the action of acyltransferase or ester synthase. So far, three different alcohol acyltransferase genes have been cloned from different yeasts. Flowers and fruits are the main organs for the synthesis of volatile esters in plants. The mature volatile esters constitute the unique aroma of flowers and fruits. This process is also related to alcohol acyltransferase. Ueda and Ogata (1977) found that the synthesis of volatile aroma components of esters in banana cell extract depends on acyl-CoA and acyltransferase. Since then, people have studied the relationship between alcohol acyltransferase and the synthesis of volatile esters in strawberry, apple, pineapple, melon and other fruits. The formation of esters in these fruits is due to the action of alcohol acyltransferase, which transfers the acyl group in the acyl CoAs to the alcohol substrate to form an ester. Perez et al. (1993) studied the AAT activity of four strawberry varieties during fruit ripening, showing that the AAT activity of each variety increased with fruit ripening, but the maximum activity of different varieties varied greatly. Shalit et al. (2001) showed that the composition of aroma volatile components was significantly different between ripe and unripe fruits of the aroma-rich melon cultivar 'Arava'.

In recent years, with the development of biotechnology, several alcohol acyltransferase genes have been cloned in flowers and fruits. The study found that these structurally and functionally similar enzymes are a new category evolved from acyltransferases (EC 2.3.1.x), and all belong to the BAHD gene family of acyltransferases. The earliest alcohol acyltransferase gene isolated in plants came from the flower of Clarkia breweri . BEAT encodes benzyl alcohol acetyltransferase, which regulates the metabolism of benzyl acetate, and belongs to the BAHD family of acyltransferases (Dudareva et al , 1998). D'Auria et al. (2002) obtained the full-length cDNA of BEBT from fairy fan by using expressed sequence tag (EST) library search combined with rapid amplification of cDNA ends (RACE). The gene has great homology with the plant acyltransferase gene sequence, and belongs to the BAHD family of acyltransferases. BEBT was expressed in both flowers and injured leaves, suggesting that benzyl benzoate also plays a role in plant defense mechanisms.

Aharoni et al. (2000) used expressed sequence tag (EST) library search combined with plant volatile component profiles for the first time to isolate a new alcohol acyltransferase gene SAAT related to aroma odor from ripe strawberry fruit using cDNA chip. It has the ability to catalyze the formation of medium-chain fatty acid acetates. Two highly homologous (87%, determined at the protein level) alcohol acyltransferase genes, CM-AAT1 (GenBank CAA94432) and CM-AAT2 (GenBank AF468022), encoding 461 amino acids, were cloned from Charentais melon. The molecular weights are 51.5KD and 51.8 KD, respectively, and the pIs are 8 and 8.5, respectively. Transformed yeast found that CM-AAT1 had the ability to synthesize volatile components of esters, while CM-AAT2 had no alcohol acyltransferase activity. Shalit et al. (2003) searched the rose flower EST database containing 1834 single genes and found 3 possible EST sequences similar to known BAHD alcohol acyltransferase genes from other sources. One of the cDNAs was named RhAAT 1, which encoded a polypeptide of 458 amino acid residues, with a molecular weight of 51.8 KD and a pI of 5.45, belonging to the BAHD acyltransferase family. Through homology comparison, this protein sequence has 69% homology with strawberry SAAT protein. Boatright et al. (2004) used functional genomics to clone the benzyl alcohol/phenyl alcohol benzoyltransferase gene ( BPBT ) in the study of petunias. The amino acid sequence of the gene has high homology with BEBT gene of tobacco and BEAT of fairy fan. Benzyl alcohol/phenylethyl alcohol benzoyltransferase uses benzoyl-CoA as a benzoyl donor and uses benzyl alcohol and phenyl ethanol as precursors to transfer benzoyl to benzyl alcohol and phenyl alcohol to form benzoic acid Benzyl esters and phenethyl benzoate. Li et al. (2006) isolated alcohol acyltransferase genes from Jonagold and Golden Delicious apples by RT-PCR and RACE-PCR, and named them MdAAT 1 and MdAAT 2, respectively. The full lengths are 1,639 bp and 1,628 bp, respectively, and the homology between the two at the amino acid level is 94.26%. Sequence alignment revealed that MdAAT 2 also contains the conserved regions shared by the BAHD gene family and other known alcohol acyltransferase genes: HXXXD and FGWG. Cluster analysis found that the MdAAT2 protein had the closest relationship with the alcohol acyltransferase protein of pear fruit, which belongs to the Rosaceae pome fruit, but had a distant relationship with the alcohol acyltransferase proteins of lemon, strawberry and rose. In addition, Yoshioka and Hashimoto (1981) found that alcohol acyltransferase is located on the cell membrane in yeast research, and carried out preliminary research on the enzymatic characteristics of the enzyme. D'Auria (2002) suggested that most members of the BAHD family are thought to be located in the cytoplasm. Fujiwara et al. (1998) conducted an immunolocalization study on 5-O-glucoside aromatic acyltransferase of Gentiana triflora and found that it was mainly distributed in the cytoplasm of epidermal cells. However, Yu et al. (2008) found that MtMaT1 of the BAHD family in Medicago truncatula ( Medicago truncatula ) was localized in the nucleus.

In recent years, genetic manipulation of secondary biosynthesis of plant floral fragrance has become a hot research area. Guterman et al. (2006) also introduced the AAT cloned from rose into petunia, resulting in the production of benzyl acetate and phenylethyl acetate in the transgenic plants. This provides an effective way for cultivating fragrant flower varieties by genetic engineering technology.

Contents of the invention

At present, domestic studies on alcohol acyltransferase and the formation of floral fragrance are rarely reported, and no report on lily flower fragrance gene was found until this patent application. The cloning of the floral fragrance gene is the premise of the study on the formation mechanism of lily fragrance, revealing the molecular mechanism of lily fragrance can increase the floral fragrance. This provides a brand-new approach for cultivating new varieties of plants with strong floral fragrance by means of genetic engineering. The purpose of the present invention is to isolate and clone a gene controlling ester floral fragrance component ethyl benzoate carried in Lilium siberia lily. the

Another object of the present invention is to provide the protein encoded by the above gene. the

Another object of the present invention is to provide a vector containing the above gene. the

Another object of the present invention is to provide a transgenic plant containing the above vector. the

Another object of the present invention is to provide the application of the above-mentioned gene in producing molecular markers and breeding varieties for flower fragrance, and the application of the protein encoded by the above-mentioned gene in the preparation of essence and medicine. the

The present invention achieves the above-mentioned purpose through the following technical solutions:

A lily ester floral fragrance gene LoAAT1 , the full-length cDNA sequence of the gene is shown in SEQ ID NO:1, and the full-length cDNA sequence is 1380bp in length; the full-length DNA sequence of the gene is shown in SEQ ID NO:2, the full-length The DNA sequence is 1472bp long.

