油茶CoALMT9基因的克隆与表达分析

Cloning and expressional analysis of CoALMT9 gene in Camellia oleifera Abel

本研究以油茶良种‘华金’扦插苗根系为材料,通过RT-PCR克隆出油茶铝激活的苹果酸转运蛋白基因家族一个成员,命名为CoALMT9,该基因的编码序列长1 761 bp,编码586个氨基酸。利用ProtParam等软件进行生物信息学分析,同时通过实时荧光定量PCR和亚细胞定位对该基因的表达模式和表达部位进行分析,结果表明:CoALMT9蛋白的分子质量是66.84 kDa,理论等电点是6.57,为不稳定的非分泌蛋白;CoALMT9蛋白含5个跨膜区,二级结构以α螺旋为主。实时荧光定量PCR结果表明,铝毒(4 mmol·L^-1 Al3+处理)诱导了CoALMT9在油茶根系中的表达,在铝毒条件下添加1 mmol·L^-1PO43–能显著提高该基因表达水平。亚细胞定位分析确定CoALMT9所编码的蛋白质定位于液泡膜。该研究表明CoALMT9基因可能在油茶磷缓解铝毒过程中起作用,为揭示其适应高铝低磷环境的分子机制提供参考。

In this study, a member of aluminum-activated malate transporter gene family was amplified from the cutting seedling root of the Camellia oleifera superior variety ‘Huajin’ by RT-PCR, which was named CoALMT9. The coding sequence of CoALMT9 was 1 761 bp and encodes 586 amino acids. Bioinformatics analysis was performed by ProtParam and other softwares, and the expression pattern and expression site of the gene were analyzed by real-time fluorescent quantitative PCR(qPCR) and subcellular localization, respectively. The results showed that the theoretical molecular weight of CoALMT9 is 66.84 kDa and the isoelectric point is 6.57, which is unstable non-secretory protein. CoALMT9 contains five transmembrane regions, and the secondary structure is mainly α-helix. The results of qPCR showed that the expression of CoALMT9 in roots was induced by aluminum toxicity(4 mmol·L^-1 Al3+ treatment), and under the condition of aluminum toxicity, adding 1 mmol·L^-1 PO43– could significantly increase its expression level. CoALMT9 is located on the vacuole membrane by subcellular localization analysis. This study indicates that CoALMT9 may play an important role in the alleviation of aluminum toxicity by phosphorus, which provides a reference for revealing the molecular mechanism of C. oleifera adapting to high aluminum and low phosphorus.