CN101985624A - Freesia 1-aminocyclopropane-1-carboxylate (ACC) synzyme FhACS1 protein coding sequence - Google Patents

Abstract

A freesia ACC synthetase FhACS1 protein coding sequence in the technical field of genetic engineering. Including the polypeptide having the nucleotide sequence from 1-1371 nucleotides shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.3, or its conservative variant polypeptide, or its active fragment , or its active derivatives. The invention provides a scientific basis for introducing the antisense FhACS1 gene into freesia by using antisense RNA technology in the future, inhibiting the synthesis of ethylene in the body of the freesia, thereby prolonging the viewing life of freesia flowers, and has great application value.

Description

Freesia ACC synthetase FhACS1 protein coding sequence

technical field

The present invention relates to a protein coding sequence in the technical field of genetic engineering, in particular to a freesia ACC synthetase FhACS1 protein coding sequence.

Background technique

As fresh cut flowers, not only are required to have good ornamental value, but also to maintain a long ornamental period (shelf life, vase period). From the perspective of mechanism, one of the initial reactions of petal senescence is autocatalysis to produce ethylene. On the one hand, ethylene is produced during the aging process, and on the other hand, the produced ethylene activates the expression of senescence-related genes to further promote its senescence and lead to The flowers eventually wilt and deteriorate. In higher plants, the biosynthetic pathway of ethylene is as follows: methionine, S-adenosylmethionine, ACC, ethylene. Methionine forms S-adenosylmethionine under the action of S-adenosylmethionine synthase, and S-adenosylmethionine forms ethylene precursor ACC, ACC under the catalysis of ACC synthase (1-aminocyclopropane-1-carboxylate synthase, ACS) Ethylene, _CO2 and HCN are formed under the catalysis of ACC oxidase. Among them, catalyzing the conversion of SAM to ACC is the rate-limiting reaction of this pathway, and ACC synthase (ACS) is the rate-limiting enzyme for plant ethylene biosynthesis. The use of antisense RNA technology to introduce the antisense ACS gene into flowers can inhibit the synthesis of ethylene and prolong the ornamental life (Halevy A H.1995).

After searching the prior art literature, it is found that the ACS multi-gene family has been found in many plants. Such as Have A Ten, WelteringE J in "Ethylene biosynthetic genes are differentially expressed during carnation flowerenescence (differences in gene expression of ethylene biosynthesis in the aging process of carnation flowers)" (Plant-Mol-Biol Plant Molecular Biology, 1997, 34: 89 -97.) Research on the ACS gene of carnations shows that ethylene regulates flower opening by changing gene expression, and there are differences in the expression of this gene in different parts of flower organs. But for the bulbous flower freesia (Freesia×hybrida), the cloning and expression pattern of ACS gene and the coding sequence of ACS protein are still unclear.

In the further search, there is no literature report related to the ACS protein sequence of freesia related to the subject of the present invention.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a freesia ACC synthetase FhACS1 protein coding sequence. The present invention discloses the protein gene closely related to Freesia FhACS1, the protein sequence of Freesia FhACS1 and its nucleotide sequence, and its expression patterns in different organs and different developmental stages of Freesia, and provides a basis for using antisense RNA technology in the future Introducing the antisense ACS gene into the freesia to inhibit the synthesis of ethylene in the body of the freesia so as to prolong the ornamental life of the flowers of the freesia has great application value.

The present invention is achieved through the following technical solutions:

The present invention includes a polypeptide having a nucleotide sequence from nucleotides 1-1371 shown in SEQ ID NO.2 and an amino acid sequence shown in SEQ ID NO.3, or a conservative variant polypeptide thereof, or Active fragments, or active derivatives thereof.

