CN113186197B - Citrus CitPITP1 gene and application of nucleotide sequence thereof in prokaryotic vector promoter modification - Google Patents

Abstract

The invention discloses a citrus CitPITP1 gene and application of a nucleotide sequence thereof in prokaryotic vector promoter modification, wherein the nucleotide sequence of the CitPITP1 gene is shown as SEQ ID NO: 1 is shown. The invention obtains the CitPITP1 gene from the genome of the citrus by screening, and discovers that a 64bp nucleotide sequence on the exon 4 of the CRAL _ TRIO structural domain of the gene coding CitPITP1 can drive the gene expression in prokaryotes from the CitPITP1 gene; therefore, the sequence containing CitPITP1_ key64 can be applied to vector promoter modification in the field of genetic engineering.

Description

Citrus CitPITP1 gene and application of nucleotide sequence thereof in prokaryotic vector promoter modification

Technical Field

The invention relates to the field of genetic engineering, in particular to a citrus CitPITP1 gene and application of a nucleotide sequence thereof in prokaryotic vector promoter modification.

Background

During the development of an organism, transcription is one of the basic processes in molecular biology, converting the genetic code in DNA into RNA molecules. Transcription initiation is a critical step that determines the location of the transcription initiation site (TSS) and the spatiotemporal expression pattern and intensity of transcription. The transcription initiation device (RNA polymerase) needs to recognize and bind to a specific DNA sequence in order to initiate transcription. This sequence, called the promoter, is a non-coding DNA sequence located generally upstream of the 5' end of the gene.

In the fields of genetic engineering and synthetic biology, promoters are one of the important elements for regulating the expression level of a target protein. Promoters can be classified into: a constitutive promoter; an inducible promoter; tissue specific promoters. Methods for cloning promoters can be roughly classified into 3 types: the genome library screening method includes screening promoter and PCR cloning promoter with promoter probe plasmid vector. In the species with the sequenced genome, specific primers can be designed to carry out PCR to directly obtain the promoter sequence of the target gene. And combining the RNA-Seq data of each tissue to obtain the space-time expression mode and strength of the promoter. The promoter sequence and reporter genes such as GUS and GFP are fused and stably transformed into plants for verification, and the cis-acting element analysis of databases such as PLACE, plantarCARE, EPD and the like is combined, so that the promoter sequence can be subjected to truncation analysis, and key elements are locked.

Promoters from different genes in an organism constitute a source of promoter data, which provides many functionally diverse promoter elements for bioengineering and synthetic biology such as: the T7 promoter to which T7 RNA polymerase specifically binds; a constitutive promoter CaMV35S, a Ubi promoter of a maize Ubiquitin gene; tissue specific promoters such as: arabidopsis floral organ development specific promoter leaf, tomato fruit specific promoter PG and ACO1, and 'TACACAT' conserved sequence of seed specific promoter; inducible promoters can be broadly classified into three main groups due to the wide variety of inducers:

1. physical factors induce such as light, temperature, trauma, etc.,

2. chemical induction such as salt concentration, estradiol, hormones and the like,

3. induction of biological factors such as pathogenic bacteria TALE response elements;

in addition to the discovery of promoters based on genes of organisms, artificial synthesis of promoters has also been reported. The promoter which meets the requirement of an accurate expression mode is synthesized by combining different types of cis-acting elements. The synthetic promoter with different response strength to TALE pathogenic factors is designed by analyzing the promoter sequences of CaMV35S, MMV, NOS, AtACT2, AtUBC9 and AtUBQ10, determining the shared cis-acting element (C-CRE), combining TALE response elements under the basic promoter frame containing TATA-box, adding different types and numbers of C-CRE and changing the relative positions of C-CRE and TATA-box.

The diversified gene functions and the expression modes thereof provide abundant resources for exploring various promoters. However, the target of promoter mining is usually focused on the upstream DNA sequence of the 5' end of the gene, and the gene sequence itself can be used as the promoter without research.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a citrus CitPITP1 gene and application of a nucleotide sequence thereof in prokaryotic vector promoter modification, the citrus CitPITP1 gene is obtained by screening, and a CDS sequence or a partial truncated sequence and a key64 sequence of the citrus CitPITP1 gene can be used as a promoter sequence in prokaryotes to drive gene expression, so that the source of promoter discovery is widened, a new visual angle is provided for promoter discovery, and theoretical support and basis are provided for selection of the promoter in vector modification in the fields of genetic engineering and synthetic biology.

In order to achieve the purpose, the invention designs a citrus CitPITP1 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1 is shown.

Further, the nucleotide sequence of the CDS sequence in the CitPITP1 gene is shown as SEQ ID NO: 2, respectively.

