摘要
【目的】鉴定5-氨基乙酰丙酸(ALA)缓解酸枣NaCl胁迫相关miRNAs,为阐明miRNA及核心靶基因参与ALA缓解NaCl胁迫的分子机制提供理论依据。【方法】以酸枣水培幼苗为试验材料,设置叶面喷施蒸馏水为对照(CK)、叶面喷施ALA处理(ALA)、营养液中添加NaCl溶液和叶面喷施蒸馏水(NaCl)及营养液中添加NaCl溶液和叶面喷施ALA(ALA+NaCl)4个处理,进行高通量测序,鉴定了响应ALA缓解酸枣NaCl胁迫过程的miRNAs及靶基因。【结果】从4个处理共12个文库中分析鉴定到149个已知的miRNA(属于31个miRNA家族)和11个novel miRNA。许多响应盐胁迫的miRNAs,如zju-miR172d、zju-miR395i和zju-miR172a均显著下调,表明这些miRNAs可能受单一盐胁迫抑制。进一步分析发现,ALA显著抑制了zju-miR408a的表达。对zju-miR408a的靶基因进行注释,结果为过氧化物酶、原卟啉原氧化酶、线粒体、木质素形成阴离子过氧化物酶、钙依赖性蛋白激酶1、漆酶等。qRT-PCR分析结果与测序结果基本一致。【结论】ALA通过抑制zju-miR408a的表达上调靶基因POD的表达量达到清除活性氧的目的,从而提高酸枣的耐盐性。该结果为更全面地解析ALA缓解酸枣盐胁迫的miRNA提供了新的信息。
【Objective】The objective of this study was to identify miRNAs related to the alleviation of NaCl stress in wild jujube by ALA(5-aminolevulinic acid),thereby providing a theoretical basis for elu-cidating the molecular mechanism by which miRNAs and core target genes participate in the alleviation of NaCl stress by ALA.【Methods】The leaves of plantlets propagated by tissue culture of wild jujube(Ziziphus jujuba var.spinosa)were used as the research materials.Four treatments were set up:spray-ing distilled water on the leaf surface as the control(CK),spraying ALA on the leaf surface(ALA),add-ing NaCl solution and spraying distilled water on the leaf surface(NaCl),and adding NaCl solution and spraying ALA on the leaf surface(ALA+NaCl).The differences in physiological indicators such as plant height,root length,root surface area,and root volume were measured and analyzed.High-through-put sequencing was performed of the leaves of the four treatments to identify key miRNAs responding to ALA alleviating NaCl stress in wild jujube seedlings,and to determine the functions of their target genes.【Results】The study showed that NaCl treatment led to water lost and wilt of the seedling leaves,significantly reduced the plant height,root length,root surface area,and root volume of wild jujube seedlings.The damage caused by NaCl stress to jujube seedlings was alleviated after exogenous application of ALA.Twelve sRNA libraries were constructed from the leaves of the four treatments,and the Q30 value of each sample was≥93%,with the reads aligned to genes accounting for 86.46%-93.12%of the total reads,indicating good data quality.A total of 149 known miRNAs belonging to 31 miRNA families and 11 novel miRNAs were identified from the 12 libraries.The length of sRNAs was mainly between 21-24 nt,with 24 nt sRNAs being the most abundant in all libraries.Compared with the CK,there were 152 differentially expressed miRNAs in the NaCl treatment group,including 76 down-regu-lated genes and 76 up-regulated genes;in the ALA+NaCl treatment group,there were 149 differentially expressed miRNAs,including 78 down-regulated genes and 71 up-regulated genes.The functional en-richment analysis of differentially expressed miRNAs based on the Kyoto Encyclopedia of Genes and Genomes database showed that the target genes of differentially expressed miRNAs were involved in metabolic pathways such as TMV resistance proteins,membrane proteins containing SPX domains,so-diumpotassium transporters,ribosomal RNA processing proteins,transcription factors such as auxin re-sponse factors(ARF)family,MYB transcription factors,WRKY transcription factors.Many miRNAs responding to salt stress,such as zju-miR172d,zju-miR395i,and zju-miR172a,were significantly down-regulated,indicating that these miRNAs might be inhibited by single salt stress.Further analysis revealed that ALA significantly inhibited the expression of zju-miR408a.The target gene annotation of zju-miR408a showed that it targeted enzymes such as the peroxidase,protoporphyrinogen oxidase,mito-chondrial anion peroxidase,lignin-forming anion peroxidase,calcium-dependent protein kinase 1,and laccase.The qRT-PCR analysis results were basically consistent with sequencing results.The expres-sion level of the target gene POD and POD enzyme activity increased more significantly after exoge-nous application of ALA than after NaCl stress.【Conclusion】The results of this study showed that ex-ogenous application of ALA alone had no significant effect on wild jujube seedlings.The growth of ju-jube seedlings was inhibited by NaCl stress,and exogenous application of ALA could alleviate NaCl stress in wild jujube seedlings.The construction of sRNA libraries from wild jujube seedlings revealed that ALA significantly induced the expression of zju-miR408a.The target gene annotation analysis of zju-miR408a mainly targeted the enzymes such as oxidases and protoporphyrinogen oxidase.The re-sults were further verified by measuring the expression level of target gene POD and POD enzyme ac-tivity.In summary,ALA would alleviate NaCl stress in wild jujube seedlings by inhibiting the expres-sion of zju-miR408a to upregulate the expression level of target gene POD,thereby scavenging reactive oxygen species and improving salt tolerance.This result would provide new information for a more comprehensive analysis of miRNAs involved in alleviating salt stress in wild jujube by ALA.
作者
朱春梅
常心怡
刘志宇
赵宝龙
孙军利
ZHU Chunmei;CHANG Xinyi;LIU Zhiyu;ZHAO Baolong;SUN Junli(Agriculture College of Shihezi University/Key Laboratory of Special Fruits&Vegetables Cultivation Physiology and Germplasm Re-sources Utilization,Xinjiang Production and Construction Crop,Shihezi 832003,Xinjiang,China)
出处
《果树学报》
北大核心
2025年第12期2828-2840,共13页
Journal of Fruit Science
基金
国家自然科学基金项目(31460495、32560698)
兵团“三农”骨干人才培养项目。
