首页 > 过刊浏览>2016年第24卷第1期 >40-49. DOI:10.11926/j.issn.1005-3395.2016.01.006
PDF HTML阅读 XML下载 导出引用 引用提醒
夜香树均一化全长cDNA文库的构建及EST分析
作者:
作者单位:

永安市农业局

基金项目:

福建农林大学科研创新团队项目(cxtd12013)资助


Construction of Normalized Full-length cDNA Library and EST Analysis from Cestrum nocturnum L.
Author:
Affiliation:

Farm Bureau of Yongan County

  • 摘要
    • | |
  • 访问统计
    • |
  • 参考文献 [25]
    • |
  • 相似文献 [20]
    • | | |
  • 文章评论
    摘要:

    为构建高质量的夜香树(Cestrum nocturnum)均一化全长cDNA文库,以其花朵为材料,采用DSN均一化技术与改进的SMART技术相结合,将连接产物进行脱盐浓缩后电转化进行构建。结果表明,未脱盐连接产物的转化效率为1 μL连接产物有7000个菌落,理论重组率为96%;脱盐后,提升为4.1×106个菌落,理论重组率为98%;蓝斑也有插入片段,实际重组率应为100%;插入片段大小平均为1.6 kb。随机挑取500个单克隆(含50个蓝斑)测序,共获得464条EST,单一序列(unigene)为426条,占91.8%,其中片段重叠群26个,单基因400个,冗余率仅为8.1%,表明构建文库的均一化效果较好,可满足后续功能基因的筛选和基因信息的研究。

    Abstract:

    In order to construct high-quality normalized full-length cDNA library of Cestrum nocturnum L., the library from its flowers was established successfully by combined modified SMART technology, DSN digestion with desalting of ligation product. The results showed that transformation efficiency of the ligation product without desalting was 7000 colonies per microliter, and theoretical recombination rate was 96%, whereas, the transformation efficiency increased up to 4.1×106 colonies per microliter, and theoretical recombination rate was 98% after the ligation product desalting. Actually, blue colonies also contained the insert fragments, and, the recombination rate was to 100%. The average size of inserts in the library was about 1.6 kb. Five hundred colonies including 50 blue colonies were randomly picked for sequencing from the library, 464 effective sequences were obtained including 426 unigenes (accounting for 91.8%), which were composed of 26 contigs and 400 single genes, redundant rate was only 8.1%. So, it was suggested that the quality of the normalized cDNA library was better than that of ordinary cDNA libraries, which could be used for further research of screening of functional genes and their related information.