The lily ester floral aroma gene LoAAT1 endows ginger flowers with agarwood and mellow aroma, and the lily ester floral aroma gene LoAAT1 is expressed in scented lily tissues, but not in non-scented lily varieties, and its expression is related to the flower development process. The coding region of the gene is 1380bp (full-length cDNA sequence), and it is presumed that it encodes 459 amino acids, and the protein molecular weight is 50 kDa. In this gene sequence, there are two conserved sequences, HXXXD and DFGWG. The full-length nucleotide sequence of the DNA is 1472bp in length, including 1 intron, located at positions 442-533, with a total of 92bp, and the cut positions all conform to the "GT -AG Law". Ethyl benzoate was produced in vitro by prokaryotic expression and reacted with reaction substrate ethanol and benzoyl-CoA.

A protein encoded by the lily ester floral fragrance gene LoAAT1 , the amino acid sequence of the protein is shown in SEQ ID NO:3.

A pair of primers for amplifying the lily ester floral aroma gene LoAAT1 , the primer sequences are shown in SEQ ID NO: 10-11.

A recombinant vector, inserted into the multi-cloning site of the departure vector, the sequence of the lily ester floral fragrance gene LoAAT1 described in claim 1. The starting vector can be all types of plant expression vectors commonly used in this technical field. Preferably, the plant expression vector used in the present invention is pMOGMON plant expression vector.

A recombinant bacterium comprising the above-mentioned recombinant vector. A cell line comprising a recombinant vector as described above. the

A transgenic plant containing the above-mentioned vector. the

As mentioned above, the application of the lily ester floral aroma gene LoAAT1 is specifically connecting the lily ester floral aroma gene LoAAT1 to a plant transformation vector, and then introducing it into lily or other plant cells to obtain a transgenic floral aroma expressing the lily ester floral aroma gene LoAAT1 Variety.

As mentioned above, the application of the lily ester floral aroma gene LoAAT1 is specifically to generate a specific molecular marker based on the gene, which is used to identify the floral aroma genotype of lily or other plants.

As mentioned above, the application of the protein encoded by the lily ester floral aroma gene LoAAT1 in the preparation of essence and medicine.

The present invention also includes the chimeric gene formed by effectively linking the main structural part of the LoAAT1 floral fragrance gene with a suitable regulatory sequence, as well as the plant containing the gene in the genome and the seeds of the plant. Such genes may be natural or chimeric, for example. For example, a fragment comprising the gene is linked to a constitutively expressed promoter which can be expressed under any condition and at any stage of cell development. Such constitutively expressed promoters include the cauliflower mosaic virus 35S promoter and the like. On the other hand, the gene can also be linked to a tissue-specific expression promoter or a developmental stage-specific expression promoter or a precise environment-induced promoter, these promoters are called inducible promoters. In this way, changes in the environment and different developmental stages can change the expression of the gene. Similarly, the expression of the gene can also be limited to a certain tissue, so that the resistance response induced by the gene can be artificially controlled. The environmental conditions include the attack of diseases and insect pests, high and low temperature and light, etc., and the tissue and developmental stages include leaves, fruits, seeds and flowers.

According to the LoAAT1 gene sequence information provided by the present invention, those skilled in the art can easily obtain the gene equivalent to LoAAT1 by the following methods: (1) obtain by searching the database; (2) use the LoAAT1 gene fragment as a probe to screen ginger flower or other (3) Design oligonucleotide primers according to the LoAAT1 gene sequence information, and use PCR amplification method to obtain from the genome, mRNA and cDNA of ginger flower or other plants; (4) LoAAT1 (5) The gene is obtained by chemical synthesis.

The lily fragrance gene LoAAT1 provided by the invention has important application value. One of the applications is to connect the LoAAT1 gene sequence to any plant transformation vector, and use any transformation method to introduce the LoAAT1 floral fragrance gene into lily or other plant cells, and obtain transgenic floral fragrance expressing the gene, so as to be applied to Production. The gene of the present invention is constructed into a plant transformation vector, the gene or its regulatory sequence can be appropriately modified, and other promoters can be used to replace the original promoter of the gene before its transcription start codon, thereby Broadens and enhances the plant's ability to produce floral fragrance and increase resistance.

Another application of the floral fragrance gene provided by the present invention is to generate specific molecular markers based on the gene sequence information, including but not limited to SNP (single nucleotide polymorphism), SSR (simple sequence repeat polymorphism), RFLP (restricted endonuclease length polymorphism), CAP (cleavage amplified fragment polymorphism). These markers can be used to identify the floral genotypes of lilies or other plants, and can be used in molecular marker-assisted selection breeding, thereby improving the selection efficiency of breeding. the

Compared with the prior art, the present invention has the following beneficial effects:

The invention transfers the cloned floral fragrance gene into the non-floral fragrance plant, which helps to produce new floral fragrance plants. In particular, transformation technology can be used to accumulate multiple floral fragrance genes in plants, without causing the linkage problem of bad genes in the genome that is accompanied by traditional breeding technology, and can shorten the breeding time. The cloning of the floral fragrance gene is the premise to overcome the problem that the floral fragrance gene cannot be transferred between plant species in traditional breeding. In addition, the present invention can further provide or use floral transgenic plants and corresponding seeds obtained using the above-mentioned DNA fragments, as well as plants transformed with the gene of the present invention or a recombinant based on the gene or seeds obtained from such plants. The gene of the present invention can be transferred to other plants by means of sexual crossing.

Instructions attached

Figure 1. The expression of LoAAT1 in lily cultivars with different release amounts of floral fragrance ethyl benzoate; A: release amount of ethyl benzoate, the main component of floral fragrance from lily petals; B: expression level of LoATT1 in lily petals; C: different lily cultivars.

Figure 2. The expression of LoAAT1 in different developmental stages of floral organs and the specificity of expression in different tissues; A: the expression level of LoAAT1 in different organs of Lily Siberia; B: the release of ethyl benzoate in different flower development stages of Lilium Siberia; C: Lilium Siberia Different flowering stages; D: the expression level of LoAAT1 in Lily Siberia petals; E: the expression level of LoAAT1 in calyx of Lily Siberia; F: the expression level of LoAAT1 in stamens of Lily Siberia; G: the expression level of LoAAT1 in pistils of Lily Siberia.

Figure 3. In vitro enzyme-catalyzed reaction of LoAAT1 recombinant protein; A: Chromatogram and mass spectrogram of pET-28a- LoAAT1 recombinant protein in vitro enzyme-catalyzed reaction product; B: Chromatogram and mass spectrometry of ethyl benzoate standard sample.

