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Abstract ［Objectives］ This study was conducted to compare the quality of total RNA extracted from different tissues of cone snail ( Conus geographus ).
［Methods］ The total RNA of four different tissues of cone snail, venom gland, venom tube, salivary gland and tooth gland was extracted by the Trizol method. The total RNA of cone snail was detected by agarose gel electrophoresis, NanoDropTM 2000 spectrophotometer and Aligent 2100 biological analyzer.
［Results］ The total RNA extracted from different tissues of cone snail showed clear band, and thus had similar concentrations and purity, and the highest yield was obtained from venom tube.
［Conclusions］ The total RNA extracted from different tissue parts of cone snail could meet the basic requirements of molecular biology, and its venom tube was the best tissue part for extracting total RNA, which lays a foundation for molecular biology research such as high-throughput transcriptome sequencing of cone snail.
Key words Cone snail; Venom gland; RNA extraction; Trizol method
Received: February 23, 2020Accepted: April 20, 2020
Supported by the Hainan Provincial Keypoint Research and Invention Program (ZDYF2018138).
Qin CHEN (1983-), female, P. R. China, lecturer, devoted to research about marine drugs. Chao PENG (1986-), male, P. R. China, engineer, devoted to research about marine drug.
#Corresponding author. E-mail: gaobingmiao@hainmc.edu.cn.
*These authors contributed equally to this work.
Cone snail ( Conus geographus ) belongs to Conidae of Stenoglossa in Prosobranchia of Gastropoda in Mollusa. Conotoxins are a type of small peptide neurotoxins secreted by cone snail for self-defense and predation. They have the characteristics of high abundance, small molecular weight, diverse structure, wide range of targets and strong specificity［1-2］. Pharmacological experimental research shows that conotoxins have cytotoxicity, analgesic activity, pesticidal activity, anti-cancer activity and other pharmacological activity by acting on different ion channels or receptors［3-6］. Therefore, it can be used not only as a molecular model for new drug design, but also directly as a drug for disease treatment. There are about 700 kinds of cone snails in the world, and there are more than 100 kinds of cone snails in China, mainly distributed in the Xisha Islands, Nansha Islands and Zhongsha Islands under the jurisdiction of Hainan Province, and tropical sea areas such as Taiwan Island［2］. Transcriptomics and proteomics methods have identified that each kind of cone snail can secrete about 200 types of conotoxins. In theory, conotoxins may exceed 140 000 species accumulatively［5］. Therefore, conotoxins are called "the treasure trove of marine drugs", and they are also an unlimited drug resource［7］. In recent years, the deterioration of the marine ecological environment and the overfishing of fishermen have destroyed cone snails habitat, which has led to the situation that the systematic development and utilization of cone snail has evolved into endangered resources. The protection and utilization of genetic resources of Hainans characteristic cone snails are more urgent. The steps of conotoxin isolation and toxin gene cloning are time-consuming, labor-consuming, inefficient, and greatly affected by resources, and some specimens are difficult to obtain［5-7］. Therefore, to study the conotoxin gene resources through high-throughput sequencing technology and to establish the Hainan insectivorous cone snail toxin gene library and peptide library can solve the problem of disappearance of functional gene caused by frequent extinction of cone snail resources, and will simultaneously lay a foundation for studying the subsequent structure and function and pharmacology and efficacy of conotoxins and the development of marine drugs.
Mollusc cone snail belongs to vertebrates, and its genomic DNA is very complex and huge, about 100 times that of protein and mRNA. The work of isolating target gene fragments of conotoxins from cone snail genomic DNA is very time-consuming and labor-consuming. The total RNA of cone snail can be extracted to synthesize cDNA by reverse transcriptase, and the conotoxin gene can be obtained by construction of a library and high-throughput sequencing［8-9］. Therefore, in this study, we compared the effects of extracting total RNA from different tissue parts of cone snail, so as to obtain high-quality, high-purity and complete integrity conotome RNA and provide a basis for high-throughput transcriptome sequencing and other molecular biological studies.
