杉木叶片原生质体分离及RNA提取体系的建立
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摘要
【目的】以杉木组培苗幼嫩叶片为材料,分析不同因素对杉木原生质体分离的影响,建立杉木叶片原生质体分离体系,以期为杉木基因功能验证提供有效的技术平台;比较不同RNA提取方法,筛选获得杉木原生质体总RNA提取的有效方法,为今后利用杉木原生质体开展分子生物学研究提供技术支持,也可为其他林木原生质体的总RNA提取研究提供参考。【方法】选取杉木组培苗最上端未分轮的幼嫩叶片为材料,通过分析5种不同酶组合、真空处理时间(0、5、10、15、20 min)、渗透压(0.3、0.4、0.5、0.6 mol·L-1甘露醇)、BSA浓度(0.1%、0.2%、0.3%、0.4%)及酶解时间(1、2、3、4、5 h)5个条件,进行杉木叶片原生质体的分离。采用改良Trizol法、改良Trizol+10μg糖原法、CTAB-Li Cl法、CTAB-Li Cl+10μg糖原法、改良CTAB-异丙醇法、改良CTAB-异丙醇+10μg糖原法、天根RNA提取试剂盒法共7种方法提取杉木原生质体RNA,通过杉木内参基因和2个特异基因进行RNA质量验证,比较不同方法的提取效果。【结果】在1.5%纤维素酶R-10、1%离析酶R-10、0.2%果胶酶Y-23、0.5 mol·L-1甘露醇以及0.3%BSA的混合酶液中,真空处理10 min,酶解2 h,可分离出高活力的纯净杉木原生质体,其产量可达到7.27×106个·g-1,活力可达到94%。7种方法皆能提取出杉木叶片原生质体的总RNA,但不同方法间存在明显差异,其中改良Trizol法、改良Trizol+10μg糖原法、改良CTAB-Li Cl法、改良CTAB-Li Cl+10μg糖原法提取的RNA有不同程度的降解现象,其余方法所提取的RNA完整性较好,且OD260/280和OD260/230值均在1.8~2.0范围内;在RNA得率方面,改良CTAB-异丙醇+10μg糖原法可高效提取杉木原生质体RNA,106个细胞RNA产量平均可达到6.89μg,分别是改良CTAB-异丙醇法和天根RNA提取试剂盒法的1.73倍和1.58倍;综合考虑,改良CTAB-异丙醇+10μg糖原法提取杉木原生质体RNA的效果最好。【结论】以杉木组培苗幼嫩叶片为材料,分离条件为纤维素酶R-10浓度1.5%、离析酶R-10浓度1%、果胶酶Y-23浓度0.2%、甘露醇浓度0.5 mol·L-1、BSA浓度0.3%,真空处理10 min,酶解2 h,可大量获得高质量的杉木原生质体;利用改良CTAB-异丙醇+10μg糖原法,可高效提取杉木原生质体RNA,106个细胞RNA产量平均可达到6.89μg。研究结果为杉木重要性状关键基因的功能研究及其在育种中的应用提供了重要基础。
【Objective】 To establish protoplast isolation system for Chinese fir( Cunninghamia lanceolata) leaves,the effects of different factors on protoplast preparation were analyzed by using young leaves of Chinese fir plantlets from tissue culture. Meanwhile,the most effective method for extraction of total RNA from Chinese fir protoplast was chosen through comparison of different RNA extraction methods. 【Method 】 The young leaves of Chinese fir plantlets were selected as explants,and the effects of major factors,such as five enzyme combinations,vacuum treatment time( 0,5,10,15,20 min),osmotic pressure( 0. 3,0. 4,0. 5,0. 6 mol·L-1 mannitol),BSA concentration( 0. 1%,0. 2%,0. 3%,0. 4%)and enzymolysis time( 1,2,3,4,5 h) were investigated to construct optimum protoplasts isolation system for Chinese fir leaves. Seven RNA extraction methods,such as improved Trizol method,improved Trizol + 10 μg glycogen method,CTAB-Li Cl method, CTAB-Li Cl + 10 μg glycogen method, improved CTAB-isopropanol method, improved CTABisopropanol + 10 μg glycogen method and TIANGEN RNA extraction kit were applied to compare the efficiency of RNAextraction from Chinese fir protoplasts. The quality and yield were detected by reference genes and two specific genes.【Result】The optimum condition for protoplast isolation from Chinese fir leaves included a mixed enzyme solution of1. 5% cellulase R-10,0. 2% pectolyase Y-23,1% macerozy R-10,0. 5 mol·L-1 mannitol and 0. 3% BSA,vacuum treatment for 10 min,and digestion for 2 h. High activity and pure protoplasts could be prepared from Chinese fir leaves by using the optimum condition. The protoplast yield could reach to 7. 26 × 106 per gram with 94% viability rate. All seven methods could extract RNA from protoplasts. However there were obvious differences between different methods.RNA degradation was found in the improved Trizol method,improved Trizol + 10 μg glycogen method,CTAB-Li Cl method and CTAB-Li Cl + 10 μg glycogen method. While,the other three methods obtained good RNA quality,of which the values of OD260/280 and OD260/230 were in the normal range of 1. 8-2. 0. Additionally,the yield of RNA was the highest in improved CTAB-isopropanol + 10 μg glycogen method,up to 6. 89 μg per 106 protoplasts,which was 1. 73 times and 1. 58 times of the improved CTAB-isopropanol and TIANGEN RNA extraction kit,respectively. In conclusion,the modified CTAB-isopropanol + 10 μg glycogen method was most efficient for RNA extraction from Chinese fir protoplasts.