The invention also provides a protein coded by the CitPITP1 gene, and the amino acid sequence of the protein is shown as SEQ ID NO: 3, respectively.

The invention also provides application of the nucleotide sequence of the CitPITP1 gene in the modification of prokaryotic vector promoters.

Furthermore, the application method is that any segment of nucleotide sequence truncated in the CitPITP1 gene sequence is used as a vector promoter for driving gene expression in prokaryotes.

Still further, the vector promoter contains a sequence of CitPITP1_ key 64.

Still further, the vector promoter is any one of the sequences of CitPITP1CDS, CitPITP1NO, CitPITP1Narrow1 and CitPITP1.1_ key64, and the nucleotide sequences thereof are shown in SEQ ID NO: 2. 4, 5 and 8.

Still further, the vector promoter is CitPITP1CDS, and the nucleotide sequence is shown in SEQ ID NO: 2, respectively.

The principle of the invention is as follows:

the invention finds that the CDS sequence of the citrus CitPITP1 gene is fused with an eGFP reporter gene, the CDS sequence is connected into a pTOPO-Blunt Simple Vector of Aidlab, E.coli DH5 alpha is transformed by heat shock, and sequencing shows that the obtained positive monoclonal sequence is correct. Positive monoclonal bacteria were enriched by centrifugation, and fluorescence emission from the bacteria was observed only in the excitation group (460-490 nm) of OLYMPUS SZX7 stereofluorescence microscope B. The CitPITP1 gene encodes a protein containing CRAL _ TRIO conserved Domain, and the result of sequence analysis and sequence truncation experiments by combining BPROM and BDGP promoter prediction analysis shows that a 64bp CitPITP1_ key64 sequence on the 4 th exon of the CitPITP1 gene is a prokaryotic promoter key sequence and is positioned in the CRAL _ TRIO coding sequence. The research results show that the CitPITP1_ key64 of the CitPITP1 can be used as a prokaryotic promoter to drive gene expression, and support is provided for vector promoter modification in the field of genetic engineering.

The invention has the beneficial effects that:

the invention obtains the CitPITP1 gene from the genome of the citrus by screening, and discovers that a 64bp nucleotide sequence on the exon 4 of the CRAL _ TRIO structural domain of the coding CitPITP1 gene can drive the gene expression in prokaryotes from the CitPITP1 gene; therefore, the sequence containing the CitPITP1_ key64 (the CitPITP1_ key64 sequence has the function of a promoter) can be applied to the transformation of a vector promoter in the field of genetic engineering.

Drawings

FIG. 1 is a schematic diagram of the gene structure of the citrus CitPITP1 gene

FIG. 2 is a diagram showing the analysis of the promoter activity of the CitPITP1 sequences with different truncated lengths.

FIG. 3 is a diagram of the analysis of the activity of the CitPITP1 prokaryotic promoter sequence CitPITP1_ key64 promoter.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1

Obtaining of citrus CitPITP1 gene sequence and CDS

Picking mature leaf of GUAN-MIAN GUAN, quick freezing with liquid nitrogen, storing in-80 deg.C ultra-low temperature refrigerator, and extracting pulp total DNA by CTAB method. According to the citrus genome sequence, PCR is carried out by using Primer5 to design amplification primers of a CitPITP1 gene, TA cloning and sequencing are carried out on a PCR product, and the gene sequence is obtained as shown in SEQ ID NO: 1, the sequence of the amplification primer, the PCR system and the reaction program are as follows:

the primer sequence is as follows:

forward primer cittpitp 1 gene F: 5'-CACAACAAAGTCCAGCGTCG-3', respectively;

reverse primer cittpitp 1 gene R: 5'-ACAGCAACAGACAGTAGTTTCACC-3' are provided.

The PCR system was as follows:

the PCR reaction procedure was as follows:

picking fruit 150 days after MiYou Mixi GUAN flower, taking out pulp, quick freezing with liquid nitrogen, and storing in refrigerator at-80 deg.C; extracting pulp total RNA by adopting a CTAB method; mu.g of total RNA was used to synthesize cDNA using Novozan HiScript II Q RT Supermix for qPCR reverse transcription kit as follows:

the first step is as follows: genomic DNA removal:

RNase free ddH2O	To 16μL
		4×gRNA wiper Mix	4μL
template RNA	1μg

Gently sucking and stirring the mixture by a pipette, and reacting the mixture for 2min at 42 ℃.

