日本落叶松种子萌发过程中18个miRNAs的表达变化

运用qRT-PCR技术,检测日本落叶松种子萌发过程中的5个阶段——干种子 (萌发培养0 d)、吸胀(1 d)、萌动(5 d)、胚根外露(9 d)和子叶展开(28 d),miR156、miR159和miR160等18个miRNAs在其种胚内的表达模式,并对其目标基因进行生物信息学分析,探讨miRNA在针叶林木种子萌发过程中的基因表达模式。结果表明:(1)从整个萌发过程分析,18个miRNAs的表达水平皆以干种子阶段最高,吸胀和萌动阶段迅速下降;干种子阶段的表达水平分别是吸胀、萌动、胚根外露和子叶展开阶段的4.8 43.5、17.2 1 000.0、45.5 1 000.0、62.5 1 862.2倍。(2)从各个阶段分析,miR894的表达水平在5个阶段均最高,miR408均最低;其余16个miRNAs在5个阶段内的表达水平各有变化,综合排序结果从高到低依次为:miR951、miR950、miR156、miR159、miR396、miR398、miR390、miR160、miR947、miR165、miR319、miR162、miR171、miR397、miR395和miR172。(3)miRNA目标基因预测结果显示:目标基因主要是ATP/DNA结合蛋白、ARF等转录因子、质膜和核糖体等细胞组分及MAP等激酶活性蛋白等蛋白编码基因。(4)miR160对其目标基因JR160237 具有切割作用,切割位点发生在miR160与目标基因互补结合位点第10与11位碱基之间。JR160237在种子吸胀阶段表达水平迅速升高,在萌动、胚根外露和成苗阶段有所回落。

18 miRNAs in Larix kaempferi During Seed Germination

The seed of Larix kaempferi (Siebold. & Zucc.) Gordon was used as material to investigate the potential molecular regulation mechanisms of miRNA during seed germination. The germination process of L.kaempferi seed includes the following: The imbibition of dry seed was completed after 24 hours of socking; the testa rupture occurred after 5 days and followed by radicle emergence (videlicet, endosperm rupture) after 9 days of germination culture; the cotyledons expanded fully after 28 days. The expression level of 18 miRNAs was relatively quantified using qRT-PCR technology among five key seed germinating stages, which including dry seed (0 day after germinating), imbibed seed (1st day after germinating), testa rupture (5th day), radicle emergence (9th day) and cotyledon extension (28th day). The results are as follows. (1)The expression levels of 18 miRNAs were the highest at dry seed stage and then went down rapidly at imbibed seed and testa rupture stage. The miRNAs were highly expressed in dry seed stage than in subsequent stages. The expression level in dry seed stage was 4.8 to 43.5 times, 17.2 to 1 000 times, 45.5 to 1 000 times and 62.5 to 1 862.2 times that of those in imbibed seed, testa rupture, radicle emergence and extension cotyledon stage, respectively. (2)The expression level of miR894 was the highest and that of miR408 was the lowest in each stage; and the others fluctuated, ranking from high to low as miR951, miR950, miR156, miR159, miR396, miR398, miR390, miR160, miR947, miR165, miR319, miR162, miR171, miR397, miR395 and miR172. (3)The target genes and their possible functions predicted using bioinformatics methods were responsible for the expression of a great number of binding proteins, cellular components and MAP kinase proteins associated with seed germination. (4) JR160237 was the target gene of miR160, and the cleaved fragments mapped exactly to the predicted cleavage site (between nucleotides 10 and 11 from the 5'end of miR160). JR160237 mRNA reached the peak at imbibed seed stage, then decreased and dynamically expressed at following stages with small fluctuation.