• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

波罗蜜柠檬酸合酶基因(AheCS)的表达及其与柠檬酸积累的相关性分析

Expression and Function of Citrate Synthase Gene in Jackfruit

  • 摘要:
      目的  分析波罗蜜柠檬酸合酶基因(Artocarpus heterophyllus citrate synthase,AheCS)的生物学功能,研究柠檬酸含量与AheCS基因相对表达量的相关性,为探究AheCS基因在波罗蜜(Artocarpus heterophyllus Lam. , Ahe)果实有机酸代谢中的可能作用提供参考。
      方法  以海大2号波罗蜜果实为材料,采用0.5 mg·L−1 1-甲基环丙烯(1-Methylcyclopropene,1-MCP)和1000 mg·L−1 外源乙烯利(ethrel , ETH )(40%)处理,研究室温(22±1) ℃、相对湿度90% 条件下波罗蜜果实成熟过程中柠檬酸的动态变化,克隆获得3个波罗蜜的CS基因(AheCS1 、 AheCS2 和 AheCS3),对其进行生物信息学分析,同时分析3个AheCS基因在不同采后处理下(自然成熟、外源乙烯催熟和1-MCP延缓成熟)的相对表达情况及其与柠檬酸含量的相关性。
      结果  随着波罗蜜果实的自然成熟,果实中柠檬酸含量呈先上升后下降的变化趋势;外源ETH处理加速了柠檬酸的合成速率,提前了峰值出现的时间;1-MCP处理抑制了贮藏前期柠檬酸含量下降速率,推迟了高峰出现时间。生物信息学分析发现,AheCS1/2/3基因的开放阅读框(ORF)长1422~1827 bp,编码蛋白都含有柠檬酸合酶保守结构域WPNVDAHS,属于柠檬酸合酶家族成员。AheCS1/2/3蛋白氨基酸序列分别与柑橘(Citrus reticulata Blanco.)CsCS(MH_048698.1)、川桑(Morus notabilis C. K. Schneid.)MnCs(XP010087965.1) 、 红掌(Anthurium andraeanum Linden.)AaCS(JAT55223.1)亲缘关系最近,相似性分别达到86.49%、97.00%、86.00%。qRT-PCR结果显示: AheCS1/2/3基因在果实自然成熟(CK)前期表达量低,而后期表达量高;外源ETH处理提前了AheCS 1的表达峰值时间,整体提高了AheCS 2/3的表达量;而1-MCP处理推迟了AheCS1/2/3表达峰值出现时间,增加了AheCS1/2/3成熟后期的表达量。相关性分析发现,波罗蜜果实成熟过程中柠檬酸含量与AheCS1/2/3基因表达呈一定相关性,其中与AheCS2相关性显著。
      结论  AheCS2是参与调控波罗蜜成熟过程中柠檬酸积累的潜在基因,可作为进一步研究波罗蜜AheCS基因的功能及遗传改良的候选基因。

     

    Abstract:
      Objective  The biological function of AheCS gene and the correlation between citric acid content and relative expression of AheCS gene were analyzed, and the possible role of AheCS gene in the metabolism of organic acids in jackfruit was discussed.
      Method   AheCS1, AheCS2, and AheCS3 from fruits of A. heterophyllus Haida 2 were cloned for a bioinformatic analysis. At room temperature(22±1℃) and 90% RH, changes on the gene expression and citric acid content in the fruits under natural ripening process or exposed to either 0.5 mg·L−1 of 1-MCP or 1,000 mg·L−1 of 40% ethylene (ETH) were determined.
      Result  The citric acid content in a naturally ripening jackfruit gradually rose and declined subsequently. It increased at an accelerated rate when exposed to ETH, but the rate was slowed down by the 1-MCP treatment. The ORFs of the three genes ranged from 1 422 bp to 1 827 bp containing conserved WPNVDAHS domain and belonging to the CS family. The amino acid sequences were phylogenetically closely related to those of CsCS (MH_048698.1) in citrus with a similarity of 86.49%, MnCS (XP010087965.1) in mulberry with a similarity of 97%, and AaCS (JAT55223.1) in anthurium with a similarity of 86%. The expressions of these genes were low in the early stage and raised subsequently during natural ripening (CK). However, the exogenous ETH hastened the increasing rate of AheCS1 expression and elevated the levels of AheCS2 and AheCS3 expressions, while 1-MCP delayed the rise but heightened the expression levels of the three genes at the stage near fruit maturity. The citric acid content of the ripening fruits generally positively correlated with the gene expressions. The correlation with AheCS2 reached a statistically significant level.
      Conclusion  AheCS2 is a potential gene involved in the regulation of citric acid accumulation during the ripening process of jackfruit, and it can be a candidate gene for further study of the function and genetic improvement of the AheCS gene in jackfruit .

     

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