The second step is that: preparing a reverse transcription reaction system:

5×HiScriptⅡqRT SuperMixⅡ	4μL
		first-step reaction solution	16μL

The PCR reaction procedure was as follows:

25℃	10min
		50℃	30min
85℃	5min
		12℃	30min

using cDNA as a template, and using Primer5 to design a CitPITP1 cDNA specific amplification Primer for PCR according to the obtained CitPITP1 gene sequence, wherein the amplification Primer sequence, a PCR system and a reaction program are as follows:

the sequence is as follows:

forward primer cittpitp 1 cDNA F: 5'-CATTCGGAGTATCTCGTTGC-3', respectively; reverse primer cittpitp 1 cDNA R: 5' -CATAAACTTCGCCTATCTGCTT-3.

The PCR system was as follows:

the PCR reaction procedure was as follows:

the PCR reaction product was separated by agarose Gel electrophoresis and purified and recovered from the DNA Gel/PCR Purification Miniprep Kit from Biomiga to obtain CitPITP1 cDNA fragment.

According to the sequence of CitPITP1 cDNA fragment, PCR is carried out by using Primer5 to design specific amplification Primer, CitPITP1 cDNA is taken as a template, PCR reaction product is separated by agarose Gel electrophoresis and purified and recovered by DNA Gel/PCR Purification Miniprep Kit of Biomiga, and TA cloning and sequencing are carried out on the PCR product, thus obtaining the CitPITP1CDS fragment (without stop codon) with the nucleotide sequence shown as SEQ ID NO: 2, and the coded amino acid sequence is shown as SEQ ID NO: 3, the sequence of the amplification primer, the PCR system and the reaction program are as follows:

forward primer cittpitp 1CDS F: 5' -CGAGCTCATGTCGAGAAAATCACGTGGATTTG-3' (the SacI cleavage site is underlined)

Reverse primer cittpitp 1CDS R: 5' -AGAGCCACCACCGCCAGAGCCACCACCGCCAGAGCCACCACC GCCTTGTACTTGTTTCGCCATTGCAGCTT-3' (Linker sequence underlined).

The PCR system was as follows:

the PCR reaction procedure was as follows:

the bioedit software was used to compare SEQ ID NO: 1 and SEQ ID NO: 2, carrying out alignment analysis to determine the CitPITP1 gene structure. Converting SEQ ID NO: 3, the sequence is submitted to a PFAM website for domain prediction, and the structure shows that the CitPITP1 contains two domains of CRA L _ TRIO _ N and CRA L _ TRIO, and the gene structure and the domain schematic diagram are shown in FIG. 1.

Example 2

Analysis of the promoter Activity of the CitPITP1CDS sequence in prokaryotes

Selecting eGFP as a reporter gene, firstly, in order to obtain a CDS sequence of the eGFP reporter gene, any carrier containing eGFP can be used as a template for amplifying eGFP CDS, according to a plasmid pHSE401-35S-eGFP sequence, a plasmid pHSE401-35S-e GFP is used as a template, a Primer5 is used for designing a specific amplification Primer to carry out PCR, and the sequence of the amplification Primer, a PCR system and a reaction program are as follows:

forward primer eGFP extension fragment F: 5'-GCACATACAAATGGACGAACG-3', respectively; reverse primer eGFP extension fragment R: 5'-GCCTGAATGGCGAATGCTA-3' are provided.

The PCR system was as follows:

the PCR reaction procedure was as follows:

the PCR reaction product was separated by agarose gel electrophoresis and purified and recovered from the DNA gel/PCR Purification Miniprep Kit from Biomiga to obtain the eGFP extension fragment.

Secondly, designing a specific amplification Primer by using Primer5 to carry out PCR according to the eGFP CDS sequence in the plasmid pHSE401-35S-eGFP, taking an eGFP expansion fragment as a template, carrying out agarose Gel electrophoresis separation on a PCR reaction product, and purifying and recovering a DNA Gel/PCR Purification Miniprep Kit of biomaga to obtain an eGFP CDS fragment, wherein the amplification Primer sequence, the PCR system and the reaction program are as follows:

forward primer eGFP CDS F: 5' -GGCGGTGGTGGCTCTGGCGGTGGTGGCT CTGGCGGTGGTGGCTCTATGGTGAGCAAGGGCGAGGAGCT-3' (bottom)Underlined is Linker sequence) reverse primer eGFP CDS R: 5' -CCCAAGCTTTTACAGCTCGTCCTTCTTG

TACAGCT-3' (underlined hindIII cleavage site).