Figure 4. PCR detection chart of LoAAT1 transgenic tobacco plants.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents and methods used in the examples are those routinely used in the art. the

Example 1 Acquisition of LoAAT1 gene cDNA and full-length DNA:

S1. Extraction of lily petal RNA: Weigh 0.2 g of lily petals in full bloom, add liquid nitrogen and quickly grind them into powder, and quickly transfer to 0.5 mL 2 % CTAB (containing 0.1% mercaptoethanol) extract stored at 4 °C. In a 2 mL centrifuge tube, shake until thoroughly mixed; place at room temperature for 5 minutes, lay the centrifuge tube flat to maximize the surface area; centrifuge at 12,000 rpm at 4°C for 1 min, and transfer the supernatant to a new RNase-free centrifuge tube. Add 0.1mL 5M NaCl, mix gently; add 0.3mL chloroform, mix upside down; centrifuge at 12000rpm at 4°C for 10min, transfer the upper aqueous phase to a new RNase-free centrifuge tube. Add pre-cooled isopropanol equal to the volume of the obtained water, invert and mix well, place at room temperature for 10 minutes, place the centrifuge tube horizontally to maximize the surface area; centrifuge at 12000 rpm at 4°C for 10 minutes, discard the supernatant, be careful not to pour out the precipitate, add Wash the RNA pellet with 1 mL of 75% ethanol (prepared with DEPC water), invert and mix well; centrifuge at 5000 rpm at 4°C for 3 minutes, pour out the liquid, and be careful not to pour out the pellet. The remaining small amount of liquid was briefly centrifuged, then sucked out with a pipette tip, and dried at room temperature for 2-3 minutes; 30 μL DEPC H ₂ O was added, and the RNA was fully dissolved by pipetting and mixing repeatedly. Store at -80°C for later use.

S2. The first-strand cDNA was synthesized with Sangon's M-MuLV First cDNA Synthesis Kit using the total petal RNA of lily at the blooming stage as a template. Primers were designed according to the relevant gene sequences reported in the GenBank nucleic acid database, upstream primer F1: TCTCCAAGGCTCTGGTGTTCTA(T/C)TA(T/C)CC(A/C/G/T)(G/T/C)T; such as SEQ ID NO:4 shown. Downstream primer R1: TGCATGAACCTGATAGCGAAG(A/G)(T/C)(A/G)AA(A/C/G/T)CC(A/C/G/T)CC; as shown in SEQ ID NO:5 . And it was synthesized by Shanghai Bioengineering Company. Using the cDNA synthesized above as a template, PCR amplification reaction was carried out using TaKaRa PCR Amplification Kit, and the specific method was carried out according to the instructions. The PCR program was: 94°C pre-denaturation for 4 minutes; 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min; then extension at 72°C for 10 min. Store at -20°C for later use. After the PCR reaction, 1.0% agarose gel electrophoresis was used to preliminarily detect whether the PCR product contained the target fragment band. After the PCR amplification product was detected by 1% agarose gel electrophoresis, the gel block containing the target fragment was cut out with a scalpel under ultraviolet light, and the DNA gel recovery kit (Agarose Gel DNA Purification Kit, TaKaRa) was used for recovery. The method basically refers to the kit instructions. Afterwards, 1% agarose electrophoresis should be carried out on the recovered product to see its recovery effect and approximate concentration, so as to ensure the subsequent test. According to the size of the recovered target fragment and its effective concentration, take an appropriate amount of the recovered and purified product and connect it to the cloning carrier. The carrier is the TaKaRa pMD18-T carrier. The molar ratio of the target DNA to the cloning carrier is controlled at about 3:1. The specific operation is carried out according to the instructions. . Connect at a constant temperature of 16°C for 3-6 hours, and the length of the connection time depends on the length of the target fragment. Take the competent cell DH5α (TaKaRa) out of the -80°C refrigerator in advance, and put it in the ice box until it melts naturally, add 10 μL of the connection solution to the centrifuge tube of the competent cell, and after ice bathing for 30 minutes, heat it in a 42°C water bath. Stimulate for 50 s, quickly place on ice for 2-5 min, then add 890 μL of SOC liquid medium pre-warmed at 37 °C to make up to 1 mL, mix well, and shake at 180 rpm at 37 °C for 1 h. Apply 30 μL of X-gal (20 mg/mL) and 30 μL of IPTG (20 mg/mL) on the surface of LB solid medium plate containing 100 μg/mL ampicillin, then apply an appropriate amount of transformation solution, and invert after the transformation solution is completely absorbed Cultivate overnight in a 37°C incubator, observe the results after about 16 hours, screen the white colonies by X-gal/IPTG blue and white spots, and initially identify the recombinant plasmid, and store the plate at 4°C. After passing the preliminary screening of blue and white spots, usually 6 plaques were selected and shaken to extract plasmids for further identification. Use a sterilized toothpick to pick a white single colony from the LB plate culture medium and inoculate it in the LB liquid medium containing 100 μg/mL ampicillin, and culture it overnight on a temperature-controlled shaking water bath shaker at 37°C and 240 rpm. The extraction kit (Shanghai Boya Biological Co., Ltd.) was used to extract the plasmid, and the steps were carried out according to the method in the manual. The extracted plasmids were detected by 1.0% agarose gel electrophoresis, the sizes of the plasmids were compared, and the plasmids that were obviously lagging behind were analyzed by double enzyme digestion ( EcoR Ⅰ/ Hind Ⅲ, TaKaRa). After digestion at 37°C for 1 h, the digested products were detected by 1.0% agarose gel electrophoresis. Recombinant plasmids containing target fragments were randomly selected and identified for DNA sequence determination. The sequencing work was completed by Shanghai Yingjun Biotechnology Co., Ltd., using the American ABI377 sequence analyzer. The resulting sequences were aligned and homology analyzed at NCBI.