The coding sequence refers to the nucleotide sequence encoding the polypeptide having the activity of the freesia polypeptide FhACS1 protein, such as the 1-1371 nucleotide sequence in SEQ ID NO.2 and its degenerate sequence. The degenerate sequence refers to a sequence generated by replacing one or more codons in nucleotides 1-1371 of SEQ ID NO.2 with degenerate codons encoding the same amino acid. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% to the 1-1371 nucleotide sequence in SEQ ID NO.2 can also encode the sequence described in SEQ ID NO.2. The term also includes a nucleotide sequence with at least 70% homology to the nucleotide sequence from nucleotides 1-1371 in SEQ ID NO.2.

The isolated polypeptide FhACS1 of the present invention includes: a polypeptide having the amino acid sequence of SEQ ID NO.3. The polypeptide is a polypeptide with SEQID NO.3. The presence or absence and activity of the polypeptide in different organs are quite different in different growth and development stages of cut flowers, and can induce activity under environmental stress, plant hormones, and chemical substances. Improve or reduce, advance or delay the aging process.

The present invention relates to an isolated DNA molecule, which comprises a nucleotide sequence of a polypeptide having the activity of Freesia FhACS1 protein, and said nucleotide sequence is the same as that of SEQ ID NO.2 from nucleotide No. The nucleotide sequence at position 1-1371 has at least 70% homology; or the nucleotide sequence can hybridize with the nucleotide sequence at position 1-1371 in SEQ ID NO.2.

In the present invention, "isolated" and "purified" DNA means that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and also means that the DNA or fragment has been combined with the accompanying nucleic acid in the natural state. The components are separated and have been separated from the proteins that accompany them in the cell.

The present invention also includes variant forms of the sequence in SEQ ID NO. 3 that can encode a protein having the same function as the natural freesia FhACS1. These variant forms include (but are not limited to): deletions, insertions and/or substitutions of usually 1-90 nucleotides, and additions of up to 60 nucleotides at the 5' and/or 3' ends.

The "Freesia ACC synthetase FhACS1 protein" in the present invention refers to the polypeptide of SEQ ID NO.3 having the activity of Freesia FhACS1 protein. The term also includes variant forms of the sequence of SEQ ID NO. 2 that have the same function as that associated with native Freesia FhACS1. These variant forms include (but are not limited to): usually 1-50 amino acid deletions, insertions and/or substitutions, and addition of one or less than 20 amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the Freesia FhACS1 protein.

The variant forms of the freesia FhACS1 polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and can be related to freesia FhACS1 under high or low stringent conditions The protein encoded by the DNA of DNA hybridization, and the polypeptide or protein obtained by using the antiserum of Freesia FhACS1 polypeptide.

In the present invention, "freesia FhACS1 conservative variant polypeptide" refers to a polypeptide formed by replacing up to 10 amino acids with amino acids with similar or similar properties compared with the amino acid sequence of SEQ ID NO.3. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1

initial residue representative replacement preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys

Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

Table 2

73% concordance across 1070 nt overlaps

Query 136 gagaaccagctctcgttcaatcttctcgagtcttggcttgagcgtcaccctcacgctgcc 195

|||||||||||||| || |||| |||| || ||||||||| || || || | ||

Sbjct 13 gagaaccagctctgctttgatctcatcgaatcgtggcttgagaaccatcccgacccagct 72

Query 196 gctttcaagacggaaggcacgtcgaagtcagttttccgggagttggctctcttccgggat 255

|| ||||||| || || | | | | | ||||||||| | ||| | |||| |||

Sbjct 73 gcattcaagaaagatggagc-----act-actattccgggagctcgctttgttccaggac 126

Query 256 taccatggcctccctgcttttaaacaagcattgacagaattcatgggtgaattgagagga 315

||||||||| | || || || || | ||||||||||||||||||||||||||

Sbjct 127 taccatggcttgccagccttcaagcgtgcattgactaaatacatgggagaagtaagagga 186

Query 316 aacaaggttgattttgaacctaacaaactggtcctcacagctggtgcaacctcggctaac 375

||||| || | ||| || || |||| || |||||||||||||||| || || || ||

Sbjct 187 aacaaagtagctttcgatcccaacaggctcgtcctcacggctggtgctacttctgccaat 246

Query 376 gagaccctcatgttctgccttgccgacccaggagaagcattcttgctccctactccatac 435

|||||||||||||||||||||||||||||||||||||||||||||||||||| ||

Sbjct 247 gagactctcatgttttgtcttgccgaacctggagaagcattccttctgcctactccatac 306

Query 436 tatccagggtttgatcgtgatctcaagtggagaacaggagtggagatcgttccgatacac 495

|| ||||| || || | || ||||||||||||||||||||||||||||||||

Sbjct 307 tacccaggattcgacagagacctcaaatggcgaaccggagcggagatcgttcccatccat 366

Query 496 tgctcgagttcgaacagcttccggatcaccaagggcgccctcgaacaagcctatcggcat 555

|| ||||| || ||| ||||| |||||||||| ||||| ||| || || | ||

Sbjct 367 tgttcgagctctaacggcttcaggatcaccaaaccggccctggaagctgcataccaagat 426

Query 556 gccggaaagtgcaatttgcgagttaagggagtgctgataaccaatccctccaatccactc 615

|| ||| | | | | |||| || || |||||| |||||| ||||||

Sbjct 427 gcgcagaagcgtagcctcagagtgaaaggtgtgctggtgaccaacccgtccaacccattg 486

Query 616 ggcacgacgataaacaaagccgagctcgacatcctcgccgactttgtcgaagccaaagac 675

|| |||||||||||||||||||||||||||||||||||||||||||||

Sbjct 487 gggacgacgctgacccgacacgaactcgacattcttgtcgactttgtcgtctccaaggac 546

Query 676 atccattagtcggtgacgaaatctacgccggaacgaacttcgacatgccaggcttcatc 735

||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 547 atccatctcatcagtgacgagatctactcagggaccaacttcgactcgccggggttcatc 606

Query 736 agtgttgcagaggcaataaaggagagaccccagatatccgaccgtgtccacattgtctat 795

|| |||||||||| | |||||| || | | | ||| | || | ||||| || |

Sbjct 607 agcattgcagaggccacaaaggacaggaacaacgtctcccatcggattcacatcgtgtgc 666

Query 796 agcctctcgaaagacttcgggctaccgggtttccgagtcggtgcaatttattccaacaat 855

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 667 agcctctctaaagatctcggcctcccaggttttcgtgtcggtgcaatctattcggagaat 726

Query 856 gatttggtcgtcgcggcggctacgaagatgtcgagcttcgggctgatatcgtcgcagact 915

|| ||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 727 gaagcagtcgtgtctgctgctactaagatgtcaagctttgggatggtctcttctcaaacc 786