The PCR system was as follows:

the PCR reaction procedure was as follows:

and finally, a CitPITP1 CDS-eGFP fragment is obtained by using a fusion PCR method by taking the CitPITP1CDS and eGFP CDS fragments as templates and taking a forward primer CitPITP1CDS F and a reverse primer eGFP CDS R as forward and reverse primers. The specific PCR system and reaction procedure were as follows:

the PCR system was as follows:

the PCR reaction procedure was as follows:

the recovered CitPITP1 CDS-eGFP fragment was ligated with Aidlab pTOPO-Blunt Simple Vector to transform DH 5. alpha. E.coli by the following procedures:

the first step is as follows: preparation of connecting system

ptopO-Blunt Simple Vector	1μL
		10×Enhancer	1μL
CitPITP1-eGFP fragment	60ng
		ddH2O	To 10μL

Preparing the system, gently sucking and beating the mixture by a pipette, uniformly mixing the mixture, centrifuging the mixture at a low speed for a short time, and incubating the mixture for 20min at the temperature of 25 ℃.

The second step is that: transformation of DH5 alpha E.coli

Adding 5 mu L of the connecting system into 50 mu L of DH5 alpha escherichia coli competence, sucking and beating gently and mixing uniformly, performing ice bath for 30min, performing 42-degree hot shock for 90s, performing ice bath for 2min, adding 400 mu L of LB without antibiotics, culturing for 45min at 37 ℃ and 200rpm, taking 200 mu L of bacterial liquid coated plate (containing 50 mu g/mL ampicillin), culturing overnight at 37 ℃, picking positive single clone for shaking bacteria, sending bacterial liquid to Scophyton Biotech Limited for sequencing, and confirming the sequence of the CitPITP1CDS sequence in the insert fragment CitPITP1 CDS-eGFP and SEQ ID NO: 2, the eGFP CDS sequence is identical to the eGFP sequence in plasmid pHSE 401-35S-eGFP.

Inoculating the monoclonal bacterial liquid into a fresh culture medium according to the ratio of 1:1000, culturing for 8h in an incubator at 37 ℃ and 200rpm, centrifugally enriching 500 microliter of positive monoclonal bacterial liquid, observing the fluorescence of the bacterial body in an OLYMPUS SZX7 stereovision fluorescence microscope B excitation group (460-490 nm), wherein the observation result is shown as CitPITP1-eGFP in figure 2, and the result shows that the CitPITP1CDS sequence can drive eGFP expression and generate fluorescence in prokaryotic escherichia coli.

Example 3 mining and application of CitPITP1 promoter key sequence

The sequence of the CitPIP 1 gene and the CDS sequence thereof are submitted to BPROM (http:// www.softberry.com/berry. phtltropic & group. programs & subgroup. gfindb) and BDGP (http:// www.fruitfly.org/SEQ _ tools/promoter. html) websites for prokaryotic promoter prediction, and the results show that the sequence contains-10 element (TGCTATAAT) and-35 element (TTGGTG) which are respectively positioned in SEQ ID NO: 2 at 396-404 and 376-381. Further adopting a promoter truncation analysis method to obtain 5 nucleotide sequences with different lengths, which are respectively positioned in the nucleotide sequences shown in SEQ ID NO: 2, 1-1008 full length CitPITP1CDS, 6-1008 CitPIP 1NO, 357-1008 CitPIP 1Narrow1, 376-1008 CitPIP 1Narrow2 and 396-1008 CitPIP 1Narrow3, wherein the gene sequences are respectively shown as 2, 4, 5, 6 and 7, and Primer5 is used for designing corresponding specific amplification primers, and the Primer sequences are as follows (the underlining is the SacI digestion site):

CitPITP1NO_F：5’-CGAGCTCGAGAAAATCACGTGGATTTG-3’，

CitPITP1Narrow_F1：5’-CGAGCTCAGAGGGGCAGATCAAGTAC-3’，

CitPITP1Narrow_F2：5’-CGAGCTCTTGGTGTATTGCATGGAAAA-3’，

CitPITP1Narrow_F3：5’-CGAGCTCTGCTATAATGAATTTGAACCCT-3’；

taking CitPITP1 CDS-eGFP as a template, taking CitPITP1NO _ F or CitPITP1Narrow _ F1 or CitPIP 1Narrow _ F2 or CitPIP 1Narrow _ F3 as a forward primer and taking eGFP CDR as a reverse primer to obtain amplicons fused with the eGFP and CitPITP1 sequences with different truncation lengths, namely CitPIP 1NO-eGFP, CitPIP 1Narrow1-eGFP, CitPIP 1Narrow2-eGFP and CitPIP 1Narrow3-eGFP respectively, and the specific PCR system and the reaction program are as follows:

the PCR system was as follows:

the PCR reaction procedure was as follows:

