烟草脱落酸受体基因NtPYR1和NtPYL9的克隆及表达模式分析

摘要

为揭示烟草脱落酸(ABA)受体基因NtPYR1和NtPYL9的生物学功能，以拟南芥PYR1和PYL9基因为探针，在NCBI中通过同源比对查找普通烟草表达序列标签（Expressed sequence tag, EST)序列，将EST序列进行拼接并设计特异性引物，从普通烟草红花大金元cDNA文库中克隆出NtPYR1和NtPYL9基因，其开放阅读框长分别为678 bp和564 bp，分别编码225个和187个氨基酸。NtPYR1和NtPYL9蛋白都具有一个保守的疏水性结构域，同属于PYR/PYL/RCAR ABA受体基因家族， NtPYR1和NtPYL9与番茄同源蛋白的相似性分别达到79.10%和95.24%。遗传聚类分析结果显示：NtPYR1和NtPYL9与番茄和马铃薯同源基因的遗传进化距离最近，说明PYR1和PYL9在茄科植物中较为保守。定量PCR对烟草不同组织表达模式分析表明，NtPYR1和NtPYL9在烟草不同组织中均有表达，分别在子叶和须根中的表达量最高。NtPYR1和NtPYL9表达均受ABA抑制，同时受H2O2诱导，说明两基因可能参与烟草的抗逆生理过程。%In order to reveal the biological function of abscisic acid (ABA) receptor genes, NtPYR1 and NtPYL9, in Nicotiana tabacum L. (N. tabacum), the encoding sequences of PYR1 and PYL9 in Arabidopsis thialianawere used as probes to find out homologous expressed sequence tags (ESTs) from N. tabacum genome in NCBI. Specific primers were designed following EST stitching, and NtPYR1 and NtPYL9 genes were cloned from cDNA library of cv. Honghuadajinyuan (N. tabacum). The open reading frames (ORFs) of NtPYR1 and NtPYL9 were 678 bp and 564 bp in length and encoded proteins of 225 and 187 amino acids, respectively. Both NtPYR1 and NtPYL9 contained a conserved hydrophobic domain and belonged to PYR/PYL/RCAR ABA receptor gene family. The similarity between NtPYR1/NtPYL9 and their homologous proteins in Solanum lycopersicum reached 79.10% and 95.24%, respectively. The results of phylogenetic analysis showed that the genetic distances between NtPYR1/NtPYL9 and their homologous genes in tomato and potato were the nearest, which indicated that PYR1 and PYL9 genes were relatively conserved in Solanaceae family. The quantitative PCR analysis of the expression patterns of NtPYR1 and NtPYL9 showed that the two could be expressed in various tissues, particularly in cotyledons and fibrous roots. The expression of NtPYR1 and NtPYL9 could be inhibited by ABA, while induced by H2O2, it implied that they might take part in the stress response process in tobacco plants.