    参考文献
    [1] Delectis Florae Reipublicae Popularis Sinicae, Agendae Academiae Sinicae Edita. Florae Reipublicae Popularis Sinicae, Tomus 67(1) [M]. Beijing: Science Press, 2005: 149-150. 中国科学院中国植物志编辑委员会. 中国植物志, 第67卷第1分册 [M]. 北京: 科学出版社, 2005: 149-150.
    [2] Thanh T, Chi V T Q, Abdullah M P, et al. Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda [J]. Mol Biol Rep, 2011, 38(1): 177-182. doi: 10.1007/s11033-010-0092-4.
    [3] Raveh E, Cohen S, Levanon D, et al. Dynamic expression of Runx1 in skin affects hair structure [J]. Mech Dev, 2006, 123(11): 842-850. doi:10.1016/j.mod.2006.08.002.
    [4] Tabata S. Impact of genomics approaches on plant genetics andphysiology [J]. J Plant Res, 2002, 115(4): 271-275. doi: 10. 1007/s10265-002-0036-8.
    [5] Jiang C C, Chen G X, Pan D M, et al. Construction and ESTs analysis of a normalized full-length cDNA library of brown Prunus salicina [J]. Chin J Trop Crops, 2013, 34(2): 292-296. 姜翠翠, 陈桂信, 潘东明, 等. (木奈)褐变果实均一化全长cDNA文库的构建及部分ESTs序列分析 [J]. 热带作物学报, 2013, 34(2): 292-296.
    [6] Jiang M, Wang J, Wen G S, et al. Construction and sequence analysis of a normalized full-length cDNA library of Dendrobium officinale [J]. Chin J Chin Mat Med, 2013, 38(4): 504-510. doi: 10.4268/ cjcmm20130408. 蒋敏, 王江, 文国松, 等. 铁皮石斛均一化全长cDNA文库的构建与序列分析 [J]. 中国中药杂志, 2013, 38(4): 504-510. doi: 10.4268/cjcmm20130408.
    [7] Zhang D, Liu Y J, Li C J, et al. Construction of normalized cDNA library and analysis of corresponding EST sequences in Picea wilsonii [J]. Biotechn Bull, 2012(6): 71-76. 张盾, 刘亚静, 李长江, 等. 青杄均一化cDNA文库构建及EST序列分析 [J]. 生物技术通报, 2012(6): 71-76.
    [8] Wang Z, Yin X M, Wang J B, et al. Construction and characterization of normalized full-length cDNA library of banana roots [J]. Acta Hort Sin, 2011, 38(9): 1667-1674. 王卓, 殷晓敏, 王家保, 等. 香蕉根系均一化全长cDNA文库的构建和鉴定 [J]. 园艺学报, 2011, 38(9): 1667-1674.
    [9] Fregonezi J N, Fernandes T, Torezan J M D, et al. Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA [J]. Genet Mol Biol, 2006, 29(1): 97-104. doi: 10.1590/S1415-47572006000100019.
    [10] Rasheed K A. Improving an in vitro propagation protocol for Cestrum nocturnum L [J]. Acta Agrobot, 2013, 66(2): 35-44.
    [11] Zhao S Y, Huang Z H, Ye H H, et al. Experimental study on K562 cell apoptosis induced by steroid saponins extracted from the flowers of Cestrum nocturnum Linn. [J]. Chin Trad Pat Med, 2013, 35(3): 445-449. 赵世元, 黄之虎, 叶海洪, 等. 夜香树花甾体皂苷诱导K562细胞凋亡机制研究 [J]. 中成药, 2013, 35(3): 445-449.
    [12] Nong Z X, Zhao S Y, Huang Z H, et al. Effects of nocturnoside B from Cestrum nocturnum on doxorubicin-induced myocardial injury in rats [J]. Chin J Exp Trad Med Form, 2013, 19(18): 251-255. doi: 10.11653/syfj2013180251. 农智新, 赵世元, 黄之虎, 等. 夜香树提取物nocturnoside B对大鼠心肌损伤的保护作用及体外抗肿瘤作用研究 [J]. 中国实验方剂学杂志, 2013, 19(18): 251-255. doi: 10.11653/syfj2013180251.
    [13] PiaoY L, Ko N T, Lim M K, et al. Construction of long-transcript enriched cDNA libraries from submicrogram amounts of total RNAs by universal PCR amplification method [J]. Genome Res, 2001, 11(9): 1553-1558. doi: 10.1101/gr.185501.