the amplicon was ligated into Aidlab pTOPO-Blunt Simple Vector, heat-shocked to transform DH 5. alpha. E.coli, the procedure was as in example 2, the positive clones were subjected to fluorescence detection, the promoter activity corresponding to the different truncated sequences was detected in fluorescence as shown in FIG. 2, the exposure time was 199ms, and the results showed that the fluorescence intensity was slightly reduced when the sequence was truncated from CitPITP1Narrow1 to CitPITP1Narrow2, and significantly reduced when the sequence was truncated from CitPITP1Narrow2 to CitPIP 1Narrow 3. Combining with the analysis of promoter prediction results, identifying the 64bp sequence of 357 and 420 as a key sequence of CitPITP1_ key64, such as SEQ ID NO: as shown in 8, CitPITP1_ key64 is independently fused with eGFP, a reverse primer is a reverse primer eGFP CDS R, and a forward primer, a specific PCR system and a specific reaction program are as follows:

the PCR system was as follows:

the PCR reaction procedure was as follows:

the CitPITP1_ key64-GFP amplicon is obtained, Aidlab pTOPO-Blunt Simple Vector is connected, DH5 alpha escherichia coli is transformed by heat shock, the specific method is the same as that in example 2, the positive clone thallus is subjected to fluorescence detection, the promoter activity fluorescence detection is shown in figure 3, and the exposure time is 999 ms. The results show that CitPITP1_ key64 also has prokaryotic promoter activity.

The results of the above analysis are combined to show that the CitPITP1_ key64 can drive eGFP expression to generate fluorescence in Escherichia coli, and the fluorescent protein is a common essential sequence of a prokaryotic promoter of the CitPITP1. The fluorescence intensity of CitPITP1 CDS-eGFP, CitPITP1NO-eGFP, CitPITP1Narrow1-eGFP and CitPITP1Narrow2-eGFP is strong, and the intensity of CitPITP1 CDS-eGFP is strongest; thus, the CitPITP1CDS sequence acts as a prokaryotic promoter with the best promoter activity.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> citrus CitPITP1 gene and application of nucleotide sequence thereof in prokaryotic vector promoter modification

<160> 8

<170> SIPOSequenceListing 1.0

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<213> orange (Citrus grandis)

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aattccaaat aattcttcgg tatttttttt ttctcattcg atttttaaaa tttggtttca 120