Materials and Methods
Materials
Cone snail materials
The cone snails used in this study were collected along the coast of Sanya, Hainan, and dissected for venom glands, venom tubes, salivary glands and tooth glands at different parts as materials for total RNA extraction. Remaining materials were stored in a refrigerator at -70 ℃ for later use.
Reagents
DNA marker D2000, Taq polymerase, Mg2+ , dNTPs and RNaseA were purchased from Tiangen Biotech (Beijing) Co., Ltd. TRIzol reagent was purchased from Invitrogen company. Liquid nitrogen and Spanish agarose (Biowest Agarose) were purchased from Hainan Hifly Industrial Co., Ltd. Nucleic acid dyes were purchased from SBS Genetech Co., Ltd. Other related reagents were produced in China. Instruments
Ultrapure water system (PURELAB Ultra, ELGA); electronic balance (Precisa, Xs365M-sc); magnetic stirrer (RET Basic C, IKA); Agilent 2100 bioanalyzer (Agilent, USA); Qubit 3.0 fluorescence quantifier (Life Invitrogen, USA); NanoDropTM 2000 spectrophotometer (Thermo Fisher, USA); electrophoresis apparatus and gel imager (Bio-Rad, USA).
Methods
Total RNA extraction method
In this experiment, the Trizol method was used to extract total RNA from the venom glands, venom tubes, salivary glands, and tooth glands. Specifically, the shells of cone snail were crushed, different tissues were taken out, including the venom gland, venom tube, salivary gland and tooth gland. Then, 100 mg of shredded cone snail was added in a 1.5 ml centrifuge tube, into which 1 ml of Trizol was added, followed by fully homogenizing and standing at room temperature for 5 min. Into the homogenate, 0.2 ml of chloroform was added, and the mixture was shaken for 15 s and stood for 2 min. After centrifugation at 4 ℃,the supernatant was added with 0.5 ml of isopropanol, and the liquid in the tube was gently mixed and stood at room temperature for 10 min. The liquid was centrifuged again at 4 ℃, and the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol to allow precipitation. The precipitate was air-dried, and added with an appropriate amount of DEPC H2O to dissolve it.
RNA agarose electrophoresis
A certain amount of each RNA sample (4 μl) was mixed with bromophenol blue loading buffer. The obtained liquid was added to 1.0% agarose gel containing Goldview, electrophoresed at 100 V for 30 min, and then photographed using a gel imager and stored.
Total RNA detection method
A certain amount of each RNA sample (1 μl) was diluted by 100 times, and the dilution was determined by a NanoDropTM 2000 spectrophotometer and a Qubit 3.0 fluorescence quantifier for the purity and concentration of the total RNA, respectively. An Agilent 2100 bioanalyzer was employed to detect the integrity of each total RNA sample.
Results and Analysis
Agarose electrophoresis analysis of total RNA
The total RNA extracted from different tissues (venom gland, tooth gland, venom tube and salivary gland) of cone snail by the Trizol method was detected by 1% agarose gel electrophoresis. The electrophoresis results are shown in Fig. 1. The total RNA bands of the venom gland, tooth gland, toxic tube and salivary gland of cone snail were clearly visible, and there was no obvious drag, indicating that the extracted RNA did not degraded. Using the Trizol method to extract the total RNA of mollusc cone snail, it was found that the 18 S bands were bright, and the 28 S and 5 S bands were darker or not extracted, which is normal. Detection results of RNA concentration
The total RNA of the venom gland, venom tube, salivary gland and tooth gland of cone snail was subjected to Nanodrop detection for purity and Qubit detection for concentration, respectively. It can be seen from Table 1 that the total RNA concentrations of the venom gland, venom tube, salivary gland and tooth gland obtained by Nanodrop detection ranged from 857.3 to 1 255.2 ng/μl, and the OD260/280 values were in the range of 1.99-2.15, and the OD260/230 values were all greater than 2.0 . Therefore, the results met the basic requirements that the OD260/280 values of non-contaminated RNA are in the range of 1.8-2.1. The total RNA concentrations of the venom gland, venom tube, salivary gland and tooth gland obtained by Qubit detection ranged from 744.0 to 1 008.0 ng/μl, and the total amounts were in the range of 8.9-13.1 μg. From the experimental data, it could be seen that the quality of the total RNA obtained from the same weight of venom gland, venom gland, salivary gland and tooth gland was different. Among them, the total RNA extracted from the venom tube had the highest concentration and better purity. Taken together, the total RNA extracted from different tissue parts of cone snail met the requirements of the library construction for subsequent transcriptome sequencing.