Query 916 cagtatcttctttcggcaatgctagctgacaaggagtttacgaggaactacttggtggag 975

||||| || || |||| || || | ||||| || || || || ||| | | |||

Sbjct 787 cagtacctcctcgcggcgttgttatccgacaaagaattcaccgataagtaccttctcgag 846

Query 976 aacaagaagaggctcaggaagaggcatggcgatcttgtcgagggtctccaaagggccggg 1035

|| ||||||| |||| || |||| | | |||||||| || || | ||| ||||

Sbjct 847 aatcagaagagactcaaagagcggcacgacatgcttgtcgaaggactgcgcaggatcggg 906

Query 1036 atcggttgcttggagagcaatgcgggcttgttctgttgggtagacatgaggcatctgctc 1095

||||| |||||| | | | ||||||||||||||||||||||||||||

Sbjct 907 atcgggtgcttgaaaggaagtgcggccttgttctgctgggtggacgtgagacacctgctg 966

Query 1096 aagtctaatagtccccaaggagaaatggagctatggaagaagatgttgtacaatgtggga 1155

||||| || | | |||||||||||||||||||||||||||||||||||

Sbjct 967 aagtccaacaccttcaaaggagcgatggagctgtggaagaagatagtgtatcaggtggga 1026

Query 1156 ctaaacatttctgcgggctcgtcgtgtcactccgacgaaccgggttggtt 1205

|||||||| || |||| || ||||||||||||||||||||||||||

Sbjct 1027 ctaaacatctcgccgggttcttcgtgccactgcgacgaaccggggtggtt 1076

Query: Coding sequence of ACC synthase FhACS1 from Freesia

Sbjct: mRNA sequence of ACC synthetase in plantain

Table 2 is a homologous comparison (GAP) table of the nucleotide sequences of the ACC synthase FhACS1 of Freesia in the present invention and the ACC synthase gene mRNA of Musa adenocarpus.