tttttatttt ctacagaaat gagcaaaact cgttgaagaa agaggtaaat tactacttcc 180

cagaattttt ttttttaaga ttccgttttg ccaaaaaaaa aaaacaaaaa tgatgttttt 240

gattcaggtt tggttgaaat tttgattatt ggatcgatac ttgtttcaga tttatttcta 300

attcagaaac gaaataatgg gaaatttttt tttcttttac ttgattttga cgtaaataaa 360

acttgatcaa ttagatgaga attgtttgag attgaagaaa atctggttga tttttctatg 420

tcaatttggc attcccataa tttaaattta tttgtgtttt tttttcgtga ttaataataa 480

tttagtaatt atggtgtaat acttgaaaca atttagctga ttattggcaa atttgtttcc 540

ctattttgat gatatcagaa gctttttgta taatccgaaa cgatgtcgag aaaatcacgt 600

ggatttggag cagagaaatc tctgtcgcct gaagagcagc aagccaaagt atgcatcaaa 660

atattgatta agatgttgtt actgacattt tttttttttg gttagttatt tggtggagat 720

tcattctttt ccaatgaatt tttctatcta tttttagttg taatttcgtg ttagttttgc 780

gcatttgcat cttcattatg gtttttccat ctaaataatt tgatatagcc tgtttgcttg 840

gatgcccaga gagttctttg tttctaaaat ctgaaaaaag gtaaatacgc agggaaatgt 900

gtctggttaa gagtttacag taataatagt tttcaattgt gaccctgaac taagtaaata 960

tcagagaaaa ataaatagtt cgttttgttg ttttctaaga tattataacc agattatatg 1020

tttgaaaaaa tctgaactgt attctacgtt ttaaagaatt gtattggcct catgaacgaa 1080

acagtctggc gattatttgg ttgaggattc ttgccctcat tagagtttga agggaaattt 1140

ttattttagt ctgtggagtc aaatgtaaaa aactctttag gagatcaact gattgaattt 1200

gatttcattc agataaatga ggttagaaag attattggtc ctattgctga taaatatcca 1260

gtactttgtt cggacgaatc aatatcaagg tacctcagag caaggaactg gcatacaaag 1320

aaggcaagca aaatgttagt agaaagcgtg aagtggaggc tggaatacaa gccagagaag 1380

atcgtatggg tatgcatata ttctttccag tagttttttc ttttttcttt tttgggtaaa 1440

ttaatctagt tttctcgaat atgtaaaaac ctttaaacaa agggaaaatg gaaagagata 1500

cttggttttg tttgtgaaaa ttcatcctat ttctgctgtt ggattgcaac atgcaatgct 1560

tgtttggcga ctagtaatct attactacaa catggatgct tagtaagctt ttgcaccata 1620

ctctttagct gtgctcttct aaactctgat ctatcaacta tgaaggataa aacaataaca 1680

ttaaagcaac ttacaagata ataattttct tttagctgct ggtactatat ctgcatattg 1740

ctgctatgta taacattaaa atcacctttt gttattgctt taggaagatg ttgctcgaga 1800

agctgaaaca gggaaacttt acagagctaa tttctgtgac aaattaggaa gacctgttct 1860

tataatgagg ccaggtttcc aggtagatag atccatattt ccatttggtt ctttcctctg 1920

ctgttattcc tatactgagt tcgatgatga gggattttaa caagttactg tgtcagaatt 1980

caagctcaac agaggggcag atcaagtact tggtgtattg catggaaaat gctataatga 2040

atttgaaccc tgatagggag caaatggtgt ggctaattga ttttcaaggg tggacaatgg 2100

ggagtgtgtc agtaaaagtg actcgggaaa cagctaatgt gttgcaaaat cattaccctg 2160

agagattggg cctggcaatc ctctataatc ccccaaaagt attcgaatct ttttggacgg 2220

tatctcactg ttcctttctt ttatataatc tttgcttgtc gagttgcggc attgtggtta 2280

tactataaat cttaatagag tcctctggag atgctcattt gctgctgagg agaacatgat 2340

tcagaccatt actgagcact gaatttgttt ttctaatgaa tgtaatttct tgttttactt 2400

gtcctctcaa caattttggc tgctgattgg aggaccactt tttcaatacc tgtctggaaa 2460

ggagtgagga atctctggtt atttccaatg tcgttgtatt gtctcgttct tttgcttcat 2520

tgatttcccc accaactttt ggccacctgg tactttcatt gctttggctt agctctgatg 2580

tgagacattg tctaagaaca ctctttaagg gtttcatgtg aattccgtat ctagagcttc 2640

ttcctctctg ttcttttggg agcattttcc atcctttatt tttctttctt ttttcatttg 2700

tatgaatgtg aagactaagc agtaatattg taaaaagatg gttagccttt gaaaatataa 2760

aacagctgag atggaaaagt cgtgccttct gctctggcaa cattttgtgt taggccactt 2820

cctgtttaca gtatgatgta ctttaccata tttataatac gagaaaagtt taagagaatt 2880

tttctttcat gcaggtggtg aaaccattcc ttgagccaaa gacatacaag aaagtgaggt 2940

ttgcctattc taatgatcct cagagccaga aaataatgga agccctcttt gatattaata 3000

aattggattc ttcttttggg ggaagaaaca gagttgggtt tgattatgaa gcatttgggc 3060

agcttatgag ggctgatgat aagaaaaagt ctgatttaat gaactccgga tgctcagtac 3120

caactgacca tctattagtt gcatctcagt catcacaatc cgagtcattg acatcagacc 3180

actgttcgga tgactctgat aacgagttag atgaggcaac ttccaccttg gaggatgttg 3240

atgagaaagt accggggctg aaactcggct atgatgatgt tccaaagagt gaagctgcaa 3300

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ccttctttac aaattctgat cataccttaa tgcaatctta ttcagttagt ttcttgaaag 3420

caagattttg accgaaatct tgctgctata ataatgagta tcccaaacta tttaagcaga 3480

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gccaaaataa atgaggttag aaagattatt ggtcctattg ctgataaata tccagtactt 120

tgttcggacg aatcaatatc aaggtacctc agagcaagga actggcatac aaagaaggca 180

agcaaaatgt tagtagaaag cgtgaagtgg aggctggaat acaagccaga gaagatcgta 240

tgggaagatg ttgctcgaga agctgaaaca gggaaacttt acagagctaa tttctgtgac 300

aaattaggaa gacctgttct tataatgagg ccaggtttcc agaattcaag ctcaacagag 360

gggcagatca agtacttggt gtattgcatg gaaaatgcta taatgaattt gaaccctgat 420

agggagcaaa tggtgtggct aattgatttt caagggtgga caatggggag tgtgtcagta 480

aaagtgactc gggaaacagc taatgtgttg caaaatcatt accctgagag attgggcctg 540

gcaatcctct ataatccccc aaaagtattc gaatcttttt ggacggtggt gaaaccattc 600

cttgagccaa agacatacaa gaaagtgagg tttgcctatt ctaatgatcc tcagagccag 660

aaaataatgg aagccctctt tgatattaat aaattggatt cttcttttgg gggaagaaac 720

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tcatcacaat ccgagtcatt gacatcagac cactgttcgg atgactctga taacgagtta 900

gatgaggcaa cttccacctt ggaggatgtt gatgagaaag taccggggct gaaactcggc 960

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<213> orange (Citrus grandis)

