CN114875060B - Use of dORF in enhancing translation of upstream encoding genes - Google Patents

Abstract

The invention discloses an application of dORF in enhancing upstream coding gene translation, and belongs to the technical field of genetic engineering. The invention discovers that a large amount of ribosome signals exist in a non-coding region of a gene by carrying out ribosome imprinting sequencing on ovules of Asian cotton in four different development periods of 0 day, 5 day, 10 day and 20 day and seedlings treated by 14 days and four abiotic stresses (ultraviolet, salt, high temperature and low temperature), and proves that an open reading frame with coding capability exists in the non-coding region of the gene and an open reading frame dORF with coding capability in a 3' UTR region downstream of the gene can enhance the translation capability of genes upstream of the ovules, and meanwhile, the same phenomenon exists in other monocotyledonous and dicotyledonous plants. Based on the function of dORF, the gene can be used as an element to regulate the translation efficiency of the gene, plays a role in a transgenic or stably expressed gene system, and has great application potential and application prospect.

Description

Use of dORFs to enhance translation of upstream coding genes

technical field

The invention relates to the technical field of genetic engineering, in particular to the application of dORF in enhancing the translation of upstream coding genes.

Background technique

In recent years, with the development of sequencing technology, great breakthroughs have been made in the study of protein level. The conclusion that each eukaryotic messenger RNA (mRNA) encodes a protein has also been revised with the continuous improvement of ribosomal imprint sequencing technology and mass spectrometry sequencing methods. Ribosomal imprinting analysis has revealed that the non-coding regions 5'UTR and 3'UTR of many genes have the potential to encode proteins, and in multiple species and viruses, it has been confirmed that long non-coding RNAs (lncRNAs) can also encode proteins. In most cases, the open reading frame with coding ability located in the 5'UTR region of the gene, called uORF (upstream openreading frame), can inhibit the translation ability of downstream genes.

The open reading frame with coding ability in the 3'UTR region of the gene is called dORF (down stream open reading frame). In plants and animals, no systematic research has been done on its related characteristics and functions. According to the data of the present invention, it is found that in plants, dORF (down stream open reading frame) can promote the translation ability of upstream genes, and regulating the translation level of genes through dORF may become a new transformation method, which can be applied to the cultivation of high-yielding plant varieties As well as improving the response to stress, it has very important biological significance.

Contents of the invention

The present invention confirms that plant endogenous dORF (down stream open reading frame) has the ability to promote the translation of upstream genes, and dORF can be used as a new way to regulate the level of gene translation. Based on this, the present invention provides dORF in enhancing the translation of upstream coding genes Applications.

In the present invention, ribose is carried out on the ovules of Asian cotton in four different development stages: 0 day, 5 days, 10 days, 20 days, and 14 days and four kinds of abiotic stress (ultraviolet, salt, high temperature, low temperature) treatment of seedlings. For body imprint sequencing, the quality of the data was first evaluated, with an obvious 3-nt periodicity, indicating that the acquisition is valid data. The next step is to comprehensively annotate the sorf (small open reading frame) of the Asian cotton genome, and find that there are a large number of open reading frames with coding potential in the non-coding region of the coding gene. In order to further understand the significance of the existence of these open reading frames with coding potential, the present invention analyzes their basic characteristics. Compared with the coding region of the gene, the upstream open reading frame uORF and the downstream open reading frame dORF have shorter amino acid length, about 100 amino acids; the translation ability is similar to the coding region of the gene. relatively weak.

In order to explore the biological significance and potential functions of Asian cotton sorf, the present invention firstly compared the translation efficiency of genes containing uORF and genes without uORF under the same transcription level. The present invention randomly selected 3000 examples, and found that the translation efficiency of genes containing uORF was significantly lower than that of genes without uORF, which was consistent with the currently known conclusions, while dORF showed the opposite trend, and the gene without dORF The translation efficiency of genes containing dORF is significantly higher than that of uORF, which indicates that dORF plays the opposite function to uORF.

In order to prove the existence of dORF and the ability to enhance the translation efficiency of upstream genes, the present invention uses the tobacco transient transformation system under in vitro conditions to fuse the commercial FLAG tag to the dORF sequence, and uses Tricine-SDS-PAGE to confirm the dORF in vitro translation. In order to prove that dORF has the function of enhancing the translation efficiency of upstream genes, the present invention compares the translation efficiency of upstream genes by deleting the dORF sequence, and the upstream genes in the present invention select the gene encoding luciferase. By detecting the activity of the fluorescence, it was found that when the dORF sequence was deleted so that the dORF could not be translated, the fluorescence activity decreased significantly, indicating that the non-translation of the dORF affected the translation level of the upstream gene. In order to determine whether the dORF affects the translation product of the gene through the transcription process, the present invention uses rt-qpcr to quantify the gene transcription level, and finds that there is no difference in the transcription level of the fluorescent gene compared with the unaltered dORF sequence. It shows that the presence of dORF can indeed enhance the translation efficiency of the upstream gene without affecting the transcription level of the upstream gene.

