An oligonucleotide pool specifically digests viral transcripts in pitaya and application thereof
By designing an oligonucleotide pool specifically for digesting dragon fruit virus transcripts and combining it with RNase H and DNase I, viral transcripts in dragon fruit RNA were successfully removed. This solved the problem of low efficiency due to viral contamination in existing technologies, enabling the acquisition of high-purity RNA and supporting molecular biology research.
Patent Information
- Application Number
- CN202510346028.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-24
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2045-03-24
AI Technical Summary
Existing methods for studying plant virus contamination in dragon fruit are inefficient, limited by organization and time constraints, and make it difficult to effectively remove virus contamination for high-quality molecular biology research.
An oligonucleotide pool was designed to specifically digest dragon fruit virus transcripts. By combining RNase H and DNase I, the oligonucleotides form a complex with the viral RNA and are then digested. The RNA is then purified using RNA Clean Beads to remove viral transcripts from the dragon fruit RNA.
This achievement enabled the acquisition of high-purity dragon fruit RNA, increasing the amount of effective data from RNA sequencing and supporting more comprehensive and accurate molecular biology research.
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Figure CN120138228B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically to an oligonucleotide pool for specifically digesting viral transcripts in dragon fruit and its applications. Background Technology
[0002] Dragon fruit, also known as pitaya, is a perennial succulent plant belonging to the genus *Hylocereus* in the family Cactaceae. With its unique appearance and rich nutritional content, dragon fruit has become one of the most popular fruits in the food market.
[0003] In recent years, RNA sequencing technology has been widely used in molecular biology research on dragon fruit. RNA sequencing has revolutionized our understanding of RNA biology, and enriching sufficient mRNA to obtain high-quality RNA-Seq data allows for a more comprehensive and accurate study of mRNA expression levels and transcriptional changes. However, in traditional plant transcriptome studies, viruses with identical poly(A) tails at their 3' ends are also enriched during sequencing, leading to low localization rates of the target genome. High levels of viral contamination have been found in several dragon fruit transcriptome datasets we previously sequenced. Viruses such as Cactus Virus X (CaVX), Pitaya Virus X (PiVX), Schlumbergera Virus X (SchVX), and Zygocactus Virus X (ZyVX) have been identified and isolated in dragon fruit and other cactus species. However, existing research on plant virus contamination mostly remains at the analytical level, with few proposals for suitable molecular biological solutions to plant virus contamination. While traditional devirulence methods (such as shoot tip devirulence and virus removal agents) can be used, these methods also limit the spatial and temporal scope of tissue analysis to some extent.
[0004] In summary, how to provide an economical and effective method to eliminate viral contamination in dragon fruit species to facilitate molecular biology research is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides an oligonucleotide pool for specifically digesting viral transcripts in dragon fruit and its application.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] An oligonucleotide pool for specifically digesting viral transcripts from dragon fruit, comprising 175 oligonucleotides, is shown in Table 1.
[0008] Furthermore, the viruses are cactus virus X, dragon fruit virus X, Christmas cactus virus X, prickly pear virus X, and Guangxi α-curved virus.
[0009] A dragon fruit transcript purification system, comprising the aforementioned oligonucleotide pool.
[0010] Furthermore, it also includes RNase H, DNase I, and RNA Clean Beads.
[0011] The application of the aforementioned oligonucleotide pool in the purification of dragon fruit transcripts.
[0012] A method for specifically digesting viral transcripts in dragon fruit, using the aforementioned oligonucleotide pool.
[0013] Further steps include the following:
[0014] (1) The 175 oligonucleotides were added to the dragon fruit RNA and annealed at high temperature to generate a viral RNA-Oligos DNA complex.
[0015] (2) Add RNase H to digest the viral RNA-Oligos DNA complex;
[0016] (3) Add DNase I to digest the unconsumed oligonucleotides;
[0017] (4) Purify the digestion products.
[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0019] Currently, there is no technology capable of specifically eliminating RNA from a multi-species mixed RNA. This invention assembles and analyzes transcripts of viruses found in dragon fruit and designs an independent and universally applicable oligonucleotide pool for dragon fruit. By applying the oligonucleotide pool, RNase H, and DNase I to mixed dragon fruit RNA, high-purity dragon fruit RNA can be obtained for further RNA sequencing. This invention is of great significance for molecular biology research on dragon fruit. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0021] Figure 1This diagram illustrates the viral contamination status of different dragon fruit samples in Example 1 of the present invention. The large circle represents the overall alignment rate, with blue representing the percentage of sequences aligned to the dragon fruit genome and pink representing sequences that were not aligned. The small circle represents the percentage of viruses in the sequences that were not aligned, including four different viruses and other data.