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

1 5 10 15

Glu Glu Gln Gln Ala Lys Ile Asn Glu Val Arg Lys Ile Ile Gly Pro

20 25 30

Ile Ala Asp Lys Tyr Pro Val Leu Cys Ser Asp Glu Ser Ile Ser Arg

35 40 45

Tyr Leu Arg Ala Arg Asn Trp His Thr Lys Lys Ala Ser Lys Met Leu

50 55 60

Val Glu Ser Val Lys Trp Arg Leu Glu Tyr Lys Pro Glu Lys Ile Val

65 70 75 80

Trp Glu Asp Val Ala Arg Glu Ala Glu Thr Gly Lys Leu Tyr Arg Ala

85 90 95

Asn Phe Cys Asp Lys Leu Gly Arg Pro Val Leu Ile Met Arg Pro Gly

100 105 110

Phe Gln Asn Ser Ser Ser Thr Glu Gly Gln Ile Lys Tyr Leu Val Tyr

115 120 125

Cys Met Glu Asn Ala Ile Met Asn Leu Asn Pro Asp Arg Glu Gln Met

130 135 140

Val Trp Leu Ile Asp Phe Gln Gly Trp Thr Met Gly Ser Val Ser Val

145 150 155 160

Lys Val Thr Arg Glu Thr Ala Asn Val Leu Gln Asn His Tyr Pro Glu

165 170 175

Arg Leu Gly Leu Ala Ile Leu Tyr Asn Pro Pro Lys Val Phe Glu Ser

180 185 190

Phe Trp Thr Val Val Lys Pro Phe Leu Glu Pro Lys Thr Tyr Lys Lys

195 200 205

Val Arg Phe Ala Tyr Ser Asn Asp Pro Gln Ser Gln Lys Ile Met Glu

210 215 220

Ala Leu Phe Asp Ile Asn Lys Leu Asp Ser Ser Phe Gly Gly Arg Asn

225 230 235 240

Arg Val Gly Phe Asp Tyr Glu Ala Phe Gly Gln Leu Met Arg Ala Asp

245 250 255

Asp Lys Lys Lys Ser Asp Leu Met Asn Ser Gly Cys Ser Val Pro Thr

260 265 270

Asp His Leu Leu Val Ala Ser Gln Ser Ser Gln Ser Glu Ser Leu Thr

275 280 285

Ser Asp His Cys Ser Asp Asp Ser Asp Asn Glu Leu Asp Glu Ala Thr

290 295 300

Ser Thr Leu Glu Asp Val Asp Glu Lys Val Pro Gly Leu Lys Leu Gly

305 310 315 320

Tyr Asp Asp Val Pro Lys Ser Glu Ala Ala Met Ala Lys Gln Val Gln

325 330 335

<210> 4

<211> 1003

<212> DNA

<213> orange (Citrus grandis)

<400> 4

gagaaaatca cgtggatttg gagcagagaa atctctgtcg cctgaagagc agcaagccaa 60

aataaatgag gttagaaaga ttattggtcc tattgctgat aaatatccag tactttgttc 120

ggacgaatca atatcaaggt acctcagagc aaggaactgg catacaaaga aggcaagcaa 180

aatgttagta gaaagcgtga agtggaggct ggaatacaag ccagagaaga tcgtatggga 240

agatgttgct cgagaagctg aaacagggaa actttacaga gctaatttct gtgacaaatt 300

aggaagacct gttcttataa tgaggccagg tttccagaat tcaagctcaa cagaggggca 360

gatcaagtac ttggtgtatt gcatggaaaa tgctataatg aatttgaacc ctgataggga 420

gcaaatggtg tggctaattg attttcaagg gtggacaatg gggagtgtgt cagtaaaagt 480

gactcgggaa acagctaatg tgttgcaaaa tcattaccct gagagattgg gcctggcaat 540

cctctataat cccccaaaag tattcgaatc tttttggacg gtggtgaaac cattccttga 600

gccaaagaca tacaagaaag tgaggtttgc ctattctaat gatcctcaga gccagaaaat 660

aatggaagcc ctctttgata ttaataaatt ggattcttct tttgggggaa gaaacagagt 720

tgggtttgat tatgaagcat ttgggcagct tatgagggct gatgataaga aaaagtctga 780

tttaatgaac tccggatgct cagtaccaac tgaccatcta ttagttgcat ctcagtcatc 840

acaatccgag tcattgacat cagaccactg ttcggatgac tctgataacg agttagatga 900

ggcaacttcc accttggagg atgttgatga gaaagtaccg gggctgaaac tcggctatga 960

tgatgttcca aagagtgaag ctgcaatggc gaaacaagta caa 1003

<210> 5

<211> 652

<212> DNA

<213> orange (Citrus grandis)