From the above, it can be known that the plant dORF has the application of enhancing the translation of its upstream coding gene.

Further, the plants include monocotyledonous plants and dicotyledonous plants.

Furthermore, said plants include Asian cotton, rice, tomato.

A method for enhancing the translation efficiency of a target gene is to insert a plant dORF into the 3'UTR region of the target gene.

Further, the dORF sequence includes but not limited to the sequences shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, and SEQ ID NO.11.

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

The present invention finds for the first time that the presence of dORF in plants can promote the translation efficiency of upstream coding genes, and proves the translation of dORF and the function of enhancing the translation efficiency of upstream genes under in vitro conditions. Based on the function of dORF, it can be used as an element to regulate the translation efficiency of genes and play a role in transgenic or stable gene expression systems, and has great application potential and application prospects. The research of the present invention shows that in both monocotyledon and dicotyledonous plants, the dORF with translation potential can improve the translation efficiency of upstream genes.

Description of drawings

Figure 1 is the analysis result of the 3-nt periodicity of ribosomal imprinting database construction data by bioinformatics.

Figure 2 is the distribution map of the Asian cotton genome through the statistical ribosome imprinting database construction data of bioinformatics.

Figure 3 is the trend of translation efficiency of genes containing uORF or dORF and genes without uORF or dORF respectively under the condition of no difference in transcription level through bioinformatics analysis.

Figure 4 is a schematic diagram of the PGX-5dual vector. In the figure, 35S: strong promoter; LUC: luciferase, firefly luciferase; Sal, spe: restriction enzyme site; REN: Renilla luciferase; HA: commercial tag.

Figure 5 shows four examples of Ga13g02057, Ga01g00361, Ga05g01077, Ga05g01995 gene structure maps and visualization of ribosomal imprinting and polyA-seq sequencing data, indicating that the 3'UTR region of these genes contains dORF (the dotted box is the annotated dORF position).

Figure 6 is a schematic diagram of the product of synthetic sequences linked to PGX-5dual. In the figure, dORF: the complete 3'UTR sequences of the four genes Ga13g02057, Ga01g00361, Ga05g01077, and Ga05g01995 are connected to the vector using the sal I and speI restriction enzyme sites respectively; delete: the four genes Ga13g02057, Ga01g00361, Ga05g01077 The annotated dORF sequence was deleted from the 3'UTR sequence of Ga05g01995, and other sequences were connected to the vector using sal I and spe I restriction enzyme sites respectively.

Figure 7 is a schematic diagram of the vector construction product of the dORF fused with the FLAG tag annotated by the gene Ga11g02302 and the results of the Tricine-SDS-page. In the figure, dORF FLAG: the dORF fusion FLAG tag annotated by Ga11g02302 was ligated to the vector using sal I and spe I restriction sites; Control: empty vector, dORF: the vector containing dORF fusion FLAG annotated by Ga11g02302.

Figure 8 is a graph showing the experimental results of dual fluorescence detection, rt-qPCR, and western blot of four genes. In the figure, LUC/RENactivity: firefly enzyme activity/renilla luciferase activity; LUC mRNA level: firefly enzyme RNA expression level; delete: delete the dORF sequence of the 3'UTR of the gene, and other sequences remain unchanged; UTR: the complete gene 3'UTR sequence; sample: protein sample; control: internal reference, Renilla luciferase.

Figure 9 shows the trend of translation efficiency of genes containing uORF or dORF and genes without uORF or dORF respectively under the condition of no difference in transcription level through bioinformatics analysis using ribosomal imprinting and RNA-seq sequencing data of rice.

Figure 10 shows the trend of translation efficiency of genes containing uORF or dORF and genes without uORF or dORF respectively under the condition of no difference in transcription level through bioinformatics analysis using ribosome imprinting and RNA-seq sequencing data of tomato.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

In the following examples, unless otherwise specified, all are conventional methods.

