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De Novo Sequencing and Analysis of the Rana Chensinensis 3 Transcriptome to Discover Putative Genes Associated 4 with Polyunsaturated Fatty Acids

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De Novo Sequencing and Analysis of the Rana Chensinensis 3 Transcriptome to Discover Putative Genes Associated 4 with Polyunsaturated Fatty Acids bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.985457; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 DE NOVO SEQUENCING AND ANALYSIS OF THE RANA CHENSINENSIS 3 TRANSCRIPTOME TO DISCOVER PUTATIVE GENES ASSOCIATED 4 WITH POLYUNSATURATED FATTY ACIDS 5 6 7 Jingmeng Sun 1, Zhuoming Wang 1 and Weiyu Zhang 1,* 8 1 College of Pharmacy, Changchun University of Chinese Medicine, 130117, 9 #Changchun, Jilin, China 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 *Corresponding author: Weiyu Zhang 28 College of Pharmacy, Changchun University of Chinese Medicine, 29 130117, Changchun, Jilin, China. 30 Cell Phone: +8613604318087 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.985457; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 31 ABSTRACT 32 Rana chensinensis (R. chensinensis) is an important wild animal found in China, and 33 a precious animal in Chinese herbal medicine. R. chensinensis is rich in 34 polyunsaturated fatty acids (PUFAS). However, information regarding the genes of R. 35 chensinensis related to the synthesis of PUFAs is limited. To identify these genes, we 36 performed Illumina sequencing of R. chensinensis RNA from the skin and Oviductus 37 Ranae. The Illumina Hiseq 2000 platform was used for sequencing, and the I-Sanger 38 cloud platform was used for transcriptome de novo sequencing and information 39 analysis to generate a database. Through the database generated by the transcriptome 40 and the pathway map, we found the pathway for the biosynthesis of R. chensinensis 41 PUFAs. The Pearson coefficient method was used to analyze the correlation of gene 42 expression levels between samples, and the similarity of gene expression in different 43 tissues and the characteristics in their respective tissues were found. Twelve 44 differentially expressed genes of PUFA in skin and Oviductus Ranae were screened 45 by gene differential expression analysis. The 12 unigenes expression levels of 46 qRT-PCR were used to verify the results of gene expression levels consistent with 47 transcriptome analysis. Based on the sequencing, key genes involved in biosynthesis 48 of unsaturated fatty acids were isolated, which established a biotechnological platform 49 for further research on R. chensinensis. 50 51 Keywords: Oviductus Ranae; polyunsaturated fatty acids; Rana chensinensis; skin; 52 Illumina sequencing; 53 54 55 56 57 58 59 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.985457; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 60 INTRODUCTION 61 Rana chensinensis (R. chensinensis) is an important wild animal in China. Oviductus 62 Ranae, a valuable Chinese crude drug, is recorded in Pharmacopoeia of the People’s 63 Republic of China as a dried oviduct of the female Chinese frog [9], R. chensinensis, 64 distributed mainly in northeastern China. Oviductus Ranae is an established and 65 highly valued food and medicine. Traditional Chinese medicine holds that Oviductus 66 Ranae can moisten the lungs, nourish yin, and replenish the kidney essence [3]. 67 Meanwhile, modern pharmacological studies have demonstrated the activity of 68 Oviductus Ranae in improving immunity, as well as its anti-fatigue, anti-oxidative, 69 anti-lipemic, and anti-aging properties [10]. Oviductus Ranae has an established 70 safety profile, it is a raw material with natural health care functions, and has great 71 potential for further use, therefore, it is widely used in food, pharmaceutical and 72 chemical industries. At present, the food developed using Oviductus Ranae involves 73 canned food, candy, yogurt and beverages. Moreover, there are various administration 74 forms (i.e., pills, capsules, and granules) produced from Oviductus Ranae. In the skin 75 care industry, the active ingredients (i.e., unsaturated fatty acids, carotene, and 76 vitamins) in Oviductus Ranae can help improve skin dryness, reduce pigmentation, 77 and offer a cosmetic effect [11]. 78 R. chensinensis is a cold-tolerant vertebrate amphibian that grows for ≤6 months in 79 hibernation [12]. Maintaining the fluidity of the cell membrane in a low-temperature 80 environment ensures that it can perform its normal physiological functions [8]. It is 81 known to all that the fluidity of the cell membrane is closely related to the 82 composition of polyunsaturated fatty acids (PUFAs), the content of PUFAs in the cell 83 membrane is very important for maintaining cell structure, membrane mobility, and 84 enzymatic activity. PUFAs cannot be ingested from the external environment by 85 hibernating animals. Therefore, we investigated the mechanism involved in the 86 survival of R. chensinensis during hibernation and changes in the content of PUFAs. 87 We believe that PUFAs, which are abundantly in R. chensinensis, may be the reason 88 for the decrease in fatty acid saturation by R. chensinensis in the low-temperature 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.985457; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 89 environment. The synthetic pathway is the presence of fatty acid desaturase (FADS) 90 in the organism, which is a key enzyme in the synthesis of PUFAs. 91 There are four main kinds of FADS in animals, namely Δ9-FAD, Δ5-FAD, Δ6-FAD, 92 and Δ4-FAD [8]. Of those, Δ6-FAD and Δ5-FAD are the first and second 93 rate-limiting enzymes. Studies have found that a low-temperature environment can 94 cause up-regulation of Δ9-FAD gene expression. Previous experimental studies have 95 found significant differences in fatty acid content in Oviductus Ranae collected in 96 different seasons. The content of PUFAs in the predation growth period and scattered 97 hibernation samples was 14.16% and 29.83%, respectively. Therefore, we 98 hypothesized that FADs is necessary for the synthesis of PUFAs in R. chensinensis, 99 which affect their own synthesis of PUFAs under low-temperature stimulation. At 100 present, genetic information regarding R. chensinensis remains unknown, and the 101 molecular mechanism of fatty acid synthesis in R. chensinensis is unclear [7]. 102 Therefore, we used non-reference transcriptome sequencing technology to obtain the 103 genetic information of R. chensinensis. The FADs gene in vivo was identified by 104 studying the changes in the content of PUFAs in R. chensinensis. Through the 105 detection of FADs gene expression in Oviductus Ranae and the skin of R. 106 chensinensis, the role of this gene in the synthesis of PUFAs was elucidated, and the 107 pathway of PUFA synthesis was determined. 108 MATERIALS AND METHODS 109 Animals and treatments 110 To ensure the space-time specificity of the sample，We removed Oviductus Ranae 111 and skin from R. chensinensis, rapidly frozen in liquid nitrogen, and stored in an 112 ultra-low temperature freezer at -80℃ . All procedures performed in this study 113 involving the handling of R. chensinensis were approved by the Animal Care and 114 Welfare Committee of Changchun University of Chinese Medicine (Jilin, China). 115 RNA isolation and reverse transcription complementary DNA (cDNA) 116 RNA was extracted from the skin and Oviductus Ranae of R. chensinensis. Detection 117 of RNA concentration and quality was performed using Nanodrop2000 (Thermo 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.10.985457; this version posted March 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 118 Scientific, U.S.A.). Total RNA integrity was determined through 1.2% agarose gel 119 electrophoresis. Sample reverse transcription was performed using Takara’s (Takara, 120 China) PrimeScriptTM RT reagent Kit with gDNA Eraser-Perfect Real Time Kit (Code 121 No. RR047A). The reaction system include the following: reaction solution 10.0μL, 122 5×PrimeScript buffer 10.0μL, PrimeScriptTM RT Enzyme Mix I 1.0μL, RT primer 123 mix 1.0 μL, and Rnase free dH2O 4.0μL, in a total volume of 20μL. The reaction 124 procedure was: 37°C for 15min, followed by 85°C for 5s. The obtained cDNA was 125 stored at −20°C. Transcriptome sequencing was performed using the Illumina Hiseq 126 2000. The data were analyzed on the free online platform of Majorbio I-Sanger Cloud 127 Platform (www.i-sanger.com). De novo transcriptome assembly was carried out using 128 the Trinity software (https://github.com/trinityrnaseq/trinityrnaseq) [1]. 129 De novo assembly and comparative analysis between two samples 130 Using the Trinity software to head assembly of all the clean data, we spliced the 131 transcript sequence (i.e., the longest transcript of each gene, defined as unigene), as a 132 basis for the follow-up bioinformatics analysis. The TransRate 133 (http://hibberdlab.com/transrate/) software of the transcriptome assembly sequence 134 filter was used and optimized from the beginning.
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