<400> 5

agaggggcag atcaagtact tggtgtattg catggaaaat gctataatga atttgaaccc 60

tgatagggag caaatggtgt ggctaattga ttttcaaggg tggacaatgg ggagtgtgtc 120

agtaaaagtg actcgggaaa cagctaatgt gttgcaaaat cattaccctg agagattggg 180

cctggcaatc ctctataatc ccccaaaagt attcgaatct ttttggacgg tggtgaaacc 240

attccttgag ccaaagacat acaagaaagt gaggtttgcc tattctaatg atcctcagag 300

ccagaaaata atggaagccc tctttgatat taataaattg gattcttctt ttgggggaag 360

aaacagagtt gggtttgatt atgaagcatt tgggcagctt atgagggctg atgataagaa 420

aaagtctgat ttaatgaact ccggatgctc agtaccaact gaccatctat tagttgcatc 480

tcagtcatca caatccgagt cattgacatc agaccactgt tcggatgact ctgataacga 540

gttagatgag gcaacttcca ccttggagga tgttgatgag aaagtaccgg ggctgaaact 600

cggctatgat gatgttccaa agagtgaagc tgcaatggcg aaacaagtac aa 652

<210> 6

<211> 633

<212> DNA

<213> orange (Citrus grandis)

<400> 6

ttggtgtatt gcatggaaaa tgctataatg aatttgaacc ctgataggga gcaaatggtg 60

tggctaattg attttcaagg gtggacaatg gggagtgtgt cagtaaaagt gactcgggaa 120

acagctaatg tgttgcaaaa tcattaccct gagagattgg gcctggcaat cctctataat 180

cccccaaaag tattcgaatc tttttggacg gtggtgaaac cattccttga gccaaagaca 240

tacaagaaag tgaggtttgc ctattctaat gatcctcaga gccagaaaat aatggaagcc 300

ctctttgata ttaataaatt ggattcttct tttgggggaa gaaacagagt tgggtttgat 360

tatgaagcat ttgggcagct tatgagggct gatgataaga aaaagtctga tttaatgaac 420

tccggatgct cagtaccaac tgaccatcta ttagttgcat ctcagtcatc acaatccgag 480

tcattgacat cagaccactg ttcggatgac tctgataacg agttagatga ggcaacttcc 540

accttggagg atgttgatga gaaagtaccg gggctgaaac tcggctatga tgatgttcca 600

aagagtgaag ctgcaatggc gaaacaagta caa 633

<210> 7

<211> 613

<212> DNA

<213> orange (Citrus grandis)

<400> 7

tgctataatg aatttgaacc ctgataggga gcaaatggtg tggctaattg attttcaagg 60

gtggacaatg gggagtgtgt cagtaaaagt gactcgggaa acagctaatg tgttgcaaaa 120

tcattaccct gagagattgg gcctggcaat cctctataat cccccaaaag tattcgaatc 180

tttttggacg gtggtgaaac cattccttga gccaaagaca tacaagaaag tgaggtttgc 240

ctattctaat gatcctcaga gccagaaaat aatggaagcc ctctttgata ttaataaatt 300

ggattcttct tttgggggaa gaaacagagt tgggtttgat tatgaagcat ttgggcagct 360

tatgagggct gatgataaga aaaagtctga tttaatgaac tccggatgct cagtaccaac 420

tgaccatcta ttagttgcat ctcagtcatc acaatccgag tcattgacat cagaccactg 480

ttcggatgac tctgataacg agttagatga ggcaacttcc accttggagg atgttgatga 540

gaaagtaccg gggctgaaac tcggctatga tgatgttcca aagagtgaag ctgcaatggc 600

gaaacaagta caa 613

<210> 8

<211> 64

<212> DNA

<213> orange (Citrus grandis)

<400> 8

agaggggcag atcaagtact tggtgtattg catggaaaat gctataatga atttgaaccc 60

tgat 64

Claims (2)

1. The application of a vector promoter in driving gene expression in prokaryotes is characterized in that: the carrier promoter is any one of sequences CitPITP1CDS, CitPITP1NO, CitPITP1Narrow1 and CitPITP1.1_ key64, and the nucleotide sequences of the carrier promoter are shown in SEQ ID NO: 2. 4, 5 and 8.

2. Use according to claim 1, characterized in that: the carrier promoter is CitPITP1CDS, and the nucleotide sequence is shown in SEQ ID NO: 2, respectively.

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