龙眼胚性愈伤组织Remorin家族成员的克隆及其在体胚发生过程中的表达分析

Cloning of the Genes of Remorin Family from Embryogenic Callus and Their Expression Analysis During Somatic Embryogenesis in Dimocarpus longan

以龙眼松散形胚性愈伤组织为材料,采用RT-PCR结合RACE法克隆Remorin家族5个成员的c DNA全长,Dl Remorin1全长2 127 bp,编码572个氨基酸;Dl Remorin2全长1 829 bp,编码444个氨基酸;Dl Remorin3全长1 937 bp,编码541个氨基酸;Dl Remorin4全长1 743 bp,编码466个氨基酸;Dl Remorin5全长1 043 bp,编码181个氨基酸。Dl Remorin1~5 DNA序列长度分别为:4 054、3 097、3 505、4 690和1 935 bp,所有内含子剪切位点均符合真核生物"GT-AG"规则。所有成员均是不稳定亲水性蛋白,都不具有信号肽,只有Dl Remorin1具有跨膜结构,都具有Remorin家族保守结构域,与其它植物的Remorin具有较高的同源性。系统进化树分析结果表明,Dl Remorin5为经典的Remorin蛋白,Dl Remorin1与Dl Remorin2属于长链Remorin。qRT-PCR结果显示,随着龙眼体胚的发育,Dl Remorin1的表达量呈类"N"状趋势,在心形胚时期和子叶形胚时期较高;Dl Remorin2在心形胚和子叶形胚时期迅速下降;Dl Remorin3在发育中后期迅速升高,并在子叶形胚时期最高;Dl Remorin4在球形胚和鱼雷形胚时期最低,而Dl Remorin5在不完全胚性紧实结构时期最低。说明Dl Remorin1~5在龙眼体胚发生过程的转录水平具有一定的组织特异性和时序性。ps RNA Target预测显示Dl Remorin还可能受到mi RNA家族的调控。

The RT-PCR combined with RACE method was used to clone the complete c DNA sequences of 5 members of Remorin family from embryogenic callus in Dimocarpus longan. The complete c DNA sequences of Dl Remorin1–5 were 2 127,1 829,1 937,1 743 and 1 043 bp,Dl Remorin1–5 encoding 572,444,541,466 and 181 amino acids,respectively. The DNA sequences of Dl Remorin1–5 were 4 054,3 097,3 505,4 690 and 1 935 bp,and all the splice sites of the introns contained were obeyed to the'GT-AG'rule. Dl Remorin1–5 belong to the instability and hydrophilous proteins,they all had the Remorin conserved domain,and had no signal peptide,only Dl Remorin1 had transmembrane structure. The sequences of both nucleotides and amino acids of the five members were high homologous with those of the known Remorin genes in other species. Anglicizing phylogenetic tree of Remorin in plants indicated that Dl Remorin5 belonged to the classical Remorin,Dl Remorin1 and Dl Remorin2 belonged to the long Remorin. The results of qRT-PCR indicated that Dl Remorin1 showed approximately an'N'curve,it expressed at the highest levels in the heart embryos and cotyledonary embryos cultures. Dl Remorin2 expressed at the lowest levels in heart embryos and cotyledonary embryos cultures. The expression of Dl Remorin3 increased rapidly at the middle and late developmental stages and peaked at the cotyledonary embryos cultures. Dl Remorin4 expressed at the lowest levels in globular embryos and topedo embryos cultures. Dl Remorin5 expressed at the lowest levels in incomplete compact pro-embryogenic cultures. Suggesting that Dl Remorin1–5 expressed with tissue specific and sequential characteristics during longan somatic embryogenesis. ps RNA Target projections show Dl Remorin may be regulated by mi RNAs.