[0022] Figure 2 For the identification and analysis of viruses in dragon fruit in Example 1 of the present invention, A represents the contamination of 111 dragon fruit transcriptomes in different tissues and regions, and the left side of the red dotted line represents the transcriptomes we sequenced previously; B represents the geographical distribution of the 111 dragon fruit transcriptomes; C represents the tissue distribution of dragon fruit; D represents the classification of different contamination types in dragon fruit, mainly viruses; E represents the abundance of five major virus types in the collected dragon fruit transcriptomes.
[0023] Figure 3 This document describes the establishment of a dragon fruit transcript purification system in Example 1 of the present invention. A is a schematic diagram of the dragon fruit virus purification system; B shows the increase in the proportion of effective data after treatment by the transcript purification system; and C is a correlation analysis between the system before purification (control) and after purification. Detailed Implementation
[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0025] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.
[0026] Example 1
[0027] 1. Materials and Methods
[0028] 1.1 Experimental Materials
[0029] Dragon fruit varieties including 'Spineless Yellow Dragon' (Hylocereus undatus), 'Red Flower Green Dragon' (H. stenopterus), 'Guanhua White' (H. undatus), 'Guanhua Red' (H. polyrhizus), and 'Bird's Nest Fruit' (H. megalanthus) were collected from the orchard of South China Agricultural University. Pericarp, pulp, and tender stems were collected and stored at -80℃ for further analysis.
[0030] 1.2 Extraction of total RNA
[0031] RNA was extracted from dragon fruit using a polysaccharide and polyphenol plant RNA extraction kit (Huayueyang, Beijing). Concentration was determined using ScanDrop. 2 (Analytikjena, Germany).
[0032] 1.3 Transcriptome analysis and viral assembly and identification
[0033] RNA quality control, mRNA enrichment, library construction, and sequencing in the RNA-seq (Illumina NovaSeq 6000) process were handled by Novogene Beijing. Post-sequencing data alignment, quantification, and differential expression analysis were performed autonomously. Data alignment was processed using Hisat2 software, and Samtools was used for data sorting and file format conversion. Gene expression statistics were generated using FeatureCounts software, and DESeq2 was used for data normalization and identification of differentially expressed genes (DEGs).
[0034] Next, viral transcripts from dragon fruit were assembled based on unmapped reads. Unmapped reads were extracted from the dragon fruit reference genome using Samtools, and the viral transcriptome was assembled using Trinity. Redundancy in the assembled transcripts was eliminated using CD-Hit, and viral sequence identification was performed by alignment with the NT database (NCBI).
[0035] 1.4 Design of virus-specific oligonucleotides (Oligo)
[0036] Oligo-ASST (https: / / mtleelab.pitt.edu / oligo) was used to design oligonucleotide pools for various viruses based on the assembled viral transcripts. The design principles were: dissolution temperature 70–80℃, length 39–40 bp, and a maximum spacing of 30 nucleotides between oligonucleotides. Specific Oligos used for purification are shown in Table 1.
[0037] Table 1 List of Oligos used in the purification system
[0038]
[0039]
[0040]
[0041]
[0042] 1.5 Dragon Fruit Virus Purification System
[0043] The single oligonucleotides were resuspended to a concentration of 10 μM, and 2 μl of each of the 175 oligonucleotides were used to prepare an oligonucleotide mixture. 1 μl of the oligonucleotide mixture was combined with 1–2 μg of total RNA (the pericarp of 'Hylocereus undatus', 'H. stenopterus', 'H. undatus', 'H. polyrhizus', and 'H. megalanthus' during the color-changing stage), heated at 95 °C for 2 min, slowly cooled (2 °C / s) to 70 °C, incubated for 30 s, and then slowly lowered to room temperature. Next, 1 U of RNase H (Beyotime, Shanghai) and 10× reaction buffer (500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl2, 100 mM DTT, (pH 8.3 at 25℃)) were added, and the mixture was incubated at 37℃ for 15 min to remove the viral RNA-Oligos DNA complex. Then, 1 U of DNase I (Novizan, Nanjing) and 10× reaction buffer (100 mM Tris-HCl (pH 7.5 at 25℃), 25 mM MgCl2, 1 mM CaCl2) were added, and the mixture was incubated at 37℃ for 5 min to remove any remaining oligonucleotides. Finally, the product was purified using RNA Clean Beads (Novizan, Nanjing). The RNA was stored at -80℃.
[0044] 2 Results and Analysis
[0045] 2.1 Discovery of viral contamination in dragon fruit transcriptome studies
[0046] Transcriptomics is the foundation and starting point for studying gene function and structure. In our previous study, we found high levels of viral RNA contamination in dragon fruit of different varieties and tissues collected from orchards. Figure 1 (Table 2) This resulted in a low yield of transcriptome data, hindering in-depth research into the molecular biology of dragon fruit.
[0047] To understand the spatial and organizational distribution of viral contaminant enrichment in dragon fruit, we collected 111 dragon fruit transcriptome datasets from different countries and regions (Table 2). By mapping RNA-seq reads to the dragon fruit reference genome, we detected the major contaminants and their distribution in these transcriptome datasets. Figure 2 A~ Figure 2 C). The results showed that high viral contamination rates were also prevalent in dragon fruit tissue samples collected outside Guangdong Province, with a relatively even spatial distribution. Interestingly, viral contamination rates were generally significantly lower in the pulp of young dragon fruit, increasing as the fruit developed.
[0048] Table 2 shows the transcriptome sources used for analysis.
[0049]
[0050]
[0051] Note: Different numbers represent the degree of fruit development; the higher the number, the more mature the fruit is.
[0052] Then, we used Trinity and CD-Hit to perform de novo assembly and redundancy removal of unmapped reads in the highly contaminated transcriptome, generating 42 non-redundant viral transcripts, mainly annotated as: Cactus virus X (CVX), Pitaya virus X (PiVX), Schlumbergera virus X (SchVX), Zygocactus virus X (ZyVX), and Guangxi alphaflexivirus (Table 3). Based on the enrichment of these five viruses, we classified and analyzed the viral abundance of the above 111 transcriptomes. Figure 2 D~ Figure 2 E). Most dragon fruit virus contamination was dominated by ZyVX and PiVX, while SchVX (nearly 100%) and Guangxi α-curved virus (approximately 25%) contamination were found in the tetraploid dragon fruit 'Yanwoguo' and the diploid 'Honghuaqinglong', respectively. This indicates a preference for high viral contamination among different dragon fruit species. Furthermore, the presence of these high viral contamination levels underscores the urgent need to develop a system for purifying viral transcripts from dragon fruit.
[0053]
[0054]
[0055]
[0056] 2.2 Development of a dragon fruit transcript purification system
[0057] Similar to viral contamination in the dragon fruit transcriptome, a high proportion of ribosomal RNA (rRNA) contamination also exists during the traditional transcriptome assembly process in organisms. Traditional and widespread dragon fruit mRNA enrichment strategies also capture viruses with the same 3' poly(A) tail. This similar structure makes existing conventional eukaryotic transcription libraries insufficient for efficient mRNA library construction, resulting in low effective data obtained from transcriptome sequencing. Therefore, we designed a dragon fruit transcript purification system based on rRNA clearance: antisense oligonucleotide (Oligo) design, viral RNA-Oligos DNA complex formation, RNase H-mediated complex clearance, DNase I clearance, and RNA purification. Figure 3 A). The main principle is as follows: Antisense oligonucleotides that can bind to various dragon fruit viruses are annealed at high temperature with dragon fruit RNA contaminated with viruses to generate a viral RNA-Oligos DNA complex. This complex is specifically digested with RNase H, followed by digestion of unconsumed Oligos with DNase I. The digested product is purified to eliminate its impact on downstream sequencing, and finally, virus-free dragon fruit RNA is obtained.
[0058] This purification system, combined with eukaryotic mRNA enrichment methods, can improve the effective data yield of dragon fruit transcriptome analysis. To find a shared pool of oligonucleotides among different viral transcripts, we used aligned sequences from different transcripts as templates to design candidate oligonucleotides, obtaining 175 oligonucleotides (Table 1).
[0059] By applying these oligonucleotides to the purification system, the yield of dragon fruit transcriptome data (dragon fruit genome alignment rate) increased from nearly 20% to 75%. Figure 3 B). Further analysis of gene expression correlation before and after purification showed that the expression levels of most genes exhibited good correlation between pre- and post-purification levels. Figure 3 C) The use of purification systems can improve data yield without changing the distribution of gene expression itself, which demonstrates the feasibility of purification systems.
[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0061] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. An oligonucleotide pool for specifically digesting viral transcripts from dragon fruit, characterized in that, It includes 175 oligonucleotides, the sequences of which are shown in SEQ ID No. 1 to SEQ ID No.
175.
2. The oligonucleotide pool as described in claim 1, characterized in that, The viruses mentioned are cactus virus X, dragon fruit virus X, Christmas cactus virus X, finger cactus virus X, and Guangxi α-curved virus.
3. A dragon fruit transcript purification system, characterized in that, Includes the oligonucleotide pool as described in claim 1.
4. The system as described in claim 3, characterized in that, It also includes RNase H, DNase I, and RNA CleanBeads.
5. The use of the oligonucleotide pool according to claim 1 in the purification of dragon fruit transcripts.
6. A method for specifically digesting viral transcripts in dragon fruit, characterized in that, Use the oligonucleotide pool as described in claim 1.
7. The method as described in claim 6, characterized in that, Includes the following steps: (1) The 175 oligonucleotides were added to the dragon fruit RNA and annealed at high temperature to generate a viral RNA-Oligos DNA complex. (2) Add RNase H to digest the viral RNA-Oligos DNA complex; (3) Add DNase I to digest the unconsumed oligonucleotides; (4) Purify the digestion products.