沙棘种子发育期茉莉酸水平变化及其信号基因表达分析

Dynamic Changes of Jasmine Acid Level and Its Signal Genes Expression at Developmental Stage of Sea Buckthorn Seeds

为探讨沙棘种子发育期茉莉酸水平变化及其信号基因的表达模式,以品系‘56’不同发育期(7月10日, 7月26日, 8月11日, 8月27日)种子为试材,液相色谱串联质谱法(LC-MS/MS)检测五种内源激素(茉莉酸JA,生长素IAA,脱落酸ABA,赤霉素GA3,细胞分裂素tZ)含量,qRT-PCR分析JA合成代谢通路基因(DAD1,LOX, AOS, AOC, OPR3,β-氧化酶基因)及相关转录因子(TCP2, MYC2)和hrhmiR319e的表达模式。结果表明:(1)种子发育初期,内源激素IAA、tZ、GA3与JA含量最高,随后呈先下降后略有上升趋势;相反,ABA含量呈持续上升趋势;种子成熟期,高含量ABA在一定程度上抑制了其他4种激素合成,进而减缓种子生长发育速度,并促进种子成熟。(2)沙棘LOX、AOS和AOC基因表达量在不同种子发育期存在明显差异,最大值分别是最小值的3.4倍、4.8倍和18.8倍,是JA合成的关键基因。(3)沙棘hrhmiR319e负调控其靶转录因子TCP2,TCP2负调控下游JA代谢通路关键基因LOX的表达;MYC2正调控JA代谢通路关键基因AOS,负调控LOX和AOC基因表达。首次揭示了"hrhmiR319-TCP2-LOX"和"MYC2-AOS"模块协同调控沙棘不同发育期种子中的JA合成。这可为后续深入研究茉莉酸信号对沙棘种子发育及其生物活性成分合成的分子调节机制提供理论基础。

To explore the dynamic changes of jasmonic acid level change and its signal genes expression at developmental stage of sea buckthorn seeds, the seeds of line ’56’ at different developmental stages(July 10, July 26,August 11 th and August 27) were selected as the test materials. The contents of five endogenous hormones(jasmonic acid JA, auxin IAA, abscisic acid ABA, gibberellin GA3 and mitogen tZ) were detected by the method of liquid chromatography tandem mass spectrometry( LC/MS/MS), and the expression pattern of JA synthetic pathway genes(DAD1, LOX, AOS, AOC, OPR3 and β-oxidase genes), related transcription factors(TCP2, MYC2)and hrhmiR319 e were analyzed using real-time fluorescent quantitative PCR technology. The results showed that in the early seed development, the contents of IAA, tZ, GA3 and JA were the highest, then they declined first and then increased slightly. On the contrary, the ABA contents continued to increase in all seed development. In the mature seed development, the high content of ABA inhibited the synthesis of the other 4 hormones to a certain extent, thus slowing down the growth and development of seeds and promoting seed maturation. The expression content of LOX, AOS and AOC genes had significant difference at different developmental stages of seeds, and their maximums were about 3.4 times, 4.8 times and 18.8 times of their minimums, respectively. This indicated that LOX, AOS and AOC genes were the key genes for JA biosynthesis. The hrhmiR319 e negatively regulated its targeted transcription factor TCP2 which negatively regulated the expression of LOX, a key gene in the downstream JA metabolic pathway. MYC2 negatively regulated LOX and AOC genes but positively regulated AOS gene, a key gene in JA metabolic pathway. This study revealed that the JA biosynthesis in the sea buckthorn seeds at developmental stages was coordinately regulated by the models of "hrhmiR319e-TCP2-LOX" and "MYC2-AOS" for the first time. This data could provide scientific basis for further study on the molecular mechanism of JA regulating seed development and biosynthesis of bioactive components in sea buckthorn seeds.