table 3

73% identity and 84% similarity in overlap

Query 1 MNLSAKATCNSHGQDSSYFLGWEEYEKNPYHPTLNRSGIIQMGLAENQLSFNLLESWLER 180

M LS KA CN HGQDSSYFLGW+EYEKNPY P N +GIIQMGLAENQL F+L+ESWLE

Sbjct 4 MLLSRKAACNIHGQDSSYFLGWQEYEKNPYDPITNPTGIIQMGLAENQLCFDLIESWLEN 63

Query 181 HPHAAAFKTEGTSKSVFRELALFRDYHGLPAFKQALTEFMGELRGNKVDFEPNKLVLTAG 360

HP AAFK+G +FRELALF+DYHGLPAFK+AL ++MGE+RGNKV F+PN+LVLTAG

Sbjct 64 HPDPAAFKKDGAL—LFRELALFQDYHGLPAFKRALAKYMGEVRGNKVAFDPNRLVLTAG 121

Query 361 ATSANETLMFCLADPGEAFLLPTPYYPGFDRDLKWRTGVEIVPIHCSSSSNSFRITKGALE 540

ATSANETLMFCLA+PGEAFLLPTPYYPGFDRD KWRTG EIVPIHCSSSN FRITK ALE

Sbjct 122 ATSANETLMFCLAEPGEAFLLPTPYYPGFDRDRKWRTGAEIVPIHCSSSSNGFRITKPALE 181

Query 541 QAYRHAGKCNLRVKGVLITNPSNPLGTTINKAELDILADFVEAKDIHLVGDEIYAGTNFD 720

AY+ A K +LRVKGVL+TNPSNPLGTT+ + ELDIL DFV +KDIHL+ DEIY +GTNFD

Sbjct 182 AAYQDAQKRSLRVKGVLVTNPSNPLGTTLTRHELDILVDFVVSKDIHLISDEIYSGTNFD 241

Query 721 MPGFISVAEAIKERPQISDRVHIVYSLSKDFGLPGFRVGAIYSNNDLVVAAATKMSSFGL 900

PGFIS+AEA K+R +S R+HIV SLSKD GLPGFRVGAIYS N+ VV+AATKMSSFG+

Sbjct 242 WPGFISIAEATKDRNNVSHRIHIVCSLSKDLGLPGFRVGAIYSENEAVVSAATKMSSFGM 301

Query 901 ISSQTQYLLSAMLADKEFTRNYLVENKKRLRKRHGDLVEGLQRAGIGCLESNAGLFCWVD 1080

+SSQTQYLL+A+L+DKEFT YL+EN+KRL++RH LVEGL+R GIGCL + +A LFCWVD

Sbjct 302 VSSQTQYLLAALLSDKEFTDKYLLENQKRLKERHDMLVEGLRRIGIGCLKGSAALFCWVD 361

Query 1081 MRHLLKSNSPQGEMELWKKMLYNVGLNISAGSSCHSDEPGWFRMCFANSQQETLNLAMDR 1260

    MRHLLKSN+ +GEMELWKK++Y VGLNIS GSSCH DEPGWFR+CFANMS++TL LAM R

Sbjct 362 MRHLLKSNTFKGEMELWKKIVYQVGLNISPGSSCHCDEPGWFRVCFANMSEDTLTLAMQR 421

Query 1261 LTSFVEL-------NYGVQRRRLPSSIVKWVLKLSPSTDRKAER 1371

L SFV+ +G QR R P + KWVL+LS STDRK+ER

Sbjct 422 LKSFVDSGDCGSNHDSGHQRPRKP-FLTKWVLRLS-STDRKSER 463

Query: Amino acid sequence of ACC synthase FhACS1 from Freesia

Sbjct: Amino Acid Sequence of ACC Synthetase from Musa microcarpa (AAQ13435.1)

Table 3 is the homology comparison (FASTA) table of the amino acid sequences of the freesia ACC synthase FhACS1 of the present invention and the ACC synthase of the plantain. Wherein, identical amino acids are marked with amino acid single characters between the two sequences.

The invention also includes analogs of the freesia ACC synthetase FhACS1 protein or polypeptide. The difference between these analogues and related polypeptides of freesia ACC synthase FhACS1 may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the expression pattern of the freesia ACC synthetase FhACS1 gene product can be analyzed by real-time fluorescent quantitative PCR, that is, the presence or absence and quantity of the mRNA transcript of the FhACS1 gene in cells can be analyzed.

In addition, the nucleic acid molecule that can be used as a probe in the present invention usually has 8-100 consecutive nucleotides of the freesia FhACS1 nucleotide coding sequence, and the probe can be used to detect whether there is a freesia FhACS1-related sequence in the sample. nucleic acid molecule.

The detection method of the present invention for detecting whether there is a freesia FhACS1-related nucleotide sequence in a sample comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. The sample is the product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence related to Freesia FhACS1, and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the freesia FhACS1 nucleotide sequence and amino acid sequence of the present invention, it is possible to screen freesia FhACS1 related homologous genes or homologous proteins on the basis of nucleic acid homology or expressed protein homology.

In order to obtain the array of genes related to Freesia FhACS1, the freesia cDNA library can be screened with DNA probes that irradiate all or part of Freesia FhACS1 with ³² P under low stringency conditions derived from active markers. A suitable cDNA library for screening is that from Freesia. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. Additionally, many such cDNA libraries are commercially available, eg, from Clontech, Stratagene, Palo Alto, Cal. This screening method can identify the nucleotide sequences of the gene family related to Freesia FhACS1.

The freesia FhACS1-related nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct synthesis of peptides using solid phase techniques (Stewart et al., (1969) Solid Phase Polypeptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

By using the freesia FhACS1 protein of the present invention, substances, or receptors, inhibitors or antagonists, etc. that interact with freesia FhACS1 can be screened out through various conventional screening methods.

The present invention clones for the first time the coding sequence of the ethylene synthesis rate-limiting enzyme ACC synthase FhACS1 protein in freesia, and uses the method of fluorescence real-time quantitative PCR to analyze the expression pattern of the FhACS1 gene, so as to use antisense RNA technology to introduce the antisense ACS gene into small Freesia, which inhibits the synthesis of ethylene in the body of freesia and thus prolongs the ornamental life of freesia flowers provides a basis for research and development. Freesia is one of the top ten cut flowers in the world and has a high ornamental value. Phenomena such as petal discoloration affect its viewing life, so the present invention has great application value.

Detailed ways

Below in conjunction with laboratory test data, the embodiments of the present invention are described in detail: the following embodiments are implemented under the premise of the technical solutions of the present invention, and detailed implementation methods and processes are provided, but the protection scope of the present invention is not limited to the following the embodiment.