According to the obtained cDNA fragment sequence, use the biological software Primer Premier 5.0 to design two specific primers near the 3′ end of the fragment sequence, 3′-RACE 1st Primer: GAGTTGCTCTTCGACGTTGAG; as shown in SEQ ID NO: 6; 3′-RACE 2nd Primer: CGCTGATCCTAATTCAGGTGACTC; as shown in SEQ ID NO:7. After being analyzed by biological software Oligo 6.0, it was synthesized by Shanghai Bioengineering Company. The first strand of cDNA was synthesized by reverse transcription with the Oligo(dT)-3' end anchor primer as a primer. The first strand of the synthesized cDNA was used as a template, and the 3' end PCR was amplified with the 3' primary primer and the 3' end anchor primer. The PCR program is: pre-denaturation at 94°C for 3 minutes; 30 cycles of denaturation at 94°C for 30 s, renaturation for 30 s (depending on the temperature), extension at 72°C for 1 min, and a total extension at 72°C for 10 min. The first PCR reaction solution was diluted 10-100 times as a template, and the second PCR reaction was carried out with 3' double nested primers. After the PCR reaction, 1.0% agarose gel electrophoresis was used to preliminarily detect whether the PCR product contained the target fragment band. After recovery and detection, the recombinant plasmids containing the target fragment after enzyme digestion and identification were randomly selected for DNA sequence determination. The sequencing work was completed by Shanghai Yingjun Biotechnology Co., Ltd., using the American ABI377 sequence analyzer. The sequencing result and the cDNA fragment sequence were spliced and analyzed to determine whether it was the 3' extension of the cDNA fragment sequence. the

Also use the known cDNA fragment sequence to design a pair of primers near its 5' end, 5'-RACE 1st Primer: CGAGCCGAACATTGGCATCAG; as shown in SEQ ID NO: 8; 3'-RACE 2nd Primer: TAGAACACCAGAGCCTTGGA; Shown in SEQ ID NO:9. The method of adding a linker at the 5' end is used. After recovery and detection, the recombinant plasmid containing the target fragment after identification by enzyme digestion is randomly selected for DNA sequence determination. The sequencing work was completed by Shanghai Yingjun Biotechnology Co., Ltd. The sequencing results and the sequence of the cDNA fragment were spliced and analyzed to determine whether it was a 5′ extension of the cDNA fragment sequence. the

After performing Blast analysis on the obtained fragment sequences, 3′ and 5′ sequences, the three fragments were compared and spliced using the SeqMan program of the DNAstar software package, and the spliced sequence was the full-length cDNA sequence of the terpene synthase gene. The full-length cDNA sequence obtained by splicing was compared and analyzed for homology of nucleotide sequence and deduced protein sequence on NCBI. the

Obtaining the full-length sequence of gene cDNA:

According to the obtained full-length gene cDNA, design and synthesize upstream and downstream primers, Hctps1-P1: ATGGCATCATCCCTCACTTTCTC; as shown in SEQ ID NO:10. Hctps1-P2: CTA GAGGGCAGAAGCAATAAAC; shown in SEQ ID NO:11. Using lily cDNA as a template, the PCR amplification reaction was carried out using TaKaRa PCR Amplification Kit. The reaction conditions are: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 s, renaturation for 30 s (depending on the temperature), extension at 72°C for 3 min, 35 cycles; and a total extension at 72°C for 10 min. Take 5 μL of PCR products for identification by electrophoresis in agarose gel containing 0.8% ethidium bromide, and observe the experimental results under ultraviolet light.

Using Agarose Gel DNA Purification Kit Ver. 2.0 (TaKaRa), according to the operating instructions, the PCR product was purified and recovered, and the recovered product was dissolved in 25 μL Lution Buffer. Take 4 μL of purified recovered product, 1 μL of pMD20-T Vector, and 5 μL of linker Solution I to form a 10 μL connection system, and connect overnight at 16°C. Transform Escherichia coli strain DH5α competent cells, spread on LB solid medium containing X-gal, IPTG, and ampicillin, and culture overnight at 37°C; then pick 20 white clonal spots and put them in LB liquid medium containing ampicillin , 37°C, 240rpm expanded culture for 16-24h; use the plasmid DNA mini-prep kit (Shanghai Boya Biological Co., Ltd.) to extract the plasmid, compare the size of the plasmid by agarose gel electrophoresis, and carry out enzyme digestion identification of the obviously lagging plasmid. Select randomly from the identified positive recombinant plasmids and send them to Shanghai Yingjun Biotechnology Co., Ltd. for sequence determination. The full-length cDNA sequence and full-length DNA sequence of LoAAT1 gene were obtained, and its protein sequence was deduced according to the cDNA sequence.

Example 2 Expression analysis of LoAAT1 gene:

Trizol method (TaKaRa) was used for RNA extraction of lily petals of different varieties, petals of different developmental stages of Siberia lily and different tissue parts of Siberia lily, and SYBR green (TaRaKa) method for fluorescence quantitative PCR. The specific principle of dye method is shown in the instruction manual. Using Primer Premier 5.0 software to design real-time fluorescent quantitative PCR primers, according to the principles of fluorescent quantitative PCR primer design, use Primer premier 5.0 to design primers respectively, and detect whether there are mismatches or primer dimers and their amplification efficiency by fluorescent quantitative PCR. Select a pair of optimal primers, P1: ATTGTGGTGCCAGTTTGCTTGC; as shown in SEQ ID NO:12. P2: CCCTTTTACCCTTCGTTGAACTTC; shown in SEQ ID NO:13. The internal reference gene ACT was designed with Primer premier 5.0 according to the design principles of Real-time PCR primers, ACT-P1: TTCGTGTTGCACCAGAAGAGC, as shown in SEQ ID NO:14, ACT-P2: TGAATGGCGACATACATGGCAG; as shown in SEQ ID NO:15. Detect by Real-time PCR, and make a standard curve to test whether the amplification efficiency (E) is within the range of 90-110%. Using the cDNA of each sample as a template, a fluorescent quantitative PCR reaction was performed on an ABI fluorescent quantitative PCR instrument. Three replicates were set up for each sample, and ddH ₂ O was used as a negative control. The reaction system was SYBR Premix Ex Taq (TaKa Ra) 10.0 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, cDNA 2.0 μL, ddH ₂ O 7.2 μL. The reaction program was 94°C, 30 s; 94°C, 15 s; 55°C, 30 s; 72°C, 30 min; 40 cycles, 94°C 15 s, 72°C 30 s, 0.4°C/s melting curve analysis. After the reaction, confirm the amplification curve and melting curve, and use the 2- ^△△Ct method (2(-Delta Delta C(T)), Livak and Schmittgen, 2001) for data analysis, and calculate the expression of Lily LoAAT1 in different samples . The results of gene expression analysis are shown in Figure 1 and Figure 2. It can be seen from Figure 1 and Figure 2 that:

LoAAT1 is only expressed in the petals of lily cultivars that release the main component of floral fragrance, ethyl benzoate, but not in the cultivars that do not release the modified component, and the expression level is significantly positively correlated with the release of floral fragrance; LoAAT1 is only expressed in the organs of lily , is not expressed in vegetative organs such as roots, stems and leaves. The expression of this gene is significantly positively correlated with different flowering stages, and the amount of ethyl benzoate released is the highest in flower petals. expression, indicating that LoAAT1 is the key gene controlling the synthesis and release of ethyl benzoate, the main component of lily fragrance.

