Library building method for large-scale determination of nucleic acid sequence translation efficiency

Abstract

The invention provides a library building method for large-scale determination of nucleic acid sequence translation efficiency, and the library building method comprises the following steps: selecting a model plant arabidopsis thaliana endogenous 5 '-leader, and respectively carrying out construction according to a 5'-leader sequence with the length of 50-150 nt and a 5 '-leader sequence with the length of more than 150 nt to obtain two 5'-leader libraries; respectively constructing the 5 '-leader libraries through carrier seamless connection to obtain two plasmid libraries; mixing the two plasmid libraries, infecting tobacco leaves, extracting total RNA (Ribonucleic Acid) of a tobacco injection part, and separating RNA components combined with different ribosomes to obtain separated RNA; and performing library building, sequencing and analysis on the separated RNA, and assigning values to the translation capabilities of different transcripts so as to obtain the translation efficiency of the nucleic acid sequence.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a library construction method for large-scale determination of nucleic acid sequence translation efficiency. Background Art

[0002] Plants are always exposed to pathogenic microorganisms in nature. In order to prevent and resist the invasion of pathogenic microorganisms, plants have formed a multi-level immune system during the long evolution process. Generally speaking, the plant immune system includes pattern triggered immunity (PTI) (Couto and Zipfel, 2016), effector-triggered immunity (ETI) (Jones and Dangl, 2006) and systemic acquired resistance (SAR) (Gaffney et al., 1993). The conserved molecular patterns (PAMPs) on the surface of pathogenic microorganisms can be recognized by pattern recognition receptors (PRRs) on plant cell membranes, quickly activating a broad spectrum of immune responses PTI. The early immune response of plants to pathogenic microorganisms includes the production of reactive oxygen species (ROS), changes in cytoplasmic calcium ion concentration, and activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Pathogenic microorganisms enter cells by secreting effectors, thus causing plant disease; specific effectors can be recognized by intracellular immune receptors (Nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) receptors, NLRs), activating specific immune responses (ETI), which are usually accompanied by programmed cell death. Systemic acquired resistance (SAR) is a broad-spectrum, long-lasting resistance state that occurs throughout the plant after a plant is attacked by a local pathogen.

[0003] Eukaryotic gene expression begins with gene transcription in the cell nucleus. The transcripts produced by transcription are broadly divided into protein-coding RNA (i.e., mRNA) and non-coding RNA, although some non-coding RNA has also been found to be translated. After transcription is complete, the primary transcript or precursor mRNA (pre-mRNA) undergoes a series of processing steps, including the addition of a 5' cap and a 3' polyadenylic acid tail, as well as splicing. Once the mature mRNA is transported out of the nucleus, it is usually ready for translation. However, in some cases, the mRNA may be degraded or stored, preventing its translation.

[0004] The translation process has been studied in detail in yeast and animal systems, including initiation, elongation, termination, and ribosome recycling (Jackson et al., 2010). Once mRNA enters the translation system, the translation machinery (mainly composed of ribosomes, tRNAs, and translation factors) performs these steps. These components are essential for the translation of mRNA. Upstream translation regulators include proteins (such as RNA binding proteins and kinases), metabolites (such as ions and ATP), and RNAs (such as small RNAs). When cells receive developmental or environmental signals, these regulators achieve translation control by regulating the availability, composition, and activity of translation machinery components. They can globally reprogram or gene-specifically change mRNA translation efficiency (TrE), ultimately determining the quantity and quality of the global proteome or specific proteins. Therefore, the translation machinery is the basis of translation control.