The experimental method that does not indicate specific conditions in the following examples is usually according to routine conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Cloning of ACC Synthetase FhACS1 Gene from Freesia

1. Acquisition of plant material

Freesia is cultivated by the School of Agriculture and Biology of Shanghai Jiaotong University. Healthy freesia bulbs of uniform size were planted in the substrate (perlite: vermiculite: peat 1:1:1), and DNA or RNA was extracted when the aboveground part was higher than 15 cm.

2. Extraction of total DNA from leaves

The total DNA in leaves was extracted by CTAB method. Take about 50 mg of young leaves, grind them into powder with a grinding rod, add 400 μL of extraction buffer, and place in a water bath at 65°C for 45 minutes; after cooling, add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert and mix to emulsify 10min; centrifuge at 12000rpm for 10min (18-20℃), draw the supernatant into another clean 1.5mL centrifuge tube; use chloroform:isoamyl alcohol (24:1) to repeat the extraction once as above, absorb the supernatant into another clean Add 2 times the volume of supernatant equal to absolute ethanol, mix upside down; centrifuge at 12000rpm for 10min (18-20°C), pour off the supernatant carefully; rinse with 200μL of 75% ethanol, white can be seen at this time flaky precipitates. After pipetting with 75% ethanol for two to three times, carefully suck out the ethanol; after a few minutes, blot the ethanol dry with a tip; dry the precipitate in a 55°C incubator for a while until translucent and add 50 μL of deionized water to dissolve it. Store at -20°C.

3. Isolation of RNA

Total RNA was extracted with the "Plant Leaf Total RNA Minor Extraction Kit" (RNA prep pure Plant Kit: product of Treasure Bioengineering (Dalian) Co., Ltd.). The RNA quality was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

4. Full-length gene sequence cloning

Utilizing the principle of homologous gene cloning, the Genome Walking method (Genome Walking Kit: Treasure Bioengineering (Dalian) Co., Ltd.) is used for DNA sequence cloning, which is carried out in three stages:

(1) Obtain the middle fragment of the gene by PCR

Using DNA as a template, using primers F1(CA(A/G)ATGGGCCTCGC(C/A)GAGAA(C/T)CA) and R1(CCA(G/A)CA(G/A)AAGAGCCCCGC(G/A) TTGC) for PCR, amplified to obtain a fragment of about 1300bp, recovered and connected to the pMD18-T Simple vector vector, using RV-M and M13-47 as universal primers, using terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA) method, sequenced on ABI 377 sequencer (Perkin-Elmer, USA). The sequencing results searched the existing database (Genebank) with the BLAST software in the GCG software package (Wisconsin group, USA), and it was known that its nucleic acid sequence and encoded protein had a high homology with the known ACC synthetase gene of Musa genus , so it is preliminarily considered to be an ACC synthetase gene.

(2) Amplify the 3' end sequence by Genome Walking method

Three rounds of nested PCR completed the amplification of the 3' end sequence.

First round: AP+3R3 (5’-ACTCGGCACGACGATAAACA-3’)

Second round: AP+3R2 (5’-AGCCAAAGACATCCATTTAGTCG-3’)

The third round: AP+3R1 (5’-CGGCGGCTACCAAGATGTCA-3’)

The FhACS1 3' end sequence was obtained, recovered, connected to the pMD18-T Simple vector vector, and RV-M and M13-47 were used as universal primers, and the terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA) was used. Sequencing was performed on an ABI 377 sequencer (Perkin-Elmer, USA). The sequencing results searched the existing database (Genebank) with BLAST software in the GCG software package (Wisconsin group, USA), and it was known that its nucleic acid sequence and encoded protein had a high homology with the ACC synthetase gene of Musa acuminata.

(3) Amplify the 5' end sequence by Genome Walking method

Three rounds of nested PCR completed the amplification of the 5' end sequence.