Example 3 Hctps1 gene prokaryotic expression:

S1. According to the obtained full-length cDNA of LoAAT1 gene, perform PCR amplification with specific primers containing Bam HI and Not Ⅰ restriction sites. The PCR product was recovered with the Takara recovery kit, and the recovered product was directly digested with BamHI and NotI restriction endonucleases, and the target fragment was recovered on 1% agarose gel. The pET-28a prokaryotic expression vector was double-digested with BamHI and NotI restriction enzymes to recover large fragments on 1% agarose gel. After ligation at 16°C overnight, the ligation product was transformed into Escherichia coli ( E. coli ) DH5α competent cells; the recombinant prokaryotic expression vector was obtained after the extracted plasmid was identified by enzyme digestion and sequencing.

S2. Transform Escherichia coli (Rosetta) competent cells with the identified recombinant plasmid DNA, pick a single colony and inoculate it in 5 mL LB (containing 25 mg/L Kan, 34 mg/L Chl) medium, and culture overnight at 37°C with shaking . Transfer 100 μL seed solution to fresh 100ml (containing 25 mg/L Kan, 34 mg/L Chl) LB medium, culture at 180 rpm at 37°C for 4-6 hours, measure the OD value to 0.4-0.6 and use a certain amount of IPTG (0.1-0.2mM) was induced at 14-18°C for 14-16h. At the same time, another control group was taken which was not induced by IPTG. Collect the cells by centrifugation, suspend the cells with 5ml lysis buffer (50mM phosphoric acid buffer pH 8.0), cool the cells, and place them on ice to sonicate the cells. Centrifuge at 12,000 rpm at 4°C for 10 min, transfer the supernatant to a new centrifuge tube, wash the precipitate once with double distilled water, and then suspend the precipitate with 5 mL of lysis buffer. Take 50 μL of supernatant and precipitate respectively, store at -20°C, and conduct SDS-PAGE electrophoresis analysis. Prepare 12.5% SDS polyacrylamide gel and load samples in order. Electrophoresis was performed on the stacking gel and the separating gel with voltages of 60V and 120V, respectively. After electrophoresis, stain with Coomassie Brilliant Blue for 30 minutes, then decolorize with decolorizing solution for 1-8 hours, observe and record the test results. the

S3. Purification of protein: Pick a single colony and inoculate it in 5ml of liquid LB medium, cultivate overnight at 37°C, 180rpm, then transfer all of them to 500mL of fresh liquid LB culture medium, cultivate for 4h at 37°C, 180rpm, and use After induction with IPTG (0.1-0.2 mM) at 18°C for 16 hours, the cells were collected. Take 200 μL of bacteria in a centrifuge tube, store at 4°C, and perform SDS-PAGE electrophoresis analysis. Suspend the cells with 5 mL of lysis buffer and transfer to a centrifuge tube, place on ice to keep the cells cool, and disrupt the cells by ultrasonic. Centrifuge at 10,000 x g for 20-30 min at 4°C, and collect the supernatant. Take 20 μL of the supernatant and store it at -20°C for SDS-PAGE analysis. Add 1-2 mL of Ni-NTA resin (nickel-nitrilotriacetic acid resin filler) to 5 mL of cell lysate, mix well, and combine on a shaker at 4 °C for 60 min at low speed. Put the fully combined cell lysate and resin mixture into the chromatography column, remove the bottom cap to collect the effluent fraction (flow-through fraction, F), take 20 μL of the effluent, store it at -20°C, and perform SDS-PAGE analyze. Use 4ml of washing buffer to elute the chromatography column twice, collect each fraction of the eluate (wash fraction, W), and take 20 μL each and store it in a -20°C refrigerator for SDS-PAGE analysis. Wash the column four times with 0.5mL of elution buffer, collect each part of the eluent (elution fraction, E) sequentially with different collection tubes, respectively marked as E1, E2, E3, E4, and take 20 μL each for SDS- PAGE analysis. According to the detection results of SDS-PAGE, concentrate the eluted fraction containing the target protein into a centrifuge tube, transfer it to a dialysis bag with a pipette, and dialyze overnight at 4°C. Collect the solution after dialysis, add glycerol to make the total glycerol content in the protein solution 20%, and divide it into 200 μL/tube, take a small amount for SDS-PAGE protein concentration detection, and store the rest in a -70°C ultra-low temperature refrigerator Save for backup. the

S4. Identification of alcohol acyl synthetase enzyme activity: Transfer 100 μL of seed solution to fresh 200 mL (containing 25 mg/L Kan, 34 mg/L Chl) LB medium, culture at 37°C and 180 rpm for 4-6 hours, and measure OD After the value reaches 0.4-0.6, a certain amount of IPTG (0.1-0.2mM) is used to induce at 14-18°C for 14-16h. Centrifuge at 5000rpm at 4°C for 5min to collect the cells, suspend the cells with 5mL buffer (10mM MOPS buffer pH 7.0 10% glycerol 1mM DTT), and place on ice to sonicate the cells. Centrifuge at 12000rpm at 4°C for 20min, and transfer the supernatant to a new centrifuge tube. the

S5. Enzymatic reaction and GC-MS analysis: Mix 80 μL of 50 μM Tris-HCl pH 8.0, 50 μL of enzyme extract, 20 μL of 0.05 μM benzoyl-CoA solution, 5.8 μL of 20 μM ethanol solution, 2 μL of mercaptoethanol solution and ddH ₂ O 842.2 μL was added to the sample bottle and sealed, and the 75 μm polydimethyloxane (PMDS) extraction fiber head was inserted into the glass bottle, and the 28 ° C water bath was performed for 1 hour, and the headspace solid phase microextraction was performed. After the reaction, the extraction fiber head was placed in the gas phase Chromatography-mass spectrometry was used for analysis, and the gas chromatography conditions were as follows: the chromatographic column was HP-1NNOWAX column (30m×0.25mm); the carrier gas was high-purity helium, the split ratio was 20:1, the pre-column pressure was 50 Pa, and the flow rate was 1 mL /min; sampling time 2min; temperature program: the initial column temperature is 45°C, keep for 2min, increase to 80°C at a speed of 5°C/min, hold for 1min, then rise to 250°C at a speed of 10°C/min, and hold for 5 minutes . The mass spectrometry conditions are: GC-MS interface temperature 220 °C, electron bombardment source EI, 350V; ion source temperature 170 °C; electron energy 70 eV; scanning mass range 35-335 aum, and the collected mass spectra were analyzed with the WILLEY/MAINLIB library . The prokaryotic expression results are shown in Figure 3. As can be seen from Figure 3: the pET-28a-Lo-AAT1 in vitro enzyme-catalyzed reaction product with ethanol and benzoyl-CoA as the substrate was identified as ethyl benzoate by mass spectrometry, and was compared with ethyl benzoate standard sample chromatography and The mass spectrograms are consistent, indicating that the gene encoded by the gene is an enzyme that synthesizes ethyl benzoate with ethanol and benzoyl-CoA as substrates, and the gene is an ethanol benzoyltransferase gene.