Detection results of RNA integrity and purity
The quality of the total RNA extracted from the venom gland, venom tube, salivary gland and tooth gland of cone snail was detected by electrophoresis using an Agilent 2100 bioanalyzer, and the results are shown in Fig. 2. The rRNA［28S/18S］ratios were all 0. The total RNA extracted from different tissues of cone snail all showed a single peak, indicating that the total RNA of cone snail was only 18 S, but not 28 S. The RIN (RNA integrity number) values detected by the Agilent 2100 bioanalyzer were 6.50, 6.20, 8.00 and 6.90, respectively. However, the RIN value is of little significance for the total RNA of cone snail that only shows a single peak.
Conclusions and Discussion
High-quality RNA is the premise and foundation for conducting various molecular biology studies［10］. For cone snail as a mollusk, its RNA extraction suffers from relatively strict experimental requirements, and is greatly affected by various factors. The extraction for total RNA samples from different tissue parts of cone snail were optimized, in order to obtain the best tissue part as the future total RNA extraction scheme of cone snail, which will provide convenience and basis for subsequent molecular biology research. In this study, the venom gland, venom tube, salivary gland and tooth gland of cone snail were selected as the research objects. The total RNA was extracted, and its quality was tested by the Trizol method. The Trizol method is an easy-to-use commercial kit method developed based on the AGPC method［11］. Its main component, guanidine isothiocyanate, is a potent protein denaturant, which can fully lyse cells and quickly inactivate endogenous RNase in cells, thereby effectively avoiding RNA degradation［12］. Therefore, the Trizol method is very effective and feasible for the extraction of total RNA from RNase-rich tissues［13］. The agarose gel electrophoresis of the total RNA extracted by the Trizol method showed clearly visible bands without obvious drag, but the total RNA extracted showed only bright 18 S bands. The results of total RNA extracted from different parts of cone snail detected by Aligent 2100 bioanalyzer showed that the total RNA of different parts of cone snail all showed a single peak, which was consistent with the results of agarose electrophoresis. Xu et al. ［14］ also used the Trizol method to extract total RNA from Oncomelania hupensis Gredler, and only obtained 18 S bands. The concentrations and purity of the total RNA from different tissue parts of cone snail obtained by Nanodrop and Qubit detection all could meet the basic requirements of molecular biology research, and the total RNA extracted from the snails venom tubes had the advantages of high concentration and good purity compared with other tissues, which accords
with the results of Quan et al. ［15］ obtained on the extraction of total RNA from the venom glands and venom tubes of 7 different species of cone snails in Hainan.
In summary, the extraction effects of total RNA from the venom gland, venom duct, salivary gland and tooth gland of cone snail could meet the basic requirements of molecular biology. Without any special tissue requirements, extracting total RNA from the salivary gland of cone snail as a material showed a higher yield, and best purity, concentration and integrity, which could meet the requirements of molecular biology experiments such as transcriptome sequencing and gene expression research, laying a foundation for carrying out studies on the molecular level of conotoxins.
References
［1］ GAO B, PENG C, YANg J, et al. Cone snails: a big store of conotoxins for novel drug discovery［J］. Toxins, 2017, 9(12): 397. ［2］ HUANG Y, PENG C, YI Y, et al. A transcriptomic survey of ion-channel based conotoxins in Chinese tubular cone snail ( Conus betulins )［J］. Marine Drugs, 2017, 15(7): 228.
［3］ QIAN J, SUN ZH, LIU YQ, et al. Cytotoxicity of α-conotoxin TxID in lung cancer［J］. Journal of Tropical Biology, 2019, 10(2): 99-105. (in Chinese)
［4］ WU XY, AN TT, BM. Study on the chemical synthesis and pesticidal activity of conotoxin ImI［J］. Natural Product Research and Development, 2018(30): 2203-2206. (in Chinese)
［5］ GAO B, PENG C, LIN B, et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail［J］. Toxins, 2017, 9(7):214.