First round: AP+5F3 (5’-CAAATTATTTATGTGCATGATGCATGC-3’)

Second round: AP+5F2 (5’-CGAGGCAGAACATGAGGGTCTCGTG-3’)

The third round: AP+5F1 (5’-CACCCATGAATTTTGCCAATGCCTG-3’)

Obtain the 5' end sequence of FhACS1, and sequence it with the same method as above after recovering the connection.

The overlapping regions of the sequencing results obtained by the above three methods were spliced, and the spliced sequence was obtained and submitted for BLAST analysis. The results proved that the FhACS1 gene newly cloned from Freesia is indeed a gene related to ACC synthetase. Combining the sequencing results with NCBI's ORFFingding (http://www.ncbi.nlm.nih.gov/gorf) webpage prediction, the start codon and stop codon of the FhACS1 gene of Freesia were found. The obtained FhACS1 gene has a full length of 2576bp (SEQ ID NO.1), 433bp in the 5' non-coding region, and 373bp in the 3' non-coding region; its start codon is located at 434nt, and the stop codon is located at 2201nt; the gene has 3 introns, 4 exons. The positions of the introns are: 578-667nt, 805-1009nt, 1174-1277nt.

(4) Acquisition of CDS open reading frame sequence of FhACS1 gene

Using freesia petal RNA as a template, cDNA was obtained by reverse transcription with "PrimeScript II 1st Strand cDNA Synthesis Kit" (Bao Biological Engineering (Dalian) Co., Ltd.). According to the gene sequence obtained above, the upstream specific primer ORF-F (5'-ATGAATCTCTCTGCAAAAGCTAC-3') was designed from the start codon, and the downstream primer ORF-R (5'- CCTCTCGGCTTTCCGATCAGTCG-3'), using Freesia cDNA as a template for RT-PCR, amplified to obtain a 1371bp long CDS open reading frame sequence, which is the coding sequence of the FhACS1 protein (SEQ ID NO.2).

Example 2

Sequence Information and Homology Analysis of Freesia FhACS1 Gene

The new freesia FhACS1 full-length CDS open reading frame sequence of the present invention is 1371bp, and the detailed sequence is shown in SEQ ID NO.2. According to the CDS open reading frame sequence, the amino acid sequence of Freesia FhACS1 was deduced, with a total of 457 amino acid residues. See SEQ ID NO.3 for the detailed sequence.

The CDS open reading frame sequence of Freesia FhACS1 and the amino acid sequence of its encoded protein were nucleated in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleic acid and protein homology search, it was found that it has 73% identity with ACS gene (AB021907.1) at the nucleotide level (attached table 2); The plantain ACS gene (AAQ13435.1) also has 73% identity and 84% similarity (Supplementary Table 3). It can be seen that there is a high homology between the FhACS1 gene of Freesia and the ACS gene of Musa adenocarpus in both nucleic acid and protein levels.

Example 3

The expression level of freesia FhACS1 gene in flowers at different developmental stages and the expression differences among petals, stamens and pistils in flowers.

1. Acquisition of materials: When buds or flowers of various developmental stages appear on the freesia inflorescence, the whole flower (mixed samples of flowers of various developmental stages) and the separated floral organs, stamens, pistils, and petals ( Mixed samples of each developmental stage), the samples were wrapped with aluminum platinum paper and put into liquid nitrogen immediately, and then transferred to -80°C ultra-low temperature freezer for storage until use.

2. Extraction of RNA: Use RNAprep pure Plant Kit (product of Treasure Bioengineering (Dalian) Co., Ltd.) to extract RNA from the whole flower of Freesia and the petals, stamens, and pistils in the flower.

3. Determination of the integrity and concentration of RNA: the integrity was detected by ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min). In the formaldehyde electrophoresis of complete RNA, two bands, 28S and 18S, can be clearly observed, and 28S is about 1.5-2.0 times wider than 18S. If the two bands are not obvious, it means that the RNA is partially degraded. For RNA with relatively high purity, A ₂₆₀ /A ₂₈₀ = about 2.0, and A ₂₆₀ /A ₂₃₀ = about 2.0. Measure the OD value with a spectrophotometer and calculate the RNA content: 1 OD ₂₆₀ =40 μg/mL.