Cotton and tobacco are cultivated according to the following methods in the following examples:

Cotton material for greenhouse planting: remove the cottonseed husks of cotton seeds, put them in small pots containing vermiculite, and move them into a greenhouse at 28°C for 14 days. Put the cotton in the incubator for 5 hours, quickly rinse it with pure water and put it into liquid nitrogen for quick freezing, and store the sample at -80°C; (2) Adjust the temperature of the incubator to 4°C, put the 14-day-old cotton in the incubator for 15 hours, Rinse quickly with pure water and put in liquid nitrogen for quick freezing, and store the sample at -80°C; (3) Prepare 500mM NaCl, pour 20mL into a small pot for cultivating seedlings, cultivate at 28°C for 24 hours, quickly rinse with pure water and put in liquid nitrogen for quick freezing , store the sample at -80°C; (4) adjust the UV intensity of the incubator to 1.24 μmol m ^-2 s ^-1 , incubate at 28°C for 2 hours, quickly rinse with pure water and freeze in liquid nitrogen, and store the sample at -80°C; ( 5) Cotton seedlings growing normally after 14 days were quickly rinsed with pure water and put into liquid nitrogen for quick freezing, and the samples were stored at -80°C.

Planting tobacco materials in the greenhouse: Spread the tobacco seeds evenly in small pots containing vermiculite, and move them into a greenhouse at 28°C for 4-5 weeks.

Example 1 Annotation and function discovery of Asian cotton dORF

(1) Ribosome imprinting data analysis

Ribosomal imprinting of cotton ovules at four different developmental stages (0 days, 5 days, 10 days, 20 days) and seedlings at 14 days and four abiotic stress treatments (UV, salt, high temperature, low temperature) Sequencing, to assess the quality of the sequencing data.

1. Raw sequencing data quality control: use the second-generation sequencing quality control software cutadapt to remove the sequencing adapter (AGATCGGAAGAG) of the raw sequencing data (rawdata) and the sequence longer than 40 bp after removing the adapter, and obtain the filtered cleandata.

2. Remove rRNA, tRNA, and snRNA contamination: use the sequence comparison software bowtie to compare the rRNA, tRNA, and snRNA in cleandata and the Rfam database (https://rfam.xfam.org/), allowing up to two mismatches , keep the unaligned sequences.

3. Align the sequence to the reference genome: Use the sequence comparison software STAR to compare the unaligned sequence with the Asian cotton reference genome, allowing up to two mismatches. The sequence and annotation files of the reference genome come from MaGenDB ( http://magen.whu.edu.cn/), to get the final comparison file.

4. Quality control of ribosomal imprinting library construction: use the read_distribution.py script of rseqc to detect the distribution of sequences on the genome, and use ribotricer to detect the periodicity of ribosomal imprinting sequencing data.

The results are shown in Figure 1. The sequencing data has obvious 3-nt periodicity, indicating that the obtained data is valid.

(2) Discovery of uORF and dORF annotations and functions

1. Prediction of open reading frames: use PRICE to predict open reading frames with potential coding ability, and get annotation information of uORF and dORF.

The results are shown in Figure 2. 75.65% of the sequencing data are distributed in the coding region of the Asian cotton genome, 0.48% are distributed in the intron region, 4.43% are distributed in the 5'UTR region, 2.96% are distributed in the 3'UTR region. 16.48% are distributed in other area.

2. Quantitative analysis of genes at transcription level and translation level and calculation of translation efficiency: use salmon to quantify RNA-seq data and ribo-seq data, calculate gene expression values at transcription level and translation level; calculate gene translation efficiency (Translation efficiency=log2(expression value of gene transcription level/expression value of gene translation level)). 3000 examples were randomly selected for the following analysis.

3. The effect of dORF on gene translation efficiency: randomly sample genes according to the expression value of RNA level, and count the differences in translation efficiency between genes with dORF and genes with similar RNA levels but without dORF.

The results are shown in Figure 3. In the randomly selected examples, the translation efficiency of genes containing uORF was significantly lower than that of genes without uORF, while dORF showed the opposite trend. Compared with genes without dORF, genes containing dORF The translation efficiency is significantly higher.

(3) Proof of dORF function

1. Construction of the carrier

The carrier selected the dual fluorescent carrier PGX (Guoyong Xu, uORF-mediated translation allowed engineered plant disease resistance without fitness costs, 2017), and carried out the following transformation: entrusted Wuhan Aoke Dingsheng Biotechnology Co., Ltd. to add sal I after the carrier LUC gene sequence and spe I restriction enzyme site, the vector was named PGX-5dual.

