杜仲愈伤组织转录组分析及其类黄酮合成相关基因的qRT-PCR分析

Analysis of transcriptome sequencing and qRT-PCR analysis of related genes of flavonoid biosynthesis from Eucommia ulmoides calli

目的分析光、暗培养的杜仲愈伤组织转录组测序结果,验证杜仲愈伤组织转录组测序结果中与类黄酮合成相关基因的表达水平。方法利用Illumina Hi SeqTM 2500测序平台对光暗培养的2种杜仲愈伤组织进行转录组高通量测序,利用Trinity软件对测序结果进行De novo组装,使用BLAST软件将Unigenes序列与NR、Swiss-Prot、GO、COG、KOG、KEGG、Pfam等数据库比对,利用FPKM值估算基因表达丰度,利用qRT-PCR验证与类黄酮合成相关基因的表达水平。结果杜仲愈伤组织转录组测序共获得13.00 Gbp的clean data。De novo组装后共获得62 030条Unigenes,平均长度为736.40 bp。使用BLAST软件将Unigenes序列与相关数据库比对,共获得25 417条Unigenes的注释结果。其中,25 167条Unigenes注释到Nr。4 794条Unigenes和它们相关的酶(enzyme commission numbers)注释到82条KEGG通路。共获得差异表达基因1 986条。在白光(光强为12 000 lx,16 h光照,8 h黑暗)培养的愈伤组织中,有1 139条基因表达上调,847条基因表达下调。在KEGG富集分析中发现,有7个Unigenes与类黄酮合成相关,其中有6个Unigenes在白光培养的杜仲愈伤组织中上调表达,它们编码的酶分别为查耳酮异构酶(EC 5.5.1.6,chalcone isomerase,CHI),查耳酮合成酶(EC 2.3.1.74,chalcome synthase,CHS),类黄酮3′-羟化酶(EC 1.14.13.21,flavonoid3′-monooxygenase,F3’H),反肉桂酸4-单加氧酶(EC 1.14.13.11,trans-cinnamate 4-monooxygenase,CYP),黄酮醇合成酶(EC 1.14.11.23,flavonol synthase,FLS),莽草酸邻羟基肉桂转移酶(EC 2.3.1.133,shikimate-O-hydroxycinnamoyl transferase,HQT)。1个Unigene下调表达,它编码的酶为无色花青素加氧酶(EC 1.14.11.19,leucocyanidin oxygenase,LDOX)。qRT-PCR分析表明,与类黄酮合成相关的7个Unigenes的表达情况与转录组测序分析结果是一致的。结论白光条件(光强为12 000 lx,16 h光照,8 h黑暗)可以促进类黄酮代谢途径中相关化合物的积累。

Objective To analyse the transcriptome sequencing results of Eucommia ulmoides calli under light and dark culture condition, and verify the expression level of the related genes of flavonoid biosynthesis from transcriptome sequencing results. Methods The transcriptome high-throughput sequencing of E. ulmoides calli was performed by using the Illumina Hi SeqTM 2500 sequencing platform, and de novo assembly of the transcriptome sequencing results was finished by Trinity software. The sequencing results of Unigenes were compared with the databases of NR, Swiss-Prot, GO, COG, KOG, KEGG and Pfam by BLAST software. The gene expression abundance was estimated by FPKM value. qRT-PCR was used to verify the expression level of the related genes of flavonoid biosynthesis. Results About 13.00 Giga base pairs（Gbp） clean data（more than 6.02 Gbp, respectively） were obtained, and de novo assembly generated 62 030 Unigenes with an average length of 736.40 bp. A total of 25 167 Unigenes were annotated to Nr. Totally 4 794 Unigenes and their associated enzymes（enzyme commission numbers） were annotated to 82 KEGG pathway. There were 1 986 genes identified as significantly and differentially expressed genes between the two calli under light and dark culture. Among them, 1 139（57.35%） were up-regulated and 847（42.65%） were down-regulated in the calli under light culture. Metabolic pathway analysis revealed that seven Unigenes were predicted to be responsible for the flavonoid biosynthesis, six Unigenes of which were up-regulated in the calli under light culture, encoding chalcone isomerase（EC 5.5.1.6）, chalcome synthase（EC 2.3.1.74）, flavonoid 3′-monooxygenase（EC1.14.13.21）, trans-cinnamate 4-monooxygenase（EC 1.14.13.11）, Flavonol synthase（EC 1.14.11.23）, shikimate-O-hydroxycinnamoyl transferase（EC 2.3.1.133）. One Unigene was down-regulated in the calli under light culture, which encoded leucocyanidin oxygenase（EC 1.14.11.19）. qRT-PCR analysis showed that the expression of the seven genes related with flavonoid biosynthesis was coincident with the transcriptome high-throughput sequencing results. Conclusion The white light（12 000 lx, 16 h light and 8 h dark） could improve the production capacity of some metabolic intermediate of flavonoid biosynthesis pathway of E. ulmoides calli.