4. Acquisition of cDNA: 500 ng of total RNA was used as a template, and reverse transcription was performed according to the operation instructions of the TaKaRa PrimeScript ^TM RT reagentKit Perfect Real Time kit from Takara Biotech Co., Ltd. to obtain cDNA for future use.

5. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in various organs and tissues. According to the obtained freesia FhACS1 gene sequence, use primer design software primer premier 5.0 to design specific primers for quantitative analysis of FhACS1 gene in Real-time PCR, the primer name is Q-F (5'-CCCAA GGAGA AATGGAGCTA-3') , Q-R(5'-ATTCG ACGAA CGACG TAAGC-3'). The internal reference gene is Actin, and its specific primers are A-F (5'-CATGA AGATC CTGACGGAGA-3'), A-R (5'-GAGTT GTAGG TGGTC TCATG-3').

6. Make the standard curve of the target gene and internal reference gene. Use EASY Dilution (provided by the kit) to serially dilute the standard cDNA solution, then use the diluted cDNA solution as a template to perform Real-time PCR amplification with specific primers for the target gene and the internal reference gene, and draw after the reaction Melting curve and standard curve. Analyze the melting curve to determine whether the melting curves of the target gene and the internal reference gene have a single peak, so as to determine whether a single PCR amplification product can be obtained using the primer.

7. Real-time fluorescence quantitative analysis of the target gene in the sample to be tested. Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were respectively amplified with specific primers for fluorescence quantitative analysis. The Real-time PCR reaction was carried out on the BIO-RAD Chromo 4 real-time quantitative instrument. The reaction system was 20 μL. The reaction adopts a three-step method, denaturation at 95°C for 20s, followed by 41 cycles: 95°C for 15s; 60°C for 15s; 72°C for 25s. After each amplification is completed, a melting curve is made to check whether the amplified product is specifically produced.

8. Use double standard curve method for relative quantitative analysis. The results showed that the expression level of FhACS1 gene in flowers at each developmental stage showed significant differences: among them, the expression level of FhACS1 gene was higher when the flowers were fully open and most of them were open, and was the highest when fully open, which was 3.29 times that of small green flower buds. It is 32.6 times of the expression level in the lowest expression stage (bud tepal color development stage), indicating that the expression of this gene has obvious correlation with the senescence of flowers. The expression levels of FhACS1 gene in stamens, pistils, and petals from more to less were stamens > pistils > petals; the expression levels of FhACS1 genes in stamens and pistils were 2.62 and 0.46 times the expression levels in petals, respectively, and the expression levels in petals There were significant differences, indicating that the expression of this gene in stamens was most closely related to the senescence of flowers.

Claims (4)

1. freesia ACC synthetic enzyme FhACS1 albumen coded sequence, it is characterized in that, comprise and have the nucleotide sequence and polypeptide shown in the SEQ ID NO.2 with the aminoacid sequence shown in the SEQ ID NO.3 from Nucleotide 1-1371 position, or its conservative property variation polypeptide or its active fragments, or its reactive derivative.

2. freesia ACC synthetic enzyme FhACS1 albumen coded sequence according to claim 1, it is characterized in that, described encoding sequence has the nucleotide sequence of the polypeptide of freesia polypeptide FhACS1 protein-active, as 1-1371 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.2.

3. freesia ACC synthetic enzyme FhACS1 albumen coded sequence according to claim 2, it is characterized in that, described degenerate sequence is meant, be arranged in the 1-1371 position Nucleotide of SEQ ID NO.2, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.

4. the nucleic acid molecule as hybridization according to claim 2 is characterized in that, described polypeptide FhACS1, and it comprises: the polypeptide with SEQ ID NO.3 aminoacid sequence.