The results are shown in Figure 4, adding sal I and spe I restriction enzyme sites after the LUC gene.

2. Select the 3'UTR sequence containing the dORF annotation gene for artificial synthesis

Through the analysis of (1) and (2), the following four genes were selected: Ga13g02057, Ga01g00361, Ga05g01077, Ga05g01995. These 4 genes all contain dORF annotations. Wuhan Aoke Dingsheng Biotechnology Co., Ltd. was commissioned to synthesize the 3'UTR sequences of the 4 genes, and delete the dORF sequences annotated according to bioinformatics, using sal I and speI restriction enzymes Spot ligation to vector PGX-5dual. The complete 3'UTR sequences of the synthetic genes were named Ga13g02057-3'UTR (SEQ ID NO.1), Ga01g00361-3'UTR (SEQ ID NO.4), Ga05g01077-3'UTR (SEQ ID NO.7) , Ga05g01995-3'UTR (SEQ ID NO.10); the sequences of dORFs annotated by bioinformatics in these sequences are respectively as SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11 shown. The sequences of dORF deletion were synthesized and named as Ga13g02057-dORF-delete (SEQ ID NO.3), Ga01g00361-dORF-delete (SEQ ID NO.6), Ga05g01077-dORF-delete (SEQ ID NO.9), Ga05g01995-dORF - delete (SEQ ID NO. 12). Both ends of the synthesized sequence contain restriction sites of sal I and spe I.

Figure 5 is the visualization of ribosomal imprinting and polyA-seq sequencing data of four genes, indicating that the four genes contain dORF annotations, and Figure 6 is a schematic diagram of the product of linking the synthetic sequence to the vector.

2. Transform the successfully ligated vector into Agrobacterium GV3101

The constructed plasmid was transformed into Agrobacterium (Vidibiology) GV3101, and the Agrobacterium strain containing the target gene was obtained by screening. The method of transferring into Agrobacterium is as follows:

(1) Take the competent Agrobacterium stored at -80°C at room temperature or in the palm of your hand for a while until it partially melts, and insert it into the ice when it is in the state of mixing ice and water.

(2) Add 1 μg of plasmid DNA per 100 μL of competent cells, beat the bottom of the tube by hand to mix, and then place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

(3) Add 700 μL of LB without antibiotics, shake and culture at 28°C for 2-3 hours.

(4) Centrifuge at 6000rpm for one minute to collect the bacteria, take about 100 μL of the supernatant and gently pipette the resuspended bacteria onto the LB plate containing the corresponding antibiotic, and place it upside down in an incubator at 28°C for 2-3 days.

3. Instant transformation of tobacco system

(1) Pick a single clone and culture it in 5 mL LB liquid medium with shaking at 28-30°C. Add 100 μg/mL gentamicin (Agrobacterium strain GV3101 carrying resistance), 50 μg/mL kanamycin (carrying vector) to LB.

(2) Transfer 1 mL of overnight cultured Agrobacterium into 25 mL of LB liquid medium (adding gentamicin and kanamycin, and adding autoclaved acetosyringone). Detect the OD value of the bacterial solution cultured overnight, and use LB medium to adjust the OD value to 0.4.

(3) Centrifuge at 5000 g for 15 minutes to collect the bacteria, and resuspend the bacteria with a resuspension solution (10 mM MgCl ₂ , 10 mM MES, 5 mM AS), and the final OD600 is 0.4.

(4) After standing at room temperature for 2-3 hours, inject tobacco grown in a greenhouse at 28°C for 4-5 weeks. Put the suspension liquid into a 5mL syringe, and press the syringe with your thumb to inject the liquid from the lower epidermis of the leaf into the tobacco leaf. After injection, culture in a 28°C incubator for 2-3 days, put the leaves in liquid nitrogen for quick freezing, and store at -80°C.

4. Extraction of tobacco protein

Pre-cool the mortar with liquid nitrogen, cut the tobacco leaves injected with bacterial liquid into the mortar, add liquid nitrogen, quickly grind to powder, transfer the powder to a 1.5mL EP tube, add 1mL protein extract (10mM Tric-Hcl PH =8.0, 20mM EDTA (PH=8.0, 1mM PMSF, 1mM DTT, 1% Triton-100), lyse on ice for 30min. Centrifuge at 12000rpm at 4°C for 10min, transfer the supernatant to a new 1.5mL EP tube, the supernatant is total tobacco protein.