[0005] 5′-leader (also known as 5′ untranslated region or 5′-UTR) is an important structural and functional region of mRNA, which has a direct or indirect effect on the translation of mRNA. Through in-depth research on the 5′-leader sequence, its mechanism in regulating the translation of mRNA can be revealed, and a new perspective can be provided for the study of plant gene function. Therefore, it is necessary to conduct in-depth research on the 5′-leader sequence in order to develop a library construction method for large-scale determination of nucleic acid sequence translation efficiency. Summary of the invention

[0006] The purpose of the present invention is to provide a library construction method for large-scale determination of nucleic acid sequence translation efficiency, which not only provides new tools and methods for further understanding of plant immune response mechanisms, but also lays a foundation for gene function research and application.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The object of the present invention is to provide a method for building a library for large-scale determination of nucleic acid sequence translation efficiency, the method comprising: Endogenous 5'-leaders of the model plant Arabidopsis thaliana were selected, and two 5'-leader libraries were constructed based on sequences with 5'-leader lengths ranging from 50 to 150 nt and sequences with 5'-leader lengths greater than 150 nt. The 5'-leaders library is constructed by seamlessly connecting the vectors to obtain two plasmid libraries; The two plasmid libraries are mixed and then infected with tobacco leaves, and then the total RNA of the tobacco injection site is extracted, and the RNA components bound to different ribosomes are separated to obtain the separated RNA; Building a library and sequencing the isolated RNA to obtain sequencing data; The sequencing data is extracted and analyzed, and the translation capabilities of different transcripts are assigned values ​​to obtain the translation efficiency of the nucleic acid sequence.

[0008] Furthermore, the sequences with 5'-leaders lengths of 50-150 nt and sequences with 5'-leaders lengths of more than 150 nt are constructed separately to obtain two 5'-leaders libraries, specifically including: For sequences with 5'-leaders lengths of 50-150 nt, 9836 different sequences were synthesized by gene chip synthesis to obtain gene chip sample solutions. The gene chip sample solutions were then PCR amplified using upstream and downstream adapter primers to obtain PCR products. The PCR products were then hybridized with the target vector pSY105-X Bsa I restriction endonuclease and T4-DNA ligase were seamlessly connected to construct the first 5'-leaders library; For sequences with 5'-leaders longer than 150 nt, 1232 types of 5'-leaders DNA were amplified by designing specific primers for each sequence, and 1232 groups of PCR products were obtained by designing primers for the 1232 types of 5'-leaders DNA. All the amplified PCR products were then mixed and Bsa I restriction endonuclease and T4-DNA ligase seamlessly ligated the 5′-leaders into the target vector pSY105-X to obtain the second 5′-leaders library.

[0009] Furthermore, the 5'-leaders library is constructed separately by seamlessly connecting the vectors to obtain two plasmid libraries, specifically comprising: The pCONT-free plasmid was digested and ligated with the 5'-leaders library to obtain two plasmid libraries.

[0010] Furthermore, the separated RNA is subjected to library construction and sequencing to obtain sequencing data, specifically including: For RNA of different components, reverse transcription is performed using specific reverse transcription primers with Fraction-index, the cDNA products after reverse transcription are mixed, and an NGS library is established to distinguish the different sources of RNA components through Fraction-index.

[0011] Furthermore, the sequencing data is extracted and analyzed, and the translation capabilities of different transcripts are assigned values ​​to obtain the translation efficiency of the nucleic acid sequence, specifically including: The MRL values ​​of different 5′-leaders transcripts are calculated, and the MRL values ​​are subjected to correlation analysis to obtain the translation efficiency of the nucleic acid sequence.

[0012] The MRL value (Mean ribosome loading) refers to the average number of ribosomes detected on a specific mRNA and is an indicator of translation efficiency. The larger the MRL value, the greater the translation efficiency.