Embodiment 4 LoAAT1 genetic transformation of tobacco:

S1. Vector construction and Agrobacterium transformation: the full-length cDNA sequence of the LoAAT1 gene was digested with Eco RI and Bam HI, and the target fragment was recovered; the vector pMOGMON was digested with Eco RI and Bam HI, purified and recovered fragments of corresponding size, ligated, Transform DH5α, extract the plasmid, identify by enzyme digestion, pick out the desired clone, sequence it, and transform it into Agrobacterium LBA4404.

S2. Tobacco transformation: Pre-cultivate tobacco “W38” leaves (0.5 cm×0.5 cm square) in the culture medium for 2-3 days, use the Agrobacterium LBA4404 bacterial solution with OD ₆₀₀ of 0.5-0.8 obtained in step S1, and dip for 5 ~10min, and then co-cultivated for 3 days. Tobacco was on a medium containing 20 mg/L hygromycin Hyg and 400 mg/L cephalosporin Cef, and petunia was on a medium containing 5 mg/L hygromycin Hyg and 400 mg /L cephalosporin Cef culture medium, under the conditions of 2000-10000Lx light and 25°C for selection culture. After 2 to 3 weeks of selective culture, the transformed cells of the explants will produce resistant callus, and the material is transferred to the corresponding selective differentiation medium for differentiation and budding culture. When the adventitious bud grows to more than 1 cm, cut off And insert it into the rooting medium containing selective pressure for rooting culture, insert 2 to 3 seedlings in each bottle, and carry out rooting induction. The roots grow to about 4-5 cm and the seedlings are strong and strong, and the seedlings are hardened for 5-6 days. After the hardening is completed, the root medium is washed with clean water, and then transplanted.

S3. Extraction of genetically modified tobacco genomic DNA:

S31. Take 0.2 g of material, add liquid nitrogen and grind it into powder, transfer the ground material into a 2 ml centrifuge tube, add 1 ml of preheated 2 % CTAB extract (containing 0.1% mercaptoethanol), and mix well , placed in a 65°C water bath for 45 minutes, and mixed once every 6-8 minutes. Centrifuge at 10000 rpm for 5 min.

S32. Cool to room temperature, take the supernatant, add an equal volume of chloroform/isoamyl alcohol (24:1), mix gently until the solution becomes emulsified, and centrifuge at 10,000 rpm for 5 min. the

S33. Take the supernatant, add 2/3 volume of pre-cooled isopropanol, and place it at -20°C for 30 minutes. Centrifuge at 10,000 rpm for 5 min, discard the supernatant, and wash the precipitate with 70% ethanol for 2 to 3 times. Add 200 μl TE solution to dissolve. the

S34. Add 1/10 volume of NaAC (3 mol/L) and 2 volumes of absolute ethanol, and place at -20°C for more than 2 hours. Centrifuge at 12000 rpm for 5 min, discard the supernatant, wash the precipitate with 70% ethanol for 2 to 3 times, and dry the precipitate at room temperature. Dissolve with 20-50 μL 1×TE and store at -20°C. the

S4. PCR detection of transgenic tobacco: The transformed bacterial liquid was used as a positive control, and the untransformed DNA was used as a negative control, and the PCR amplification system was carried out.

Upstream primer F2: CTT CTA CAC AGC CAT CGG TCC AGA; as shown in SEQ ID NO:16. the

Downstream primer R2: GAT GTA GGA GGG CGT GGA TAT GTC; as shown in SEQ ID NO:17. the

Reaction system (20μL): transgenic plant DNA 1μL (20ng~50ng); 10×buffer 2μL; MgCl ₂ (2.5mM) 2μL; Taq enzyme 0.2μL; dNTP (2.5mM) 2μL; to 25 μL.

Reaction conditions: 94°C, 4 minutes; 94°C, 30 seconds; 54°C, 30 seconds; 72°C, 1 minute; 30 cycles; 72°C extension for 10 minutes. PCR-positive plants were screened out (Figure 4). the

S5. SPME-GC/MS analysis of volatile substances in transgenic tobacco plants: analysis by solid-phase microextraction-gas chromatography-mass spectrometry. Tobacco seedlings were taken out from the substrate, washed, and placed in a 2.5 L sealed box, and 10 μL of 31.6 ng/μL (100 μM SNP treatment was 316 ng/μL) ethyl caprate was added as an internal standard, and polytetrafluoroethylene Fluorine-lined silicone rubber gasket was sealed, and a 100 μm polydimethylsiloxane (PMDS) extraction fiber head was inserted, and headspace sampling was carried out at 25 °C for 30 min. Using FINNIGAN TRACE MS mass spectrometer (USA) for analysis. The chromatographic conditions were a DB-5 quartz capillary column with a length of 30 m, an inner diameter of 0.25 mm, a liquid film thickness of 0.25 μm, a carrier gas of He, a column head pressure of 68.974 kPa, and a programmed split/splitless (LSS) inlet temperature of 250 °C. Programmed temperature rise: 50°C, keep for 2 min; increase to 250°C at a rate of 3°C/min, keep for 30 min. In the SPME analysis, the PSS inlet was set as splitless injection, the splitless time was 2 min, the liner was a glass tube with an inner diameter of 1.5 mm, and the desorption time was 3 min; in the DHS inlet, the split ratio was 20:1, the injection volume is 2.0 μL. The temperature of the GC/MS transmission line is 250°C, the mass scanning range is 30-350 aμm, the scan time is 0.3 s, the scan interval is 0.2 s, the EI ion source temperature is 170°C, the EI electron energy is 70 eV, and the photomultiplier tube (PMT) voltage is 230 V , the collected mass spectra were analyzed with WILEY, MAINLIB, REPLIB and NISTDEMO, and the chemical components of tobacco volatile substances were determined according to the ratio of the peak area of each component to the peak area of the internal standard ethyl caprate. The results showed that the main volatiles in wild-type tobacco were leaf alcohol acetate and leaf alcohol, the contents were 0.241 μg/gFW.h and 0.263 μg/gFW.h; Esters and methyl 2-hydroxybenzoate, their volatile amounts are 0.020 μg/gFW.h, 0.349 μg/gFW.h and 0.044 μg/gFW.h, respectively, and ethyl benzoate is the main volatile component. This shows that positive expression of alcohol acyltransferase gene LoAAT1 in tobacco changes the composition of volatile components in tobacco leaves, and the volatilization of ethyl benzoate is greatly increased.