5. Tricine-SDS-page proves the translation of dORF in vitro

In order to prove the translation of the dORF, Wuhan Aoke Dingsheng Biotechnology Co., Ltd. was commissioned to synthesize the 3'UTR sequence of the gene Ga11g02302, and fused the FLAG tag (SEQ ID NO.13) before the stop codon of the annotated dORF sequence, using sal I and The spe I restriction site was connected to the PGX-5dual vector, and the protein was extracted by the tobacco transient transformation method (same as 3, 4), using Tricine-SDS-page (Hermann Tricine–SDS-PAGE, 2006) method, using the primary antibody as FLAG-Tag (Wuhan Dion Bio FLAG-Tag mouse mAb), the secondary antibody as Goat anti-mouse (Agrisa), using the empty vector as the control, to prove the dORF real translation.

The schematic diagram of the vector construction product of the Gal1g02302-annotated dORF fused with the FLAG tag and the results of the Tricine-SDS-page are shown in Figure 7. The dORF fused with the FLAG tag can indeed be translated.

6. Double fluorescence detection

Dual fluorescence detection was performed using the Dual Luciferase Reporter Assay Kit of Nanjing Nuoweizyme.

Take 50 mg of tobacco leaves, grind with liquid nitrogen, lyse with 200 μL lysate, centrifuge at 12000 rpm for 5 min, and take the supernatant. Take 20 μL of the supernatant and add it to a black microplate, first add 100 μL Luciferase Assay Reagent II, measure with a microplate reader after 2 seconds, then add 100 μL Stop&Glo Reagent for measurement. Through double fluorescence detection, it was found that compared with the complete 3'UTR sequence, the activity of the upstream LUC gene with the dORF sequence deleted was significantly reduced (Figure 8).

7. Western blot detection of Luciferase protein content

The extraction method of tobacco protein was as described in 4, and the protein amount of LUC gene was detected by western blot. The internal reference is PGX-5dual fused HA-tagged REN Renilla protein, the internal reference primary antibody is HA-Tag (Wuhan Dion Bio HA-Tag mouse mAb), the secondary antibody is Goat anti-mouse (Agrisa), internal reference western blot is the same as LUC protein The method is the same, the method is as follows:

(1) Mix 20 μL protein sample with 4 μL 5×SDS-Loding buffer (Beijing Quanshijin), and denature at 99°C for 5 minutes;

(2) Perform polyacrylamide gel electrophoresis on the denatured sample, 90V voltage for 30min, 150V voltage for 1 hour;

(3) Cut a piece of nitrocellulose membrane the same size as the polyacrylamide gel, six pieces of filter paper the same size as the sponge, and place the filter paper and sponge in the transfer solution for later use;

(4) Treat the nitrocellulose membrane in absolute ethanol for 10-20s, transfer it to pure water for 2-3min, and place the transfer solution (see below for the formula);

(5) One layer of sponge, three layers of filter paper, nitrocellulose membrane, polyacrylamide gel, three layers of filter paper, and one layer of sponge;

(6) Put the clip into the transfer tank, with the black side of the clip facing the black side of the tank, and the white side facing the red side of the tank, add the transfer solution, cover the tank, put the tank in an ice box, run 200mA constant current, electrophoresis for 2h ;

(7) Seal with 5% liposuction milk powder for 2 hours, and wash the membrane 3 times, 5 minutes each time;

(8) Put in the primary antibody working solution (Agrisa, LUC|Luciferase), incubate at room temperature for 2 hours, wash the membrane 3 times with TBST, 5 minutes each time;

(9) Put in the secondary antibody working solution (Agrisa, Goat anti-Rabbit), incubate at room temperature for 2 hours, wash the membrane 3 times with TBST, 5 minutes each time;

(10) Spread the developing solution evenly on the film, and close the developing box;

(11) Open the developing box in the developing room, take out a negative film, fold it up, as a mark, first close the developing box and time exposure for 1 minute, then turn the negative upside down, close the developing box, expose for 2 minutes, put the film in Developing device, waiting for the result of film development.

Recipe for transfer solution: Weigh 8.8618g of glycine into a 1L beaker, add 60mL of absolute ethanol into the beaker, stir well to dissolve and add ddH ₂ O to dilute the solution to 600mL for later use.

TBST formula: Weigh 8.8g NaCl into a 1L beaker, add 20mL 1M Tris-HCl (pH=8) and 800mL ddH ₂ O into the beaker, stir well to dissolve, add 0.5mL Tween-20 and mix well, add ddH ₂ O dilute the solution to 1 L for later use.