[0013] Furthermore, through the polysome profiling test, 10 components can be detected in each sample, and the distribution of specific mRNA in each component can be detected. After standardization, the MRL value of each mRNA is finally obtained to reflect the degree of its translation efficiency.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides a library construction method for large-scale determination of nucleic acid sequence translation efficiency. The present invention analyzes 5'-leaders and constructs a library, assigns values ​​to the translation capabilities of different transcripts, and thus obtains the translation efficiency of nucleic acid sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 for the distribution of endogenous 5′-leaders in Arabidopsis thaliana; Figure 2 A library strategy for screening 5′-leaders of plant immune response; Figure 3The process of building a library for NGS; Figure 3 a, Schematic diagram of Race-TS-seq (pCONT-free-N50) NGS library construction. RNA was reverse transcribed using specific primer OSY373-RT and connected to the reverse transcription end via TSO to introduce the adapter sequence. The NGS library was obtained by PCR amplification using upstream primers OWH118 and OSY359 and downstream primers OSY367 and OGY440. Figure 3 b, Schematic diagram of CE-NGS (pCONT-free-Endo 5′-leaders) NGS library construction. Endogenous RNA was reverse transcribed using reverse transcription primers with UMI and Fraction-index. NGS library was obtained by PCR amplification using upstream primers OWH118 and OSY433 and downstream primer OGY440.

[0017] Figure 4 For component separation and data correlation analysis of Polysome profiling; Figure 4 a: The total RNA of samples from the Mock, ETI and SAR experimental groups was separated by polysome profiling components. Figure 4 b Correlation analysis of the MRL values ​​of the translation group of biological replicate samples of Mock, ETI, and SAR.

[0018] Figure 5 Verification of MRL values ​​in the translation group database. Figure 5 a represents 5 transcripts with upregulated MRL values ​​(numbered 208, 209, 211, 212 and 215), 2 transcripts with unchanged MRL values ​​(numbered 225 and 227) and 4 transcripts with downregulated MRL values ​​(numbered 228, 229, 230 and 231) selected from the translation group database under ETI and SAR treatment conditions, respectively. Figure 5 b: The dual luciferase reporter system was used to detect the translation efficiency of the transcript in Figure a. DETAILED DESCRIPTION

[0019] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0020] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0022] The general idea of ​​the present invention is as follows: As an important structural and functional region of mRNA, 5′-leader directly or indirectly affects the translation of mRNA. First, the present invention analyzed the distribution of endogenous 5′-leaders in Arabidopsis thaliana. Figure 1 As shown. Based on the tair10 database, the present invention analyzed a total of 38,624 transcripts. The length of the 5′-leaders of the transcripts ranged from 1 to 3972 nt; the average length was 235 nt; the quartile size was 97 nt, the bisector size was 179 nt, and the third quartile size was 301 nt. The above results show that the length of most 5′-leaders is within 300 nt, which is much smaller than the length of the general CDS.

[0023] In order to identify immune response factors with high throughput and high stability, the present invention needs to design a reasonable experimental strategy. Figure 1 As shown, the present invention selects the endogenous 5′-leaders of the model plant Arabidopsis as the research object. We selected a specific reporter system, namely the 35S:target 5′-leaders-EGFP-terminator vector (pSY105-X). The selected promoter is derived from the constitutive 35S original promoter (344 bp) of cauliflower mosaic virus to ensure a high level of mRNA transcription. The selected tag protein is the universal EGFP, and the DNA sequence size is 720 bp. The terminator is the CaMV poly(A) signal, and the DNA sequence size is 175 bp. The 5′-leaders selected in the present invention are constructed according to two different strategies according to their length.

[0024] First, for sequences with 5′-leaders lengths of 50-150 nt, the present invention adopts a gene chip synthesis method to prepare, and a total of 9836 different sequences are synthesized. For sequences with 5′-leaders lengths greater than 150 nt, we selected 5′-leaders of Arabidopsis transcription factors and plant hormone-related genes. The second library has a total of 1232 different sequences, and by designing specific primers for each sequence, the 1232 sequences are amplified one by one. DNA containing 1232 5′-leaders sequences is transferred into tobacco cells, and a total of 2135 transcripts are transcribed.

[0025] The present invention constructs plasmid libraries by seamlessly connecting the above two 5′-leaders libraries. Subsequently, the two plasmid libraries are mixed and injected into tobacco leaves, which are treated with ETI or SA, respectively, and the untreated group is used as a control. The total RNA of the tobacco injection site is extracted, and the RNA components bound to different ribosomes are separated using the polysome profiling method, and a total of 10 components are separated. The separated RNA is library-built and sequenced. The sequencing data is extracted and analyzed, and the translation capabilities of different transcripts are assigned. Finally, by comparing the data of the ETI, SAR treatment group and the control group, the 5′-leaders that respond to ETI or SAR can be separated.