SEQUENCE LISTING

the

<110> South China Agricultural University

the

<120> A lily ester floral fragrance gene LoAAT1 and its application

the

<130>

the

<160> 17

the

<170> PatentIn version 3.3

the

<210> 1

<211> 1380

<212> DNA

<213> LoAAT1 gene full-length cDNA sequence

the

<400> 1

atggcatcat ccctcacttt ctctgtccaa agccgccagc ccgagctcat cgcaccggca 60

the

gccccgaccc cccacgagct caagcgcctc tccgacattg acgaccaaga gggtctccgg 120

the

ttccagatac cggtcatcca attctaccgc cacgaccctt tactaggcgg tgcaagagac 180

the

cctgtgatgg ttatccgaga ggcacttgct aaggctctgg tgttttacta ccccttggcc 240

the

ggccgcctca gggaggggggc tgaccgcaag ctcgcggtgg agtgcactgg tgagggtgtt 300

the

gttttcatcg aggctgatgc caatgttcgg ctcgaacagt ttggtgatgc tctgcagccg 360

the

cctttcccat gcctggagga gttgctcttc gacgttgagg gctccagcgg gatactcggc 420

the

tgcccgctga tcctaattca ggtgactcgc ttgagttgcg gtggcttcat ctttgccctc 480

the

cgtctcaacc accacatctc ggacgctgcc ggcctcgtac agttcatgac cgccgtcggc 540

the

gagctcgccc gtggcgcaat cgcaccaaca gtccaaccag tctgggcgcg ccacctcctc 600

the

gaggctcgct ccccgccctg ccccaccttc gcccatcgcg agtacgatgt catccctgat 660

the

acaaagaaca ccctcatccc cttggacgac atggtccacc gctcattctt tttcggtcgc 720

the

cgcgagatcg ctgccttgcg tcgccgtgtc ccccaccacc tccgcagctc ctccaccttt 780

the

gagatcctca ccgcctccct ctggaagtgt cgcacatcct ccatccaagt cgacccatac 840

the

gaagaggtcc gcatcatctg cattgtcaac tcacgcggga agttcaaccc accgctgccg 900

the

acagggtact atgggaatgc tttagcatta cccgtggccg tggcggaggc aggaaaggtg 960

the

gctaacaacc ctcttgggta cgcattggag ttggtgaaga aagcgaaggc agaggtgact 1020

the

gaggagtata tgaaatcggt tgctgacttg atggtaatca ggggacgacc tcacttcacg 1080

the

gcggtgagaa cttacttggt ttcggacttg aaaagggcgg ggttcaggga tgtggatttc 1140

the

ggatggggaa aggcagtata tggtggagcc gcgaagggag gggttggggt gattcccggg 1200

the

gtcataagct tctttgtacc attcaagaat aggaatggag aggatgggat tgtggtgcca 1260

the

gtttgcttgc caggtccagc aatggagaag tttttggtgg agattgaaag gctcacgaag 1320

the

gtgtccgaga tggaggaaga gcatggcacg aacgcgcagt ttattgcttc tgccctctag 1380

the

the

<210> 2

<211> 1472

<212> DNA

<213> Full-length DNA sequence of LoAAT1 gene

the

<400> 2

atggcatcat ccctcacttt ctctgtccaa aggcgccagc ccgagctcat cgcaccggca 60

the

gccccgaccc cccacgagct caagcgcctc tccgacattg acgaccaaga gggtctccgg 120

the

ttccagatac cggtcatcca attctaccgc cacgaccctt tactaggcgg tgcaagagac 180

the

cctgtgatgg ttatccgaga ggcacttgct aaggctctgg tgttttacta ccccttggcc 240

the

ggccgcctca gggaggggggc tgaccgcaag ctcgcggtgg agtgcactgg tgagggtgtt 300

the

gttttcatcg aggctgatgc caatgttcgg ctcgaacagt ttggtgatgc tctgcagccg 360

the

cctttcccat tactggagga gttgctcttc gacgttgagg gctccagcgg gatactcggc 420

the

tgcccgctga tcctaattca ggtacctacc agattattt accattgtga acacactgga 480

the

tcttcatata ccatctacag tacgctaaaa actcaagcct tgttgttatg taggtgactc 540

the

gcttgagttg cggtggcttc atctttgccc tccgtctcaa ccacaccatc tcggacgctg 600

the

ccggcctcgt acagttcatg acagccgtcg gcgagctcgc ccgtggcgca atcgcaccaa 660

the

cagtccaacc agtctgggcg cgccacctcc tcgaggctcg ctccccgccc tgccccacct 720

the

tcgcccatcg cgagtacgaa gtcatccctg atacaaagaa caccctcatc cccttggacg 780

the

acatggtcca ccgctcattc tttttcggtc gccgcgagat cgctgccttg cgtcgccgtg 840

the

tcccccacca cctccgcagc tcctccacct ttgagatcct caccgcctcc ctctggaagt 900

the

gtcgcacatc ctccatccaa gtcgacccat acgaagaggt ccgcatcatc tgcattgtca 960

the

actcacgcgg gaagttcaac ccaccgctgc cgacagggta ctatgggaat gctttagcat 1020

the

tacccgtggc cgtggcggag gcaggaaagg tggctaacaa ccctcttggg tacgcattgg 1080

the

agttggtgaa gaaagcgaag gcagaggtga cggaggagta tatgaaatcg gttgctgact 1140

the

tgatggtaat caggggacga cctcacttca cggcggtgag aacttacttg gtttcggact 1200

the

tgaaaagggc ggggttcagg gatgtggatt tcggatgggg aaaggcagta tatggtggag 1260

the

ccgcgaaggg aggggttggg gtgattcccg gggtcataag cttctttgta ccattcaaga 1320

the

ataggaatgg agaggatggg attgtggtgc cagtttgctt gccaggtcca gcaatggaga 1380

the

agtttttggt ggagattgaa aggctcacga aggtgtccga gatggaggaa gagcatggca 1440

the

cgaacgcgca gtttatgct tctgccctct ag 1472

the

the

<210> 3

<211> 459

<212> PRT

<213> Protein sequence encoded by LoAAT1

the

<400> 3

the

Met Ala Ser Ser Leu Thr Phe Ser Val Gln Ser Arg Gln Pro Glu Leu

1 5 10 15

the

the

Ile Ala Pro Ala Ala Pro Thr Pro His Glu Leu Lys Arg Leu Ser Asp

20 25 30

the

the

Ile Asp Asp Gln Glu Gly Leu Arg Phe Gln Ile Pro Val Ile Gln Phe

35 40 45 45

the

the

Tyr Arg His Asp Pro Leu Leu Gly Gly Ala Arg Asp Pro Val Met Val

50 55 60 60

the

the

Ile Arg Glu Ala Leu Ala Lys Ala Leu Val Phe Tyr Tyr Pro Leu Ala

65 70 75 80

the

the

Gly Arg Leu Arg Glu Gly Ala Asp Arg Lys Leu Ala Val Glu Cys Thr

85 90 95

the

the

Gly Glu Gly Val Val Phe Ile Glu Ala Asp Ala Asn Val Arg Leu Glu

100 105 110

the

the

Gln Phe Gly Asp Ala Leu Gln Pro Pro Phe Pro Cys Leu Glu Glu Leu

115 120 125

the

the

Leu Phe Asp Val Glu Gly Ser Ser Ser Gly Ile Leu Gly Cys Pro Leu Ile