［6］ TOSTI E, BONI R, GALLO A. μ-Conotoxins modulating sodium currents in pain perception and transmission: A therapeutic potential［J］. Mar. Drugs, 2017(15): E295.
［7］ LUO SL, ZHANG B, ZHANGSUN DT. Conotoxins［J］. Bulletin of Biology, 2003, 38(4):7. (in Chinese)
［8］ FENG JC, ZHANGSUN DT, CHEN Q, et al. Construction of cDNA library of barrel-shaped cone snail toxins from Hainan［J］. Chinese Journal of Marine Drugs, 2009, 28(3): 1-6. (in Chinese)
［9］ GAO B, PENG C, ZHU Y, et al. High throughput identification of novel conotoxins from the vermivorous oak cone snail ( Conus quercinus ) by transcriptome sequencing［J］. Int. J. Mol. Sci., 2018(19): 3901.
［10］ FENG ZF, WANG L, LI WX, et al. Extraction of total RNA from marine invertebrate tissue［J］. Marine Sciences, 2014, 38(11):24-28. (in Chinese)
［11］ CHOMCZYNSKI P, SACCHI N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction［J］. Anal Biochem, 1987, 162(1):156-159.
［12］ CHOMCZYNSKI P, SACCHI N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on［J］. Nat Protoc, 2006, 1(2): 581-585.
［13］ SIMMS D, CIZDZIEL PE, CHOMCZYNSKI P. TRIzolTM: A new reagent for optimal single step isolation of RNA［J］. Focus, 1992, 15(4): 99-102.
［14］ XU SJ, WANG K, ZHANG MH, et al. Comparison of three different methods for isolating RNA from Oncomelania hupensis ［J］. Chinese Journal of Schistosomiasis Control, 2017, 29(3): 334-337. (in Chinese)
［15］ QUAN YR, LUO SL, LIN QJ, et al. Conotoxin RNA isolation and its cDNA synthesis［J］. Chinese Journal of Marine Drugs, 2005(2): 1-5. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
［Methods］ The total RNA of four different tissues of cone snail, venom gland, venom tube, salivary gland and tooth gland was extracted by the Trizol method. The total RNA of cone snail was detected by agarose gel electrophoresis, NanoDropTM 2000 spectrophotometer and Aligent 2100 biological analyzer.
［Results］ The total RNA extracted from different tissues of cone snail showed clear band, and thus had similar concentrations and purity, and the highest yield was obtained from venom tube.
［Conclusions］ The total RNA extracted from different tissue parts of cone snail could meet the basic requirements of molecular biology, and its venom tube was the best tissue part for extracting total RNA, which lays a foundation for molecular biology research such as high-throughput transcriptome sequencing of cone snail.
Key words Cone snail; Venom gland; RNA extraction; Trizol method
Received: February 23, 2020Accepted: April 20, 2020
Supported by the Hainan Provincial Keypoint Research and Invention Program (ZDYF2018138).
Qin CHEN (1983-), female, P. R. China, lecturer, devoted to research about marine drugs. Chao PENG (1986-), male, P. R. China, engineer, devoted to research about marine drug.
#Corresponding author. E-mail: gaobingmiao@hainmc.edu.cn.
*These authors contributed equally to this work.
