低氮胁迫灯盏花全植株的转录组文库构建及其测序  

Transcriptome library construction in whole plant of Erigeron breviscapus stressed by low nitrogen and its sequencing

作　　者：田宝强[1,2] 李玥[1,3] 赖思晨[1] 木佳[1,3] 应宇翔 严胜柒[1,3] 张云峰[1,2,3] 

机构地区：[1]云南师范大学生命科学学院,云南昆明650500 [2]生物能源持续开发利用教育部工程研究中心,云南昆明650500 [3]云南师范大学-台湾新竹清华遗传-环境互作联合实验室,云南昆明650500

出　　处：《中草药》2015年第21期3235-3241,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(30660075;31360263);云南科技厅社会发展科技计划基础研究面上项目(2009CD052);云南省应用基础研究计划(2011FA016)

摘　　要：目的为探寻药用植物灯盏花Erigeron breviscapus的遗传背景,利用低氮胁迫的灯盏花全植株构建了转录组文库,并利用新一代测序技术进行测序。方法采用改良异硫氰酸胍-CTAB法,提取低氮胁迫灯盏花植株及其对照植株的总RNA,经富集m RNA、打断、构建测序用c DNA文库。结果通过测序,低氮和正常样本分别获得3 587万条和2 582万条测序读长(raw reads),总数据量超过6 G,碱基错误率低于1%(Q20)的数据分别为98.37%和98.67%。经de novo组装,总共得到101、156条Unigene,平均读长768 bp,N50为1 290 bp,其中44.39%长度超过500 bp。101、156条Unigene中,58.86%在公共数据库中比对到相似序列,89.08%Unigene在Nr数据库比对到相似序列。得到灯盏花黄酮类合成途径中包括苯丙氨酸解氨酶(PAL)、肉桂酰-4-羟化酶(C4H)、查耳酮合成酶(CHS)、查耳酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)、类黄酮3′-羟化酶(F3′H)、花色素还原酶(ANS)等序列。结论灯盏花的转录组信息得到较好的保存,为下一步灯盏花遗传环境互作研究及分子辅助育种奠定基础。Objective To explore the genetic background of Erigeron breviscapus, a very important herb, we used the plantlets under low nitrogen and normal condition cultured in vitro as material to construct the transcriptome library, and to sequence the library via next generation sequencing technique. Methods Modified guanidinium isothiocyanate-CTAB method was used to isolate the total RNA from low nitrogen and normal condition cultured plantlets. The m RNA was enriched from the total RNA and broken into short fragments, and then the c DNA library was established for RNA-Seq. Results In total, 35.87 million and 25.82 million raw reads were generated from LD and CK libraries via next generation sequencing, respectively. The overall sequencing outputs were over 6 Gb. Among all of the raw reads, more than 98.37% and 98.67% had Phred-like quality scores at Q20 level（an error probability of 1%）, respectively. After filtered to remove low quality reads, the high quality sequencing sequence was used for de novo assembling. Unigenes of 101 and 156 pieces with the average length of 768 bp（N50 1 290 bp） were obtained, and the length of 44 908 pieces（about 44.39%） is more than 500 bp. Among 101 and 156 Unigenes, 59 538（58.86%） showed the significant BLAST hits in the public databases. Many sequences concerning flavanoids bio-synthesis which included PAL, C4 H, CHS, CHI, F3 H, F3’H, and ANS were obtained from the experiment. Conclusion Transcriptome information of E. breviscapus has been better preserved, which provides the foundation for the further analysis in genetic-environment interaction and molecular assistant breeding.

关 键 词：灯盏花 低氮胁迫 转录组文库 测序 de novo组装 UNIGENE 

分 类 号：R282.12[医药卫生—中药学]