离体培养蛇足石杉叶状体对外源赖氨酸和天冬氨酸的差异响应

Different Response of Culturing Thallus of Huperzia serrata in vitro to Exogenous Lysine and Aspartic Acid

为了研究添加不同氨基酸溶液培养蛇足石杉(Huperzia serrata)离体叶状体对累积石杉碱甲(huperzine A,Hup A)的差异响应,利用转录组测序比较分析影响Hup A累积显著不同的天冬氨酸(D)、赖氨酸(K)处理的叶状体差异表达基因,以及实时荧光定量PCR验证差异转录本,探究Hup A生物合成的相关酶。结果表明,添加1 mmol/L D溶液处理会显著促进叶状体累积Hup A,其含量为84.05μg/g(干重),是对照(含量为65.15μg/g(干重))的1.29倍;添加4 mmol/L K溶液处理显著抑制叶状体累积Hup A,其含量为48.42μg/g(干重),是对照(CK)的0.75倍。转录组测序差异表明,GO比对统计有16258个unigene被注释功能;对处理样品间差异表达基因(differential expressed genes,DEGs)统计得知,CK vs D处理总DEGs为1046个、CK vs K处理总DEGs为782个、D处理vs K处理总DEGs最多为1586个;KEGG代谢通路富集的部分DEGs被荧光定量PCR鉴定的转录本有:Ⅰ型泛酸激酶基因(PANK1)、L-抗坏血酸过氧化物酶基因(APX)、H+转运-ATP酶基因(HA1)、NADH泛醌还原酶亚基4基因(ND4L)、细胞色素C氧化亚基2基因(COX1)、谷氨酸脱氢酶基因(GDH2)。以上结果说明D和K不同处理的叶状体累积Hup A及其DEGs发生了明显的不同响应,基因表达差异是影响离体培养叶状体累积Hup A的分子基础,其中PANK1和GDH2的产物为合成生物碱中间代谢产物的酶,故PANK1和GDH2可能是Hup A生物合成途径中间代谢的关键酶。本研究为离体培养蛇足石杉叶状体提高Hup A产量提供了技术方法及其分子基础。

To investigate the differential responses in huperzine A(Hup A)accumulation when different amino acids solutions were used for in vitro thallus culture of Huperzia serrata.Transcriptome sequencing was used for analysis of differentially expressed genes(DEGs)from thallis cultured with lysine and aspartic acid,and RT-qPCR was carried out to explore enzymes related to Hup A biosynthesis.Adding 1 mmol/L aspartic acid(D)could significantly promote the accumulation of Hup A,and its content was 84.05μg/g dry weight(DW),which was 1.29-fold than that of the control(65.15μg/g DW);the addition of 4 mmol/L lysine(K)solution significantly inhibited Hup A accumulation,and its content was 48.42μg/g DW,which was 0.75-fold than that of the control.Transcriptome sequencing-bioinformatics alignment analysis of the aforementioned materials showed that in GO alignment analysis,functions were annotated for 16258 unigenes.From the statistical analysis of the DEGs of the three groups,we found that there were 1046,782 and 1586 DEGs for CK vs D,CK vs K,and D vs K,respectively,with D vs K having the most DEGs.DEGs that were enriched in KEGG metabolic pathways and validated by fluorescence quantitative PCR included PANK1,GDH2,APX,HA1,ND4 L,and COX1.The above results indicated that Hup A content and DEGs of thallus had significant different respone under the D and K treatments.Gene expression differences are the molecular basis that affects Hup A accumulation in in vitro thallus cultures.PANK1 and GDH2 encode enzymes that synthesize an alkaloid intermediate.Therefore,PANK1 and GDH2 may be key enzymes involved in the biosynthesis of Hup A precursors.This paper provided a technical method and molecular basis for the culture of H.serrata thallus in vitro to increase the yield of Hup A.