大蒜体细胞胚发生mRNA及miRNA qPCR内参基因的筛选和验证  

Identification and Verification of Somatic Embryogenesis mRNA and miRNA qPCR Reference Genes in Garlic(Allium sativum)

作　　者：李梦倩 刘敏[1] 张蒙 白云赫 李萍 魏寒玉 周蓉[1] 蒋芳玲[1] 吴震[1] LI Meng-Qian;LIU Min;ZHANG Meng;BAI Yun-He;LI Ping;WEI Han-Yu;ZHOU Rong;JIANG Fang-Ling;WU Zhen(Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China,Ministry of Agriculture,College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学园艺学院/农业部华东地区园艺作物生物学与种质创制重点实验室,南京210095

出　　处：《农业生物技术学报》2021年第12期2449-2464,共16页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31372056;31872125);中央高校基本科研业务费-科技扶贫专项(KJFP201702);江苏高校优势学科建设工程资助项目-现代园艺科学(PAPD)。

摘　　要：大蒜(Allium sativum)是无性繁殖的重要蔬菜作物,其体细胞胚发生分子机理的研究还比较薄弱。筛选稳定的内参基因有助于解析体细胞发生过程中基因和miRNA的差异表达。为筛选大蒜体细胞胚发生mRNA和miRNA qPCR检测体系中稳定的内参基因,本研究分别选取大蒜7个mRNA候选内参基因,包括肌动蛋白(actin,AsACTIN)、甘油醛三磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,AsGAPDH)、18S核糖体RNA(18S ribosomal RNA,As18S r RNA)、多聚泛素酶(polyubiquitinase,AsUBQ)、α微管蛋白(α-tubulin,AsTUA)、转录因子LATE ELONGATED HYPOCOTYL(AsLHY)和Brassinosteroid resistant 1(AsBES1)及5个miRNA候选内参基因包括AsU6 snRNA、As5.8S rRNA、AsmiR159a-1、AsmiR168a和AsmiR168a-5p,采用qPCR技术分别检测各内参基因在不同基因型、不同外植体和不同2,4-D浓度作用下不同体细胞胚发育阶段的Ct值,利用Delta CT、BestKeeper、NormFinder和GeNorm 4种方法分析候选内参基因稳定值,并结合RefFinder综合评价候选内参基因的表达稳定性。结果表明:对于mRNA qPCR表达分析,AsACTIN是最适用于不同基因型的内参基因,AsBES1是不同外植体和不同2,4-D浓度处理下最稳定的内参基因,对所有样品进行综合评价,稳定性最好的内参基因是AsBES1。对于miRNA qPCR表达分析,不同基因型中最稳定的内参基因是AsmiR159a-1,AsmiR168a-5p是不同外植体中最稳定的内参基因,As5.8S rRNA是不同2,4-D浓度处理下最稳定的内参基因,综合评价所有样本,最理想的内参基因是AsU6snRNA。为验证所筛选内参基因的稳定性,以7个mRNA候选内参基因和5个miRNA候选内参基因分别对大蒜体细胞胚发育过程中差异表达的植物激素相关转录因子髓细胞组织增生蛋白(myelocytomatosis protein 2,AsMYC2)和AsmiR167d-1进行qPCR分析。结果显示,以不同外植体中稳定性最好的AsBES1和AsmiR168a-5p为内参时,AsMYC2和AsmiR167d-1的表达趋势与测序数据保持一致,�Garlic(Allium sativum)is an important vegetable crop for asexual reproduction,and the molecular mechanism research of somatic embryogenesis is still relatively weak.Selection of the stable reference genes will help clarify the differential expression of genes and miRNAs in somatic cell formation,help lay the foundation for the research to reveal the mechanism of garlic somatic embryogenesis.To select the most stable reference genes for m RNA and miRNA qPCR detection system during garlic somatic embryogenesis,this study chose 7 mRNA candidate reference genes,including the actin(AsACTIN),glyceraldehyde-3-phosphate dehydrogenase(AsGAPDH),18 S ribosomal RNA(As18 S r RNA),polyubiquitinase(AsUBQ),α-tubulin(AsTUA),transcription factors LATE ELONGATED HYPOCOTYL(AsLHY)and Brassinosteroid resistant 1(AsBES1).5 miRNA candidate reference genes,including AsU6 snRNA,As5.8 S rRNA,AsmiR159 a-1,AsmiR168 a and AsmiR168 a-5 p.qPCR technology was used to detect their Ct value at different stages of somatic embryo development in different genotypes,explants,and 2,4-D concentrations.Delta CT,BestKeeper,NormFinder,and GeNorm were applied to analyze the expression stability of candidate reference genes.The results were combined with RefFinder to evaluate the expression stability of candidate reference genes comprehensively.The results showed that for mRNA qPCR expression analysis,AsACTIN was the most suitable reference gene for different genotypes.AsBES1 was the most stable reference gene under different explants and different 2,4-D concentrations.The most stable reference gene in all samples was AsBES1.For miRNA,the most stable reference gene in different genotypes was AsmiR159 a-1,AsmiR168 a-5 p was the most stable reference gene in different explants,and As5.8 S rRNA was the most stable reference gene under different 2,4-D concentration treatments.The ideal reference gene in all samples was AsU6 snRNA.Using the 7 mRNA candidate reference genes and 5 miRNA candidate reference genes,plant hormone related transcription factors myelocyt

关 键 词：大蒜 体细胞胚发生 内参基因 MRNA MIRNA qPCR 

分 类 号：S-3[农业科学] Q94-336[生物学—植物学]