Cone snail ( Conus geographus ) belongs to Conidae of Stenoglossa in Prosobranchia of Gastropoda in Mollusa. Conotoxins are a type of small peptide neurotoxins secreted by cone snail for self-defense and predation. They have the characteristics of high abundance, small molecular weight, diverse structure, wide range of targets and strong specificity［1-2］. Pharmacological experimental research shows that conotoxins have cytotoxicity, analgesic activity, pesticidal activity, anti-cancer activity and other pharmacological activity by acting on different ion channels or receptors［3-6］. Therefore, it can be used not only as a molecular model for new drug design, but also directly as a drug for disease treatment. There are about 700 kinds of cone snails in the world, and there are more than 100 kinds of cone snails in China, mainly distributed in the Xisha Islands, Nansha Islands and Zhongsha Islands under the jurisdiction of Hainan Province, and tropical sea areas such as Taiwan Island［2］. Transcriptomics and proteomics methods have identified that each kind of cone snail can secrete about 200 types of conotoxins. In theory, conotoxins may exceed 140 000 species accumulatively［5］. Therefore, conotoxins are called "the treasure trove of marine drugs", and they are also an unlimited drug resource［7］. In recent years, the deterioration of the marine ecological environment and the overfishing of fishermen have destroyed cone snails habitat, which has led to the situation that the systematic development and utilization of cone snail has evolved into endangered resources. The protection and utilization of genetic resources of Hainans characteristic cone snails are more urgent. The steps of conotoxin isolation and toxin gene cloning are time-consuming, labor-consuming, inefficient, and greatly affected by resources, and some specimens are difficult to obtain［5-7］. Therefore, to study the conotoxin gene resources through high-throughput sequencing technology and to establish the Hainan insectivorous cone snail toxin gene library and peptide library can solve the problem of disappearance of functional gene caused by frequent extinction of cone snail resources, and will simultaneously lay a foundation for studying the subsequent structure and function and pharmacology and efficacy of conotoxins and the development of marine drugs.
Mollusc cone snail belongs to vertebrates, and its genomic DNA is very complex and huge, about 100 times that of protein and mRNA. The work of isolating target gene fragments of conotoxins from cone snail genomic DNA is very time-consuming and labor-consuming. The total RNA of cone snail can be extracted to synthesize cDNA by reverse transcriptase, and the conotoxin gene can be obtained by construction of a library and high-throughput sequencing［8-9］. Therefore, in this study, we compared the effects of extracting total RNA from different tissue parts of cone snail, so as to obtain high-quality, high-purity and complete integrity conotome RNA and provide a basis for high-throughput transcriptome sequencing and other molecular biological studies.
Materials and Methods
Materials
Cone snail materials
The cone snails used in this study were collected along the coast of Sanya, Hainan, and dissected for venom glands, venom tubes, salivary glands and tooth glands at different parts as materials for total RNA extraction. Remaining materials were stored in a refrigerator at -70 ℃ for later use.
Reagents
DNA marker D2000, Taq polymerase, Mg2+ , dNTPs and RNaseA were purchased from Tiangen Biotech (Beijing) Co., Ltd. TRIzol reagent was purchased from Invitrogen company. Liquid nitrogen and Spanish agarose (Biowest Agarose) were purchased from Hainan Hifly Industrial Co., Ltd. Nucleic acid dyes were purchased from SBS Genetech Co., Ltd. Other related reagents were produced in China. Instruments
Ultrapure water system (PURELAB Ultra, ELGA); electronic balance (Precisa, Xs365M-sc); magnetic stirrer (RET Basic C, IKA); Agilent 2100 bioanalyzer (Agilent, USA); Qubit 3.0 fluorescence quantifier (Life Invitrogen, USA); NanoDropTM 2000 spectrophotometer (Thermo Fisher, USA); electrophoresis apparatus and gel imager (Bio-Rad, USA).
Methods
Total RNA extraction method
In this experiment, the Trizol method was used to extract total RNA from the venom glands, venom tubes, salivary glands, and tooth glands. Specifically, the shells of cone snail were crushed, different tissues were taken out, including the venom gland, venom tube, salivary gland and tooth gland. Then, 100 mg of shredded cone snail was added in a 1.5 ml centrifuge tube, into which 1 ml of Trizol was added, followed by fully homogenizing and standing at room temperature for 5 min. Into the homogenate, 0.2 ml of chloroform was added, and the mixture was shaken for 15 s and stood for 2 min. After centrifugation at 4 ℃,the supernatant was added with 0.5 ml of isopropanol, and the liquid in the tube was gently mixed and stood at room temperature for 10 min. The liquid was centrifuged again at 4 ℃, and the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol to allow precipitation. The precipitate was air-dried, and added with an appropriate amount of DEPC H2O to dissolve it.
