关键词: 山杏, MATE家族, 苦杏仁苷转运, 基因克隆, 表达分析

Abstract:

The development and utilization of Prurus sibirica seeds oil and related products are greatly limited by amygdalin in the seeds of P. sibirica. Multidrug and toxic compound extrusion（MATE）proteins play an important role in the transport of amygdalin. Bioinformatics methods were used to predict and analyze the PsMATE family genes in P. sibirica seed. Meanwhile,qRT-PCR was used to analyze their expression patterns in different developmental stages of P. sibirica seeds. qRT-PCR was applied to analyze the tissue specific expression of PsMATE40,and the GFP fusion protein expression method was employed to analyze their subcellular localization. As results,17 members of PsMATE gene family from the seeds of P. sibirica were obtained through functional annotation and bioinformatics analyses based on the previous transcriptomics data of P. sibirica seeds in our lab,they might be divided into three subfamilies（subfamily 1/2/4）. The secondary structure of the encoded proteins mainly contained α-helix and multiple phosphorylation sites. Except for PsMATE23,PsMATE33,PsMATE35A and PsMATE35B,the other 13 proteins were all alkaline. Fifteen PsMATE proteins were highly hydrophobic transmembrane transporters and located in plasma membrane. qRT-PCR analysis showed that all members of the PsMATE family were expressed in the developing seeds of P. sibirica,but the expressions varied significantly. The expression of PsMATE40 gene expressed the highest among PsMATE family genes. Further analysis using phylogenetic trees revealed that PsMATE40 was clustered with MATE protein that was identified as amygdalin transporter. Therefore,it is suggested that PsMATE40 gene plays an important role in amygdalin transport in the seeds of P. sibirica. The PsMATE40 gene was successfully cloned. The qRT-PCR analysis showed that PsMATE40 presented the highest transcription level in the seeds of P. sibirica,whereas the lowest in the roots,emphasizing that PsMATE40 was specifically responded to different tissues of P. sibirica. The plant expression vector pBI121-GFP-PsMATE40 was constructed and transiently transformed into leaves of Nicotiana benthamiana,and the result by fluorescence microscopy indicated that the PsMATE40 transport protein was localized in the plasma membrane. In conclusion,these results lay the important foundation for understanding the transmembrane transport mechanism of amygdalin in the seeds of P. sibirica and molecular orientation cultivation of oil plants with low amygdalin content.

Key words: Prunus sibirica, MATE family, amygdalin transport, gene cloning, expression analysis