【Conclusion】 In this study,by using the young leaves of Chinese fir plantlets,a protoplast isolation technique was established with the condition of 1. 5% cellulase R-10,0. 2% pectolyase Y-23,1% macerozy R-10,0. 5 mol·L-1 mannitol and 0. 3% BSA,10 min vacuum treatment,and 2 h enzymolysis. Meanwhile,the improved CTAB-isopropanol +10 μg glycogen method could be used to extract total RNA from Chinese fir protoplasts with the average yield of 6. 89 μg per 106 protoplasts. All the results of this study provided an important basis for the functional analysis of key genes and their application in breeding of Chinese fir.
【Objective】 To establish protoplast isolation system for Chinese fir( Cunninghamia lanceolata) leaves,the effects of different factors on protoplast preparation were analyzed by using young leaves of Chinese fir plantlets from tissue culture. Meanwhile,the most effective method for extraction of total RNA from Chinese fir protoplast was chosen through comparison of different RNA extraction methods. 【Method 】 The young leaves of Chinese fir plantlets were selected as explants,and the effects of major factors,such as five enzyme combinations,vacuum treatment time( 0,5,10,15,20 min),osmotic pressure( 0. 3,0. 4,0. 5,0. 6 mol·L-1 mannitol),BSA concentration( 0. 1%,0. 2%,0. 3%,0. 4%)and enzymolysis time( 1,2,3,4,5 h) were investigated to construct optimum protoplasts isolation system for Chinese fir leaves. Seven RNA extraction methods,such as improved Trizol method,improved Trizol + 10 μg glycogen method,CTAB-Li Cl method, CTAB-Li Cl + 10 μg glycogen method, improved CTAB-isopropanol method, improved CTABisopropanol + 10 μg glycogen method and TIANGEN RNA extraction kit were applied to compare the efficiency of RNAextraction from Chinese fir protoplasts. The quality and yield were detected by reference genes and two specific genes.【Result】The optimum condition for protoplast isolation from Chinese fir leaves included a mixed enzyme solution of1. 5% cellulase R-10,0. 2% pectolyase Y-23,1% macerozy R-10,0. 5 mol·L-1 mannitol and 0. 3% BSA,vacuum treatment for 10 min,and digestion for 2 h. High activity and pure protoplasts could be prepared from Chinese fir leaves by using the optimum condition. The protoplast yield could reach to 7. 26 × 106 per gram with 94% viability rate. All seven methods could extract RNA from protoplasts. However there were obvious differences between different methods.RNA degradation was found in the improved Trizol method,improved Trizol + 10 μg glycogen method,CTAB-Li Cl method and CTAB-Li Cl + 10 μg glycogen method. While,the other three methods obtained good RNA quality,of which the values of OD260/280 and OD260/230 were in the normal range of 1. 8-2. 0. Additionally,the yield of RNA was the highest in improved CTAB-isopropanol + 10 μg glycogen method,up to 6. 89 μg per 106 protoplasts,which was 1. 73 times and 1. 58 times of the improved CTAB-isopropanol and TIANGEN RNA extraction kit,respectively. In conclusion,the modified CTAB-isopropanol + 10 μg glycogen method was most efficient for RNA extraction from Chinese fir protoplasts.【Conclusion】 In this study,by using the young leaves of Chinese fir plantlets,a protoplast isolation technique was established with the condition of 1. 5% cellulase R-10,0. 2% pectolyase Y-23,1% macerozy R-10,0. 5 mol·L-1 mannitol and 0. 3% BSA,10 min vacuum treatment,and 2 h enzymolysis. Meanwhile,the improved CTAB-isopropanol +10 μg glycogen method could be used to extract total RNA from Chinese fir protoplasts with the average yield of 6. 89 μg per 106 protoplasts. All the results of this study provided an important basis for the functional analysis of key genes and their application in breeding of Chinese fir.