The results are shown in Figure 8. Compared with the complete 3'UTR sequence, the protein content of the upstream LUC gene with the deleted dORF sequence was significantly reduced.

8. Detect the expression level of LUC gene by RT-PCR

The plant total RNA of tobacco plants was extracted using the polysaccharide polyphenol plant total RNA extraction kit (purchased from Tiangen Company, RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich), and the reverse transcription kit (purchased from Beijing Quanshijin Company ( EasyScript One-Step gDNA Removal and cDNA SynthesisSuperMix) was reverse-transcribed to obtain cDNA. Utilize primers LUC-F, LUC-R, REN-F, REN-R (Table 1) to detect the expression of LUC by PCR. The results showed that dORF was deleted Compared with the complete 3'UTR sequence, there was no significant difference in the transcription level of the LUC gene (Fig. 8).

Table 1

Primer name Primer sequence (5'-3') LUC-F GGATTACAAGATTCAAAGTGCG LUC-R TGATACCTGGCAGATGGAAC REN-F CCTGGGACGAGTGGCCTGACA REN-R AGTTGCGGACAATCTGGACGAC

Example 2 Analysis of uORF and dORF functions in other plant species

In order to prove that dORF enhances the translation of upstream coding genes in a wide range of plant species, the published ribosomal imprinting sequencing data and RNA-seq data of other plants (tomato data source: Hsin-Yen Larry Wu, Gaoyuan Song, Justin W .Walley, Polly Yingshan Hsu(2019)The Tomato Translational Landscape Revealed by Transcriptome Assembly and Ribosome Profiling.PlantPhysiol.Sep;181(1):367–380.Published online 2019Jun 27.doi:10.1104/pp.19.00541; Rice data source: Yang X, Cui J, Song B, Yu Y, Mo B and Liu L(2020) Construction of High-Quality Rice Ribosome Footprint Library.Front.PlantSci.11:572237.doi:10.3389/fpls.2020.572237), using (1 ) and (2), the translation efficiency of uORF and dORF in rice and tomato was analyzed, and the results were consistent with those of Asian cotton.

The specific results are shown in Figures 9 and 10: the translation efficiency of genes containing uORF is significantly lower than that of genes without uORF; while dORF shows the opposite trend, compared with genes without dORF, the translation efficiency of genes containing dORF is significantly lower high.

sequence listing

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aacatttctg acatttcaag ggctgctgcc tggttcccta tctgtttgca gtttgtggtt 780

tctgtgttgc tgtggaatat tcagaagctg ctgccctcca ctgtttgaac cagggccacc 840

tcctccgtag atctgcattc ttttttctct ttttggaatg tatgagtatt ttacaaggat 900

ttaattattt ggaacatgat gattggttct tgtggtttgt cattcttctt attatctgct 960

aatctatgta gcaactgaga ttagtgatgc aaaatttatg aatttatagc tttcttctac 1020

tag 1024

<210> 8

<211> 278

<212>DNA

<213> Asian cotton (Asiatic cotton)

<400> 8

atggatccac ctcctctacc tccaactcca ccaccgacga caccgctgct agctccgtca 60

cctccagaag gagggttatg ggaatcatgg taagatgttc tctccatgttttgcattt 120

tacttgtgca tgaagaaaca aacagggaaa catttctgac atttcaaggg ctgctgcctg 180

gttccctatc tgtttgcagt ttgtggtttc tgtgttgctg tggaatattc agaagctgct 240

gccctccact gtttgaacca gggccacctc ctccgtag 278

<210> 9

<211> 746

<212>DNA

<213> Asian cotton (Asiatic cotton)

<400> 9

gtcgaccatg ctatatacac aagttagcca ttggattttt gagaaaatat ttctttgtta 60

ttttatttgt tctggagagt atttttggcg gaataggcca aacataggta ggttgtgtta 120

gtactaggtc acagtttatt ccttgattgg atttcatgcg tttgtacaga aaatgaaaaa 180

tcaaatgaag tttgtagatg agagtgcatt ggtcctcttt cagctttctt gatggacctc 240

actagcttgt tcctaagcaa atatatgctg gtaattttgt catatttctc ctcattttag 300

ctttaaacca gatgagttgt catcaaatat tcaactccca agttttaaat tgtgtctcac 360

tccacctatg ctctataaaa ataattttaa ccagtacaaa gttcctgtgttagttgggtg 420

tcatttacta ttaattattgc agagagttga aatctccctt tgaatggtag ttgtcatatg 480

actttgagaa aggatggtgt aattcatcaa cttatataac aatctcaccg gatataagat 540

atctttccaa agtcttatct cctcaaccaa caatctgcat tcttttttct ctttttggaa 600

tgtatgagta ttttacaagg atttaattat ttggaacatg atgattggtt cttgtggttt 660

gtcattcttc ttattatctg ctaatctatg tagcaactga gattagtgat gcaaaattta 720

tgaatttata gctttcttct actagt 746

<210> 10

<211> 1977

<212>DNA

<213> Asian cotton (Asiatic cotton)