RNA agarose electrophoresis
A certain amount of each RNA sample (4 μl) was mixed with bromophenol blue loading buffer. The obtained liquid was added to 1.0% agarose gel containing Goldview, electrophoresed at 100 V for 30 min, and then photographed using a gel imager and stored.
Total RNA detection method
A certain amount of each RNA sample (1 μl) was diluted by 100 times, and the dilution was determined by a NanoDropTM 2000 spectrophotometer and a Qubit 3.0 fluorescence quantifier for the purity and concentration of the total RNA, respectively. An Agilent 2100 bioanalyzer was employed to detect the integrity of each total RNA sample.
Results and Analysis
Agarose electrophoresis analysis of total RNA
The total RNA extracted from different tissues (venom gland, tooth gland, venom tube and salivary gland) of cone snail by the Trizol method was detected by 1% agarose gel electrophoresis. The electrophoresis results are shown in Fig. 1. The total RNA bands of the venom gland, tooth gland, toxic tube and salivary gland of cone snail were clearly visible, and there was no obvious drag, indicating that the extracted RNA did not degraded. Using the Trizol method to extract the total RNA of mollusc cone snail, it was found that the 18 S bands were bright, and the 28 S and 5 S bands were darker or not extracted, which is normal. Detection results of RNA concentration
The total RNA of the venom gland, venom tube, salivary gland and tooth gland of cone snail was subjected to Nanodrop detection for purity and Qubit detection for concentration, respectively. It can be seen from Table 1 that the total RNA concentrations of the venom gland, venom tube, salivary gland and tooth gland obtained by Nanodrop detection ranged from 857.3 to 1 255.2 ng/μl, and the OD260/280 values were in the range of 1.99-2.15, and the OD260/230 values were all greater than 2.0 . Therefore, the results met the basic requirements that the OD260/280 values of non-contaminated RNA are in the range of 1.8-2.1. The total RNA concentrations of the venom gland, venom tube, salivary gland and tooth gland obtained by Qubit detection ranged from 744.0 to 1 008.0 ng/μl, and the total amounts were in the range of 8.9-13.1 μg. From the experimental data, it could be seen that the quality of the total RNA obtained from the same weight of venom gland, venom gland, salivary gland and tooth gland was different. Among them, the total RNA extracted from the venom tube had the highest concentration and better purity. Taken together, the total RNA extracted from different tissue parts of cone snail met the requirements of the library construction for subsequent transcriptome sequencing.
Detection results of RNA integrity and purity
The quality of the total RNA extracted from the venom gland, venom tube, salivary gland and tooth gland of cone snail was detected by electrophoresis using an Agilent 2100 bioanalyzer, and the results are shown in Fig. 2. The rRNA［28S/18S］ratios were all 0. The total RNA extracted from different tissues of cone snail all showed a single peak, indicating that the total RNA of cone snail was only 18 S, but not 28 S. The RIN (RNA integrity number) values detected by the Agilent 2100 bioanalyzer were 6.50, 6.20, 8.00 and 6.90, respectively. However, the RIN value is of little significance for the total RNA of cone snail that only shows a single peak.
Conclusions and Discussion
High-quality RNA is the premise and foundation for conducting various molecular biology studies［10］. For cone snail as a mollusk, its RNA extraction suffers from relatively strict experimental requirements, and is greatly affected by various factors. The extraction for total RNA samples from different tissue parts of cone snail were optimized, in order to obtain the best tissue part as the future total RNA extraction scheme of cone snail, which will provide convenience and basis for subsequent molecular biology research. In this study, the venom gland, venom tube, salivary gland and tooth gland of cone snail were selected as the research objects. The total RNA was extracted, and its quality was tested by the Trizol method. The Trizol method is an easy-to-use commercial kit method developed based on the AGPC method［11］. Its main component, guanidine isothiocyanate, is a potent protein denaturant, which can fully lyse cells and quickly inactivate endogenous RNase in cells, thereby effectively avoiding RNA degradation［12］. Therefore, the Trizol method is very effective and feasible for the extraction of total RNA from RNase-rich tissues［13］. The agarose gel electrophoresis of the total RNA extracted by the Trizol method showed clearly visible bands without obvious drag, but the total RNA extracted showed only bright 18 S bands. The results of total RNA extracted from different parts of cone snail detected by Aligent 2100 bioanalyzer showed that the total RNA of different parts of cone snail all showed a single peak, which was consistent with the results of agarose electrophoresis. Xu et al. ［14］ also used the Trizol method to extract total RNA from Oncomelania hupensis Gredler, and only obtained 18 S bands. The concentrations and purity of the total RNA from different tissue parts of cone snail obtained by Nanodrop and Qubit detection all could meet the basic requirements of molecular biology research, and the total RNA extracted from the snails venom tubes had the advantages of high concentration and good purity compared with other tissues, which accords
with the results of Quan et al. ［15］ obtained on the extraction of total RNA from the venom glands and venom tubes of 7 different species of cone snails in Hainan.