引文
傅星星.2007.杉木精油提取及其深加工研究.福州:福建农林大学硕士学位论文.(Fu X X.2007.Study on essential oil extraction from Cunninghamia lanceolata(Lamb.)Hook and deep-processing.Fuzhou:MS thesis of Fujian Agriculture and Forestry University.[in Chinese])
郭萍,武瑶,李嘉,等.2014.利用水稻原生质体快速分析miRNA靶标RNA.生物工程学报,30(11):1751-1762.(Guo P,Wu Y,Li J,et al.2014.Efficient transient expression to analyze miRNA targets in rice protoplasts.Chinese Journal of Biotechnology,30(11):1751-1762.[in Chinese])
李妮娜,丁林云,张志远,等.2014.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立.作物学报,40(2):231-239.(Li N N,Ding L Y,Zhang Z Y,et al.2014.Isolation of mesophyll protoplast and establishment of gene transient expression system in cotton.Acta Agronomica Sinica,40(2):231-239.[in Chinese])
彭邵锋,陆佳,陈永忠,等.2013.木本植物原生质体培养体系研究进展.中国农学通报,29(1):1-6.(Peng S F,Lu J,Chen Y Z,et al.2013.Research progress of protoplast culture in woody plants.Chinese Agricultural Science Bulletin,29(1):1-6.[in Chinese])
谢传胜,宋国琦,王兴军,等.2009.拟南芥和烟草幼嫩种子RNA不同提取方法的比较.中国农学通报,25(23):78-81.(Xie C S,Song G Q,Wang X J,et al.2009.RNA extraction from Arabidopsis thaliana and Nicotiana tobaccum immature seeds using different methods.Chinese Agricultural Science Bulletin,25(23):78-81.[in Chinese])
王鹏凯,吕伟光,胡雪怡,等.2013.北美鹅掌楸原生质体的分离与培养.西北植物学报,33(2):254-260.(Wang P K,LüW G,Hu X Y,et al.2013.Isolation and cultivation of protoplasts of Liriodendron tulipifera.Acta Botanica BorealiOccidentalia Sinica,33(2):254-260.[in Chinese])
王莉,姚占军.2009.植物原生质体的制备与活力检测研究进展.生物技术通报,(S1):46-50.(Wang L,Yao Z J.2009.Advance on the preparation and vitality testing for plant protoplast.Biotechnology Bulletin,(S1):46-50.[in Chinese])
Bai Y,Han N,Wu J,et al.2014.A transient gene expression system using barley protoplasts to evaluate microRNAs for posttranscriptional regulation of their target genes.Plant Cell Tissue and Organ Culture,119(1):211-219.
Burris K P,Dlugosz E M,Collins A G,et al.2016.Development of a rapid,low-cost protoplast transfection system for switchgrass(Panicum virgatum L.).Plant Cell Rep,35(3):693-704.
Chang S,Puryear J,Cairney J.1993.A simple and efficient method for isolating RNA from pine trees.Plant Molecular Biology Reporter,11(2):113-116.