<400> 10

gtcgacgaat gagatgtgtg tacggaaaag aatcgatcaa atgttcaata acaagtattc 60

atgcatattg aagatggata gaaagctttg ttggggtgat atatcagatt atcagagtcg 120

aagggaaaag cggtgtaaat aaaaacacct aacagaagcg tgtaacagtg tcattcggac 180

cggctggttt tgcagtggtg aatggatatt tttttgttgg gttagtgcac actttaatgt 240

agtataaaat gttgttttgt tttgtaatac aaatatttgt gttatatctt tttgttcatt 300

gtgtttatgc tatatataca tatcaggttt aaagaattat ttattaacat attaataatt 360

tatgctacat aaataatgca tttattttat ttatattaaa atatattata ttaaaaatgt 420

taatcactgt tagtaaaatt gattaaggtg ttaaaaaaat tgtaataaat aattaccttt 480

tttgtgtaaa taatattata tttgtgtaat agttttcctt ttttctaaag aaagccaaaa 540

agaaagtaaa tcgttggtga ataaattatc aataaatgaa aaaagggtca ataatatttg 600

ctggactccc tccctgaccg tcggtgtttc aaacagaagc agagcaactg ttttggtcgg 660

actcttttgg tggtttgttc cgatgttcaa agattgtggg tattggatct cgttttcttc 720

tcaatgttga cgcgtctgca gagtaacaaa cttttctcca aggtttgtat agttaaatgg 780

ggtttccttt ctacactttc gcttgcgtcg tattatctga cttattgttc tcgcgatctg 840

cgtttttatt tttaggttct tctgagtcgt tacaaaaacg ggtttcccaa tcttatctct 900

gggtctcaat ttttaagagg ttttaacgag attacaaagc ataggttttc tgtaatgtcg 960

ggtgaaaatc taagaagcct atctaacagt gcttccccga ggaattaccg agtcgtagtg 1020

gctgcaactc gtgaaatggg gatagggaaa gatggcaagt tgccatggag attgccttct 1080

gatctcaaat tcttcaagga acttacagtg acaacatcag atcctgagaa gaagaatgct 1140

gtagtaatgg gtagaaaaac ctgggagagt attccacttg agtttagacc tttacccggt 1200

cgcctgaatg ttgtccttac tcgttcccag agttctgata ttacaactgg agaaaatgtt 1260

gtaatatgtg ggagcattcc atcagctttg gaactattag ccgaggttcc ttatgtttt 1320

gcaatagaga aggtgtttgt cattggcggt ggccagatat tcaggtagat tttttagtaa 1380

aagttgttga atttcattgc atatttgttg gggaaagata atgttttgat gttttacaat 1440

gcagggaaac actcaatgct tcaggttgcg aagccattca cattactgaa attgggacaa 1500

gcattgaatg cgataccttc attccttcaa ttgactcgtc ttgtttccag ctgtggtact 1560

cttcgaagcc attggaagaa aataatgtcc ggttttcatt tgcaacttat gttcgtgtta 1620

gatctaggac aactgataat tatgaggtaa aagacttgag tttcttgccc aggatgattg 1680

ttgagagacg agatgaatga gtcaattagt aaataatact gagacttgtt catgaaacta 1740

tctcaagtga caagcaagac ccttgactgg ttagatacag aattcattg cctagtagt 1800

cttaaacctt ggttctctat gactctactg ctctttgcct tacattttat tgttgttgct 1860

gatgtttaaa tgtaaatatg agcatcatcc ccagtccacg ttgtgatttt gtacaatgta 1920

agtctatctt tgcagatctt ttcatcttct tatataacta aggtatttac tactagt 1977

<210> 11

<211> 48

<212>DNA

<213> Asian cotton (Asiatic cotton)

<400> 11

atgttcaaag attgtgggta ttggatctcg ttttcttctc aatgttga 48

<210> 12

<211> 1929

<212>DNA

<213> Asian cotton (Asiatic cotton)