[0026] The present application will be described in detail below in conjunction with the embodiments and experimental data. If no specific techniques or conditions are specified in the embodiments, the technical methods or conditions described in the technical materials such as the literature, standards, etc. in the art or the product instructions are used. The methods used described in this section, unless otherwise specified, are conventional methods known in the art, and the consumables and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those known to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be used for the implementation of the related technologies involved in the present invention.

[0027] [Example 1] A library construction method for large-scale determination of nucleic acid sequence translation efficiency S1. Construction of two 5'-leaders libraries For sequences with 5′-leaders in the range of 50-150 nt, a full-length sequence of 170 nt was synthesized using a gene chip (see Table 1). The target 5′-leader sequence range was 50-118 nt (for transcripts with 5′-leader lengths of more than 118 nt but less than 150 nt, the 118 nt near the CDS was selected as the representative 5′-leader). The upstream sequence of the target 5′-leader was the upstream linker and Bsa I recognition and cleavage site; the target 5′-leader downstream sequence is Bsa I recognition, cleavage site and downstream adapter. If the length of the synthesized sequence is less than 170 nt, several bases are added upstream of the sequence to ensure that all sequences synthesized on the gene chip are of equal length. The upstream and downstream adapter primers are used to PCR amplify the gene chip sample solution to enrich the 5′-leader sequence of the target transcript. The PCR product is combined with the target vector pSY105-X by Bsa I restriction endonuclease and T4-DNA ligase were seamlessly connected to construct the first 5'-leaders library.

[0028] For sequences with 5′-leaders longer than 150 nt, we selected 5′-leaders of Arabidopsis transcription factors and plant hormone-related genes, a total of 1232 genes. We designed primers for each of the 1232 genes and amplified the target 5′-leader interval. All amplified PCR products were mixed and Bsa I restriction endonuclease and T4-DNA ligase seamlessly ligated the 5′-leaders into the target vector pSY105-X, which was the second 5′-leaders library.

[0029] For transcript library samples with variable 5′-leader sequences, the present invention develops Race-TS-seq and CE-NGS based on the optimized 5′-Race method.

[0030] like Figure 3 As shown, Race-TS-seq takes pCONT-free-N50 as an example, uses specific primer OSY373-RT (sequence see Table 1) to reverse transcribe RNA, and connects it to the reverse transcription end through TSO (sequence see Table 1) to introduce the adapter sequence.

[0031] In the first round of PCR, primers OSY370 and OSY367 were used for amplification; In the second round of PCR, upstream primers OWH118 and OSY359 and downstream primer OGY440 were used for amplification; Through two rounds of PCR, an NGS library containing different 5′-leader sequences can be obtained, and the 5′ end distribution of different 5′-leaders can be obtained by second-generation sequencing.

[0032] After determining the 5′ end of the mRNA driven by the 35S promoter, the present invention developed a CE-seq method ( Figure 3 b). Use reverse transcription primers with UMI and Fraction-index to reverse transcribe endogenous RNA. Since the 5′ end has been confirmed, the shared region can be used upstream of the PCR instead of the TSO sequence. The NGS library is amplified by upstream primer OWH118 (see Table 1), primer OSY433 (see Table 1) and downstream primer OGY440 (see Table 1). The 5′-leader sequence can be qualitatively and quantitatively analyzed by next-generation sequencing. Table 1

[0033] S2. The 5'-leaders library is constructed by seamlessly connecting the vector to obtain two plasmid libraries. The recipient vector used, pSY105, contains the major construct 35S- Bsa I-ccdB- Bsa I-intron- EGFP-terminator. Both 5′-leaders library PCR products have Bsa I enzyme recognition site. Bsa Under the action of restriction endonuclease I, the two ends of the 5′-leaders library PCR product and the vector pSY105 can form complementary sticky ends; under the action of T4-DNA ligase, the target fragment is connected to the pSY105 vector; the enzyme cutting and connection process in this process are completed in the same reaction system, which is the so-called "cutting and connecting" process. Bsa I belongs to Type IIs restriction endonuclease, and the vector construction system is the Golden Gate construction system.

[0034] S3, mixing the two plasmid libraries and then infecting tobacco leaves, extracting total RNA from the tobacco injection site, and separating RNA components bound to different ribosomes to obtain separated RNA; Specifically, the two plasmid libraries are mixed and then infected into tobacco leaves, which are treated with Mock (blank control), ETI (effector-triggered immunity) and SAR (systemic acquired resistance), respectively, and RNA of tobacco leaves treated with Mock, ETI and SAR is extracted, respectively, and RNA bound to different ribosomes is separated using the polysome profiling method, such as Figure 4 As shown in a.

[0035] S4, constructing a library and sequencing the separated RNA to obtain sequencing data; The specific steps of building the library are: For RNA of different components, specific reverse transcription primers with Fraction-index were used for reverse transcription (primer sequences are shown in Table 2, and the sequences are RTS1-RTS66 shown in SEQ ID NO.1-SEQ ID NO.66, respectively). The cDNA products after reverse transcription were mixed and an NGS library was established. Different sources of RNA components were distinguished by Fraction-index, and the NGS library was sequenced by the second generation.

[0036] Table 2

[0037]

[0038]

[0039]

[0040] S5. Extracting and analyzing the sequencing data, and assigning values ​​to the translation capabilities of different transcripts, thereby obtaining the translation efficiency of the nucleic acid sequence.

[0041] The MRL (mean ribosome loading) values ​​of different 5′-leaders transcripts are calculated using the formula: (n is a specific transcript, which is an integer greater than 1; m is the number of ribosomes; and R is the number of reads.) According to the above formula, the final calculated MRL value has a data range of 0 to 10.

[0042] In order to evaluate the feasibility of using MRL values ​​to assess translation efficiency, a correlation analysis was performed on the MRL values ​​of the translation groups of biological replicate samples of Mock, ETI, and SAR. The specific steps of the correlation analysis were as follows: (1) Collect relevant data; (2) Check the normality and linearity of the data; (3) Fit the curve and calculate the coefficient of determination; (4) Explain the correlation between variables based on the size of the coefficient of determination. The results of the above correlation analysis are as follows: Figure 4 As shown in b, the calculated coefficient of determination R 2 (ranging from 0 to 1), according to the size of the coefficient of determination, the principle of the relationship between the explanatory variables is R 2 The larger the value, the more relevant it is, indicating the feasibility and repeatability of the method.

[0043] [Example 2] Verifying whether the MRL value can truly reflect the translation ability of the transcript 1. Verification methods and steps In order to verify the accuracy of the MRL value, the present invention selected 5 transcripts with enhanced translation, 2 transcripts with unchanged translation, and 4 transcripts with downregulated translation under ETI and SAR treatment conditions from the database. The 5′-leaders of the corresponding transcripts were connected to the dual luciferase reporter system and transformed into tobacco leaves. The fluorescence ratio of FLUC and RLUC was detected by an ELISA instrument and compared with the corresponding mRNA level. The results are as follows: Figure 5 As shown, the translation efficiency of different samples was calculated according to the method of Example 1.

[0044] 2. Although the correlation of the MRL values ​​of the translation group is in line with expectations, the present invention selects some transcripts for separate experiments to verify whether the MRL values ​​can truly reflect the translation ability of the transcripts. Figure 5 As shown in a, under the conditions of ETI and SAR treatment, 5 MRL value up-regulated transcripts (numbering: 208, 209, 211, 212 and 215), 2 MRL value unchanged transcripts (numbering: 225 and 227) and 4 MRL value down-regulated transcripts (numbering: 228, 229, 230 and 231) were selected from the translation group database respectively. Using the dual luciferase reporter system, the transcript selected by the present invention was consistent with the MRL value up-regulated, unchanged and down-regulated under ETI and SAR treatment, which shows the accuracy of the MRL value of the translation group database.