Davey M R,Anthony P,Power J B,et al.2005.Plant protoplasts:status and biotechnological perspectives.Biotechnology Advances,23(2):131-171.
Eeckhaut T,Lakshmanan P S,Deryckere D,et al.2013.Progress in plant protoplast research.Planta,238(6):991-1003.
Endo M,Shimizu H,Araki T.2016.Rapid and simple isolation of vascular,epidermal and mesophyll cells from plant leaf tissue.Nature Protocols,11(8):1388-1395.
Guo J,Morrell-Falvey J L,LabbéJ L,et al.2012.Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.PLo S One,7(9):e44908.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.BMC Genomics,13(1):648.
Jiang F,Zhu J,Liu H L.2013.Protoplasts:a useful research system for plant cell biology,especially dedifferentiation.Protoplasma,250(6):1231-1238.
Jones A M,Chattopadhyay A,Shukla M,et al.2012.Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility,protoplast isolation,and facilitates sustained cell division in American elm(Ulmus americana).BMC Plant Biology,12(1):75.
Jung H I,Yan J,Zhai Z,et al.2015.Gene functional analysis using protoplast transient assays.Methods in Molecular Biology,1284:433-452.
Li X,Su Q,Zheng R,et al.2015.ClRTL1 encodes a Chinese fir RNase III-like protein involved in regulating shoot branching.International Journal of Molecular Sciences,16(10):25691-25710.
Lin Y C,Li W,Chen H,et al.2014.A simple improved-throughput xylem protoplast system for studying wood formation.Nature Protocols,9(9):2194-2205.
Lionetti V,Cervone F,De Lorenzo G.2015.A lower content of demethylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.Phytochemistry,112(1):188-194.
Mathur J,Koncz C,Szabados L.1995.A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts.Plant Cell Reports,14(4):221-226.
Nanjareddy K,Arthikala M K,Blanco L,et al.2016.Protoplast isolation,transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris:tools for rapid gene expression analysis.BMCBiotechnology,16(1):1-14.
Pati P K,Sharma M,Ahuja P S.2008.Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film.Protoplasma,233(1):165-171.
Tan B,Xu M,Chen Y,et al.2013.Transient expression for functional gene analysis using Populus protoplasts.Plant Cell,Tissue and Organ Culture,114(1):11-18.
Wan L C,Wang F,Guo X,et al.2012.Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing.BMC Plant Biology,12(1):146.
Wang G,Gao Y,Yang L,et al.2007.Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir(Cunninghamia lanceolata)by suppression subtractive hybridization.Genome,50(12):1141-1155.
Woo J W,Kim J,Kwon S I,et al.2015.DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.Nature Biotechnology,33(11):1162-1164.
Wu J Z,Liu Q,Geng X S,et al.2017.Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava(Manihot esculenta Crantz).BMC Biotechnology,17(1):29.
郭萍,武瑶,李嘉,等.2014.利用水稻原生质体快速分析miRNA靶标RNA.生物工程学报,30(11):1751-1762.(Guo P,Wu Y,Li J,et al.2014.Efficient transient expression to analyze miRNA targets in rice protoplasts.Chinese Journal of Biotechnology,30(11):1751-1762.[in Chinese])
李妮娜,丁林云,张志远,等.2014.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立.作物学报,40(2):231-239.(Li N N,Ding L Y,Zhang Z Y,et al.2014.Isolation of mesophyll protoplast and establishment of gene transient expression system in cotton.Acta Agronomica Sinica,40(2):231-239.[in Chinese])
彭邵锋,陆佳,陈永忠,等.2013.木本植物原生质体培养体系研究进展.中国农学通报,29(1):1-6.(Peng S F,Lu J,Chen Y Z,et al.2013.Research progress of protoplast culture in woody plants.Chinese Agricultural Science Bulletin,29(1):1-6.[in Chinese])
谢传胜,宋国琦,王兴军,等.2009.拟南芥和烟草幼嫩种子RNA不同提取方法的比较.中国农学通报,25(23):78-81.(Xie C S,Song G Q,Wang X J,et al.2009.RNA extraction from Arabidopsis thaliana and Nicotiana tobaccum immature seeds using different methods.Chinese Agricultural Science Bulletin,25(23):78-81.[in Chinese])
王鹏凯,吕伟光,胡雪怡,等.2013.北美鹅掌楸原生质体的分离与培养.西北植物学报,33(2):254-260.(Wang P K,LüW G,Hu X Y,et al.2013.Isolation and cultivation of protoplasts of Liriodendron tulipifera.Acta Botanica BorealiOccidentalia Sinica,33(2):254-260.[in Chinese])
王莉,姚占军.2009.植物原生质体的制备与活力检测研究进展.生物技术通报,(S1):46-50.(Wang L,Yao Z J.2009.Advance on the preparation and vitality testing for plant protoplast.Biotechnology Bulletin,(S1):46-50.[in Chinese])
Bai Y,Han N,Wu J,et al.2014.A transient gene expression system using barley protoplasts to evaluate microRNAs for posttranscriptional regulation of their target genes.Plant Cell Tissue and Organ Culture,119(1):211-219.