130 135 140

the

the

Leu Ile Gln Val Thr Arg Leu Ser Cys Gly Gly Phe Ile Phe Ala Leu

145 150 155 160

the

the

Arg Leu Asn His Thr Ile Ser Asp Ala Ala Gly Leu Val Gln Phe Met

165 170 175

the

the

Thr Ala Val Gly Glu Leu Ala Arg Gly Ala Ile Ala Pro Thr Val Gln

180 185 190

the

the

Pro Val Trp Ala Arg His Leu Leu Glu Ala Arg Ser Pro Pro Cys Pro

195 200 205

the

the

Thr Phe Ala His Arg Glu Tyr Asp Val Ile Pro Asp Thr Lys Asn Thr

210 215 220

the

the

Leu Ile Pro Leu Asp Asp Met Val His Arg Ser Phe Phe Phe Gly Arg

225 230 235 240

the

the

Arg Glu Ile Ala Ala Leu Arg Arg Arg Val Pro His His Leu Arg Ser

245 250 255

the

the

Ser Ser Thr Phe Glu Ile Leu Thr Ala Ser Leu Trp Lys Cys Arg Thr

260 265 270

the

the

Ser Ser Ile Gln Val Asp Pro Tyr Glu Glu Val Arg Ile Ile Cys Ile

275 280 285

the

the

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

290 295 300

the

the

Gly Asn Ala Leu Ala Leu Pro Val Ala Val Ala Glu Ala Gly Lys Val

305 310 315 320

the

the

Ala Asn Asn Pro Leu Gly Tyr Ala Leu Glu Leu Val Lys Lys Ala Lys

325 330 335

the

the

Ala Glu Val Thr Glu Glu Tyr Met Lys Ser Val Ala Asp Leu Met Val

340 345 350

the

the

Ile Arg Gly Arg Pro His Phe Thr Ala Val Arg Thr Tyr Leu Val Ser

355 360 365

the

the

Asp Leu Lys Arg Ala Gly Phe Arg Asp Val Asp Phe Gly Trp Gly Lys

370 375 380

the

the

Ala Val Tyr Gly Gly Ala Ala Lys Gly Gly Val Gly Val Ile Pro Gly

385 390 395 400

the

the

Val Ile Ser Phe Phe Val Pro Phe Lys Asn Arg Asn Gly Glu Asp Gly

405 410 415

the

the

Ile Val Val Pro Val Cys Leu Pro Gly Pro Ala Met Glu Lys Phe Leu

420 425 430

the

the

Val Glu Ile Glu Arg Leu Thr Lys Val Ser Glu Met Glu Glu Glu His

435 440 445

the

the

Gly Thr Asn Ala Gln Phe Ile Ala Ser Ala Leu

450 455

the

the

<210> 4

<211> 31

<212> DNA

<213> Upstream primer F1

the

the

<220>

<221> misc_feature

<222> (29)..(29)

<223> n is a, c, g, or t

the

<400> 4

tctccaaggc tctggtgttc taytayccnb t 31

the

the

<210> 5

<211> 32

<212> DNA

<213> downstream primer R1

the

the

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

the

<220>

<221> misc_feature

<222> (30)..(30)

<223> n is a, c, g, or t

the

<400> 5

tgcatgaacc tgatagcgaa gryraanccn cc 32

the

the

<210> 6

<211> 21

<212> DNA

<213> 3′-RACE 1st Primer

the

<400> 6

gagttgctct tcgacgttga g 21

the

the

<210> 7

<211> 24

<212> DNA

<213> 3′-RACE 2nd Primer

the

<400> 7

cgctgatcct aattcaggtg actc 24

the

the

<210> 8

<211> 21

<212> DNA

<213> 5′-RACE 1st Primer

the

<400> 8

cgagccgaac attggcatca g 21

the

the

<210> 9

<211> 20

<212> DNA

<213> 3′-RACE 2nd Primer

the

<400> 9

tagaacacca gagccttgga 20

the

the

<210> 10

<211> 23

<212> DNA

<213> Hctps1-P1

the

<400> 10

atggcatcat ccctcacttt ctc 23

the

the

<210> 11

<211> 22

<212> DNA

<213> Hctps1-P2

the

<400> 11

ctagaggggca gaagcaataa ac 22

the

the

<210> 12

<211> 22

<212> DNA

<213> P1

the

<400> 12

attgtggtgc cagtttgctt gc 22

the

the

<210> 13

<211> 24

<212> DNA

<213> P2

the

<400> 13

cccttttacc cttcgttgaa cttc 24

the

the

<210> 14

<211> 21

<212> DNA

<213> ACT-P1

the

<400> 14

ttcgtgttgc accagaagag c 21

the

the

<210> 15

<211> 22

<212> DNA

<213> ACT-P2

the

<400> 15

tgaatggcga catacatggc ag 22

the

the

<210> 16

<211> 24

<212> DNA

<213> Upstream primer F2

the

<400> 16

cttctacaca gccatcggtc caga 24

the

the

<210> 17

<211> 24

<212> DNA

<213> downstream primer R2

the

<400> 17

gatgtagggag ggcgtggata tgtc 24

Claims (1)

1. a lily ester floral fragrance gene LoAAT1, is characterized in that, the full-length cDNA sequence of described gene is as shown in SEQ ID NO:1; The full-length DNA sequence of gene is as shown in SEQ ID NO:2. 2. A protein encoded by lily ester floral fragrance gene LoAAT1, characterized in that the amino acid sequence of said protein is as shown in SEQ ID NO:3. 3. A pair of primers for amplifying the lily ester floral fragrance gene LoAAT1 according to claim 1, characterized in that the primer sequence is as shown in SEQ ID NO:10～11. 4. a recombinant vector, is characterized in that, inserts at the multiple cloning site of departure carrier, the sequence of lily ester floral fragrance gene LoAAT1 described in claim 1. 5. A recombinant bacterium comprising the recombinant vector according to claim 4. 6. A cell line comprising the recombinant vector according to claim 4. 7. the application of lily ester floral fragrance gene LoAAT1 described in claim 1, is characterized in that, lily ester floral fragrance gene LoAAT1 is connected in the plant transformation vector, then imports in lily cell, obtains and expresses described lily ester floral fragrance gene LoAAT1 transgenic floral varieties. 8. the application of the protein encoded by lily ester floral fragrance gene LoAAT1 described in claim 2 in the preparation of essence.