[0045] Finally, it should be noted that the terms "comprises", "includes" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device includes a series of elements. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A library construction method for large-scale determination of nucleic acid sequence translation efficiency, It is characterized in that The library construction method comprises: Endogenous 5'-leaders of the model plant Arabidopsis thaliana were selected, and two 5'-leader libraries were constructed based on sequences with 5'-leader lengths ranging from 50 to 150 nt and sequences with 5'-leader lengths greater than 150 nt. The 5'-leaders library is constructed by seamlessly connecting the vectors to obtain two plasmid libraries; The two plasmid libraries are mixed and then infected with tobacco leaves, and then the total RNA of the tobacco injection site is extracted, and the RNA components bound to different ribosomes are separated to obtain the separated RNA; Building a library and sequencing the isolated RNA to obtain sequencing data; The sequencing data is extracted and analyzed, and the translation capabilities of different transcripts are assigned values, thereby obtaining the translation efficiency of the nucleic acid sequence.

2. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 1, It is characterized in that The method comprises constructing two 5'-leaders libraries based on sequences with a 5'-leaders length of 50-150 nt and sequences with a 5'-leaders length of more than 150 nt, respectively, and specifically comprising: For sequences with 5'-leaders lengths of 50-150 nt, 9836 different sequences were synthesized by gene chip synthesis to obtain gene chip sample solutions. The gene chip sample solutions were then PCR amplified using upstream and downstream adapter primers to obtain PCR products. The PCR products were then hybridized with the target vector pSY105-X Bsa I restriction endonuclease and T4-DNA ligase were seamlessly connected to construct the first 5'-leaders library; For sequences with 5'-leaders longer than 150 nt, 1232 types of 5'-leaders DNA were amplified by designing specific primers for each sequence, and 1232 groups of PCR products were obtained by designing primers for the 1232 types of 5'-leaders DNA. All the amplified PCR products were then mixed and Bsa I restriction endonuclease and T4-DNA ligase seamlessly ligated the 5′-leaders into the target vector pSY105-X to obtain the second 5′-leaders library.

3. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 1, It is characterized in that By designing specific primers for each sequence, 1232 5'-leaders DNA were amplified, including 758 transcription factors and 549 plant hormone-related genes.

4. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 1, It is characterized in that The 5'-leaders library is constructed by seamlessly connecting the vectors to obtain two plasmid libraries, specifically comprising: The pCONT-free plasmid was incubated with the 5'-leaders library by restriction digestion and ligation to obtain two plasmid libraries.

5. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 1, It is characterized in that The separated RNA is subjected to library construction and sequencing to obtain sequencing data, specifically comprising: For RNA of different components, reverse transcription is performed using specific reverse transcription primers with Fraction-index, the cDNA products after reverse transcription are mixed, and an NGS library is established to distinguish the different sources of RNA components through Fraction-index.

6. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 5, It is characterized in that The specific reverse transcription primers specifically include: RTS1-RTS66, whose sequences are shown as SEQ ID NO.1-SEQ ID NO.66 respectively.

7. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 1, It is characterized in that The sequencing data is extracted and analyzed, and the translation capabilities of different transcripts are assigned values ​​to obtain the translation efficiency of the nucleic acid sequence, specifically including: The MRL values ​​of different 5′-leaders transcripts are calculated, and the MRL values ​​are subjected to correlation analysis to obtain the translation efficiency of the nucleic acid sequence.

8. A method for building a library for large-scale determination of nucleic acid sequence translation efficiency according to claim 7, It is characterized in that The MRL value is subjected to correlation analysis to obtain the translation efficiency of the nucleic acid sequence, specifically comprising: Through the polysome profiling test, the distribution of specific mRNA in each component is detected, and through standardization, the MRL value of each mRNA is finally obtained, thereby reflecting the degree of its translation efficiency.

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