<400> 12

gtcgacgaat gagatgtgtg tacggaaaag aatcgatcaa atgttcaata acaagtattc 60

atgcatattg aagatggata gaaagctttg ttggggtgat atatcagatt atcagagtcg 120

aagggaaaag cggtgtaaat aaaaacacct aacagaagcg tgtaacagtg tcattcggac 180

cggctggttt tgcagtggtg aatggatatt tttttgttgg gttagtgcac actttaatgt 240

agtataaaat gttgttttgt tttgtaatac aaatatttgt gttatatctt tttgttcatt 300

gtgtttatgc tatatataca tatcaggttt aaagaattat ttattaacat attaataatt 360

tatgctacat aaataatgca tttattttat ttatattaaa atatattata ttaaaaatgt 420

taatcactgt tagtaaaatt gattaaggtg ttaaaaaaat tgtaataaat aattaccttt 480

tttgtgtaaa taatattata tttgtgtaat agttttcctt ttttctaaag aaagccaaaa 540

agaaagtaaa tcgttggtga ataaattatc aataaatgaa aaaagggtca ataatatttg 600

ctggactccc tccctgaccg tcggtgtttc aaacagaagc agagcaactg ttttggtcgg 660

actcttttgg tggtttgttc cgcgcgtctg cagagtaaca aacttttctc caaggtttgt 720

atagttaaat ggggtttcct ttctacactt tcgcttgcgt cgtattatct gacttattgt 780

tctcgcgatc tgcgttttta tttttaggtt cttctgagtc gttacaaaaa cgggtttccc 840

aatcttatct ctgggtctca atttttaaga ggttttaacg agattacaaa gcataggttt 900

tctgtaatgt cgggtgaaaa tctaagaagc ctatctaaca gtgcttcccc gaggaattac 960

cgagtcgtag tggctgcaac tcgtgaaatg gggataggga aagatggcaa gttgccatgg 1020

agattgcctt ctgatctcaa attcttcaag gaacttacag tgacaacatc agatcctgag 1080

aagaagaatg ctgtagtaat gggtagaaaa acctgggaga gtattccact tgagtttaga 1140

cctttacccg gtcgcctgaa tgttgtcctt actcgttccc agagttctga tattacaact 1200

ggagaaaatg ttgtaatatg tgggagcatt ccatcagctt tggaactatt agccgaggtt 1260

ccttattgtt ttgcaataga gaaggtgttt gtcattggcg gtggccagat attcaggtag 1320

attttttagt aaaagttgtt gaatttcatt gcatatttgt tggggaaaga taatgttttg 1380

atgttttaca atgcagggaa acactcaatg cttcaggttg cgaagccatt cacattactg 1440

aaattgggac aagcattgaa tgcgatacct tcattccttc aattgactcg tcttgtttcc 1500

agctgtggta ctcttcgaag ccattggaag aaaataatgt ccggttttca tttgcaactt 1560

atgttcgtgttagatctagg acaactgata attatgaggt aaaagacttg agtttcttgc 1620

ccaggatgat tgttgagaga cgagatgaat gagtcaatta gtaaataata ctgagacttg 1680

ttcatgaaac tatctcaagt gacaagcaag acccttgact ggttagatac agaattcatt 1740

tgcctaggta gtcttaaacc ttggttctct atgactctac tgctctttgc cttacatttt 1800

attgttgttg ctgatgttta aatgtaaata tgagcatcat ccccagtcca cgttgtgatt 1860

ttgtacaatg taagtctatc tttgcagatc ttttcatctt cttatataac taaggtattt 1920

actactagt 1929

<210> 13

<211> 63

<212>DNA

<213> Artificial Sequence

<400> 13

atgatgaaga cgactatgac tttggtgtgg attatggatt acaaggacga cgatgacaag 60

tga 63

<210> 14

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 14

ggattacaag attcaaagtg cg 22

<210> 15

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 15

tgatacctgg cagatggaac 20

<210> 16

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 16

cctgggacga gtggcctgac a 21

<210> 17

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 17

agttgcggac aatctggacg ac 22

Claims (2)

1. Use of a plant's dORF for enhancing translation of an upstream coding gene, characterized in that:

the plant is selected from Asian cotton, rice and tomato;

the sequence of dORF is selected from the sequences shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8 and SEQ ID NO. 11.

2. A method for enhancing the translation efficiency of a target gene, comprising: the method is to insert the dORF of the plant into the 3' UTR region of the target gene;

the plant is selected from Asian cotton, rice and tomato;

the sequence of dORF is selected from the sequences shown in SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8 and SEQ ID NO. 11.