    [14] Dai Z M, Zhu X J, Chen Q, et al. PCR-suppression effect: Kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples [J]. J Biotechn, 2007, 128(3): 435-443. doi: 10.1016/j.jbiotec.2006.10.018.
    [15] Dai Z M, Zhu X J, Yang W J. Full-length noramalization subtractive hybridization: A novel method for generating differentially expressed cDNAs [J]. Mol Biotechn, 2009, 43(3): 257-263. doi: 10.1007/s12033-009-9198-0.
    [16] Zhuang Y Z. Study of MYB transcription factors involved in fragrance biosynthesis in Phalaenopsis orchids [D]. Taiwan: National Cheng Kung University, 1997: 9-15. 庄育祯. 参与蝴蝶兰香味合成之MYB转录因子研究 [D]. 台南: 台湾成功大学, 1997: 9-15.
    [17] Ben Z M M, Negre-Zakharov F, Masci T, et al. Interlinking showy traits: Co-engineering of scent and colour biosynthesis in flowers [J]. Plant Biotechn J, 2008, 6(4): 403-415. doi: 10.1111/j. 1467-7652.2008.00329.x.
    [18] Fang G, Hammar S, Grumet R. A quick and inexpensive method for removing polysaccharides from plant genomic DNA [J]. Biotechniques, 1992, 13(1): 52-54,56.
    [19] Wang S H, Xu Q, Xu X, et al. Extraction of total RNA from the leaves of Platycladus orientalis rich in polysaccharides and polyphenol [J]. J Jilin Agri Univ, 2012, 34(1): 76-80,89. 王暑辉, 徐倩, 徐筱, 等. 富含多糖多酚的侧柏叶片总RNA提取方法 [J]. 吉林农业大学学报, 2012, 34(1): 76-80,89.
    [20] Guo C Y, Wang Y J, Liu Y L, et al. Research of total RNA extraction methods from polysaccharide-riched petals of common grape hyacinth [J]. Acta Agri Boreali-Occid Sin, 2007, 16(3): 188- 191. doi: 10.3969/j.issn.1004-1389.2007.03.046. 郭翠英, 王跃进, 刘雅丽, 等. 富含多糖葡萄风信子花瓣总RNA提取方法研究 [J]. 西北农业学报, 2007, 16(3): 188-191. doi: 10.3969/j.issn.1004-1389.2007.03.046.
    [21] Zhu J, Wang M, Jia Z W, et al. An effective method for extracting total RNA from young ears of maize [J]. Chin Bull Bot, 2007, 24(5): 624-628. doi: 10.3969/j.issn.1674-3466.2007.05.010. 朱昀, 王猛, 贾志伟, 等. 一种从富含多糖的玉米幼穗中提取RNA的方法 [J]. 植物学通报, 2007, 24(5): 624-628. doi: 10.3969/ j.issn.1674-3466.2007.05.010.
    [22] El-Deiry W S, Downey K M, So A G. Molecular mechanisms of manganese mutagenesis [J]. PNAS, 1984, 81(23): 7378-7382.
    [23] Kreader C A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein [J]. Appl Environ Microbiol, 1996, 62(3): 1102-1106.
    [24] Zhang X N, Xiao Y, Huang Y, et al. Discussion on factors affecting electro transformation efficiency of E. coli [J]. China J Mod Med, 2008, 18(4): 466-468. doi: 10.3969/j.issn.1005-8982. 2008.04.023. 张晓楠, 肖玉, 黄勇, 等. 影响大肠杆菌电转化效率因素的探讨 [J]. 中国现代医学杂志, 2008, 18(4): 466-468. doi: 10.3969/j.issn. 1005-8982.2008.04.023.
    [25] Wang F L, Zhao A Z, Han Y H, et al. Effects of T4 DNA ligase on the electroporation [J]. China J Mod Med, 2011, 21(17): 1974- 1976. 王福利, 赵爱志, 韩月恒, 等. T4 DNA连接酶对电转化效率的影响 [J]. 中国现代医学杂志, 2011, 21(17): 1974-1976.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

郑鸿昌,刘涛,吕恃衡,陈建军,何水林,潘东明,陈桂信.夜香树均一化全长cDNA文库的构建及EST分析[J].热带亚热带植物学报,2016,24(1):40~49

复制
分享
文章指标
  • 点击次数:1744
  • 下载次数: 2271
  • HTML阅读次数: 0
  • 引用次数: 0
历史
  • 收稿日期:2015-04-07
  • 最后修改日期:2015-06-27
  • 录用日期:2015-09-06
  • 在线发布日期: 2016-01-21
文章二维码
您是第16785781 位访问者
主管单位：中国科学院
主办单位：中国科学院华南植物园 广东省植物学会
地址：广州市天河区兴科路723号 中国科学院华南植物园 《热带亚热带植物学报》编辑部
电话：86 (0)20-3725 2514
热带亚热带植物学报 ® 2025 版权所有