In summary, the extraction effects of total RNA from the venom gland, venom duct, salivary gland and tooth gland of cone snail could meet the basic requirements of molecular biology. Without any special tissue requirements, extracting total RNA from the salivary gland of cone snail as a material showed a higher yield, and best purity, concentration and integrity, which could meet the requirements of molecular biology experiments such as transcriptome sequencing and gene expression research, laying a foundation for carrying out studies on the molecular level of conotoxins.
References
［1］ GAO B, PENG C, YANg J, et al. Cone snails: a big store of conotoxins for novel drug discovery［J］. Toxins, 2017, 9(12): 397. ［2］ HUANG Y, PENG C, YI Y, et al. A transcriptomic survey of ion-channel based conotoxins in Chinese tubular cone snail ( Conus betulins )［J］. Marine Drugs, 2017, 15(7): 228.
［3］ QIAN J, SUN ZH, LIU YQ, et al. Cytotoxicity of α-conotoxin TxID in lung cancer［J］. Journal of Tropical Biology, 2019, 10(2): 99-105. (in Chinese)
［4］ WU XY, AN TT, BM. Study on the chemical synthesis and pesticidal activity of conotoxin ImI［J］. Natural Product Research and Development, 2018(30): 2203-2206. (in Chinese)
［5］ GAO B, PENG C, LIN B, et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail［J］. Toxins, 2017, 9(7):214.
［6］ TOSTI E, BONI R, GALLO A. μ-Conotoxins modulating sodium currents in pain perception and transmission: A therapeutic potential［J］. Mar. Drugs, 2017(15): E295.
［7］ LUO SL, ZHANG B, ZHANGSUN DT. Conotoxins［J］. Bulletin of Biology, 2003, 38(4):7. (in Chinese)
［8］ FENG JC, ZHANGSUN DT, CHEN Q, et al. Construction of cDNA library of barrel-shaped cone snail toxins from Hainan［J］. Chinese Journal of Marine Drugs, 2009, 28(3): 1-6. (in Chinese)
［9］ GAO B, PENG C, ZHU Y, et al. High throughput identification of novel conotoxins from the vermivorous oak cone snail ( Conus quercinus ) by transcriptome sequencing［J］. Int. J. Mol. Sci., 2018(19): 3901.
［10］ FENG ZF, WANG L, LI WX, et al. Extraction of total RNA from marine invertebrate tissue［J］. Marine Sciences, 2014, 38(11):24-28. (in Chinese)
［11］ CHOMCZYNSKI P, SACCHI N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction［J］. Anal Biochem, 1987, 162(1):156-159.
［12］ CHOMCZYNSKI P, SACCHI N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on［J］. Nat Protoc, 2006, 1(2): 581-585.
［13］ SIMMS D, CIZDZIEL PE, CHOMCZYNSKI P. TRIzolTM: A new reagent for optimal single step isolation of RNA［J］. Focus, 1992, 15(4): 99-102.
［14］ XU SJ, WANG K, ZHANG MH, et al. Comparison of three different methods for isolating RNA from Oncomelania hupensis ［J］. Chinese Journal of Schistosomiasis Control, 2017, 29(3): 334-337. (in Chinese)
［15］ QUAN YR, LUO SL, LIN QJ, et al. Conotoxin RNA isolation and its cDNA synthesis［J］. Chinese Journal of Marine Drugs, 2005(2): 1-5. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU