基于转录组分析筛选牛心朴子响应低温胁迫的转录因子家族

Cold-responsive transcription factor family in Cynanchum komarovii based on transcriptome data

【目的】从转录水平分析牛心朴子在低温胁迫下的差异表达基因,筛选响应低温胁迫的转录因子家族,鉴定出牛心朴子低温胁迫响应的关键调控基因,为全面解析逆境胁迫响应分子调控网络及有效挖掘关键调控基因提供理论参考。【方法】通过高通量测序技术对低温胁迫(CT组)和常温处理(对照,CK组)的牛心朴子cDNA文库进行转录组测序分析,对差异表达基因进行功能注释和富集,鉴定出响应低温胁迫的转录因子家族,并选取4个差异表达的转录因子基因进行实时荧光定量PCR检测,以验证转录组测序结果的可信度。【结果】从CK组和CT组共获得30.50 Gb的原始数据,Cycle Q20平均值在96%以上,经数据过滤及去冗余后,拼接组装获得100006条Unigenes,但其功能注释率较低,有47082条至少在一个数据库中被功能注释,占Unigenes总数的47.07%;而在NR、NT、KO、SwissProt、PFAM、GO和KOG数据库中均被注释的Unigene有7070条,仅占Unigenes总数的7.06%。GO功能富集分析筛选到5545个差异表达基因,其中,上调表达基因2039个,下调表达基因3506个,分别富集到生物过程、细胞组分和分子功能三大类别中。从牛心朴子Unigene中共鉴定到83个转录因子家族的1826个转录因子,其中,以MYB转录因子家族成员数目最多,为136个(占7.45%)。从差异表达基因中筛选到与低温胁迫有关的66个转录因子家族的550个转录因子,其中MYB、C3H、bHLH、AP2-EREBP、C2H2、NAC、bZIP、CCAAT和WRKY等转录因子家族均有大量转录因子能被低温胁迫诱导表达。基于实时荧光定量PCR的牛心朴子低温胁迫下转录因子基因表达水平检测结果与转录组测序分析结果基本一致。【结论】MYB、C3H、b HLH、AP2-EREBP、C2H2、NAC、bZIP、CCAAT和WRKY等转录因子家族成员在牛心朴子响应低温胁迫时发挥主导作用,同时各家族转录因子间存在共表达性或协同作用,通过复杂的转录调控网络发挥重要调节作用,进而提高牛心朴子对低温胁迫的耐受性。

【Objective】In order to identify the cold-responsive transcription factors and screen the key regulatory genes of Cynanchum komarovii in response to cold stress,the transcriptome analysis on the differentially expressed genes(DEGs)in C.komarovii was analyzed,which provided a theoretical reference for the comprehensive analysis of the stress response molecular regulation network and the effective mining of key regulatory genes.【Method】High-throughput sequencing technology was applied to detect the transcriptome sequencing data of the cDNA library of C.komarovii under cold stress treatment(CT)and normal atmospheric temperature treatment(control,CK).The DEGs were functionally annotated and enriched,and the cold-responsive transcription factor families were identified.Fourdifferentially expressed transcription factor genes were selected using real time fluorescent quantitative PCR for the determination to check the reliability of transcriptome sequencing.【Result】30.50 Gb of sequencing data were obtained from CK and CT groups with the average Cycle Q20 over 96%.A total of 100,006 unigenes were assembled after data filtering and elimination of redundancy,but the functional annotation rate was low.47082 unigenes had functional annotation in at least one database,accounting for 47.07%of total unigenes.By comparing the unigenes with all the public protein databases of NR,NT,KO,SwissProt,PFAM,GO,and KOG,only 7,070(7.06%of the total)were functionally annotated.GO enrichment analysis was performed and 5,545 differentially expressed genes were identified in three main GO terms including biological process,cellular component,and molecular function,among which 2,039 were up-regulated and 3,506 were down-regulated under cold stress conditions.A total of 1,826 transcription factors belonging to 83 families were identified in C.komarovii unigenes.The most abundant transcription factors families detected in the transcriptome was the MYB(136),accounting for 7.45%.Moreover,550 transcription factors in 66 transcription factor families related to cold stress were selected from differentially expressed genes,MYB,C3H,bHLH,AP2-EREBP,C2H2,NAC,bZIP,CCAAT,and WRKY families had abundant transcription factors that could be induced to express under cold stress.The transcription factor gene expression detection results of C.komarovii under cold stress based on real time fluorescent quantitative PCR was consistent with that by transcriptome sequencing analysis.【Conclusion】The transcription factors families MYB,C3H,bHLH,AP2-EREBP,C2H2,NAC,bZIP,CCAAT,and WRKY in C.komarovii play a leading role in response to cold stress.There might have co-expression or synergistic effects among transcription factors of these family members,they regulate through complex transcription regulatory network,which can improve the tolerance of C.komarovii to cold stress.