Burris K P,Dlugosz E M,Collins A G,et al.2016.Development of a rapid,low-cost protoplast transfection system for switchgrass(Panicum virgatum L.).Plant Cell Rep,35(3):693-704.
Chang S,Puryear J,Cairney J.1993.A simple and efficient method for isolating RNA from pine trees.Plant Molecular Biology Reporter,11(2):113-116.
Davey M R,Anthony P,Power J B,et al.2005.Plant protoplasts:status and biotechnological perspectives.Biotechnology Advances,23(2):131-171.
Eeckhaut T,Lakshmanan P S,Deryckere D,et al.2013.Progress in plant protoplast research.Planta,238(6):991-1003.
Endo M,Shimizu H,Araki T.2016.Rapid and simple isolation of vascular,epidermal and mesophyll cells from plant leaf tissue.Nature Protocols,11(8):1388-1395.
Guo J,Morrell-Falvey J L,LabbéJ L,et al.2012.Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.PLo S One,7(9):e44908.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.BMC Genomics,13(1):648.
Jiang F,Zhu J,Liu H L.2013.Protoplasts:a useful research system for plant cell biology,especially dedifferentiation.Protoplasma,250(6):1231-1238.
Jones A M,Chattopadhyay A,Shukla M,et al.2012.Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility,protoplast isolation,and facilitates sustained cell division in American elm(Ulmus americana).BMC Plant Biology,12(1):75.
Jung H I,Yan J,Zhai Z,et al.2015.Gene functional analysis using protoplast transient assays.Methods in Molecular Biology,1284:433-452.
Li X,Su Q,Zheng R,et al.2015.ClRTL1 encodes a Chinese fir RNase III-like protein involved in regulating shoot branching.International Journal of Molecular Sciences,16(10):25691-25710.
Lin Y C,Li W,Chen H,et al.2014.A simple improved-throughput xylem protoplast system for studying wood formation.Nature Protocols,9(9):2194-2205.
Lionetti V,Cervone F,De Lorenzo G.2015.A lower content of demethylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.Phytochemistry,112(1):188-194.
Mathur J,Koncz C,Szabados L.1995.A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts.Plant Cell Reports,14(4):221-226.
Nanjareddy K,Arthikala M K,Blanco L,et al.2016.Protoplast isolation,transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris:tools for rapid gene expression analysis.BMCBiotechnology,16(1):1-14.
Pati P K,Sharma M,Ahuja P S.2008.Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film.Protoplasma,233(1):165-171.
Tan B,Xu M,Chen Y,et al.2013.Transient expression for functional gene analysis using Populus protoplasts.Plant Cell,Tissue and Organ Culture,114(1):11-18.
Wan L C,Wang F,Guo X,et al.2012.Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing.BMC Plant Biology,12(1):146.
Wang G,Gao Y,Yang L,et al.2007.Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir(Cunninghamia lanceolata)by suppression subtractive hybridization.Genome,50(12):1141-1155.
Woo J W,Kim J,Kwon S I,et al.2015.DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.Nature Biotechnology,33(11):1162-1164.
Wu J Z,Liu Q,Geng X S,et al.2017.Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava(Manihot esculenta Crantz).BMC Biotechnology,17(1):29.