大豆肌醇加氧酶基因响应线虫胁迫的表达分析

Expression analysis of GmMIOX gene response to SCN stress

肌醇加氧酶通常与植物细胞壁的合成以及诱导合胞体发育相关。为确定大豆胞囊线虫侵染大豆根部后,肌醇加氧酶基因表达水平的变化,以感病品种辽豆15和抗病品种灰皮支黑豆、哈尔滨小黑豆和小粒黑豆为试验材料,在人工接种大豆胞囊线虫3号生理小种后,利用实时荧光定量PCR检测肌醇加氧酶家族基因表达量。结果表明,抗病品种中GmMIOX2基因均在接种后5d表达量达到最大,而感病品种变化并不明显。GmMIOX4基因在抗病品种灰皮支黑豆、哈尔滨小黑豆和小粒黑豆中表达量分别在接种后5d,20d和10d显著高于感病品种的表达量。表明GmMIOX2和GmMIOX4基因与大豆胞囊线虫发育调控相关。通过扩增Gm MIOX基因的CDS序列,构建GFP融合表达载体p CAMBIA1303-MIOX2和p CAMBIA1303-MIOX4,农杆菌浸润法注射本氏烟叶片进行亚细胞定位观察,GmMIOX2蛋白主要分布在细胞膜,GmMIOX4蛋白主要分布在细胞质和细胞膜。生物信息学分析表明,GmMIOX2和GmMIOX4蛋白结构域主要由α螺旋构成,蛋白序列进化分析发现GmMIOX2和GmMIOX4分别与拟南芥At MIOX1、At MIOX4亲缘性较近。

Inositol oxygenase is usually associated with plant cell wall synthesis and induced syncytium development. A susceptible cultivar Liaodou 15 and the resistant cultivars were Huipizhi Xiaoheidou,Harbin Xiaoheidou and Xiaoli Heidou were used to study the gene expression of inositol oxygenase family gene from soybeans roots after SCN3 infection. The results showed that in both resistant cultivars the expression level of GmMIOX2 gene reached the maximum on the fifth day,but the changes of susceptible cultivar was not obvious. The expression levels of GmMIOX4 gene in Huipizhi Heidou,Harbin Xiaoheidou and Xiaoli Heidou were significantly higher than that of susceptible cultivar Liaodou 15 on fifth day,twentieth day and tenth day,indicating that GmMIOX2 and GmMIOX4 genes were related to nematode development. The complete GmMIOX2 and GmMIOX4 genes were amplified into binary expression vector to construct p CAMBIA1303-MIOX2 and p CAMBIA1303-MIOX4. The result of subcellular localization showed that GmMIOX2 protein was located on the cytomembrane,and GmMIOX4 protein was located on cytoplasm and cytomembrane. Bioinformatic analysis of GmMIOX2 and GmMIOX4 protein domains was mainly composed of alpha helices. Phylogenetic analysis of protein sequences showed that GmMIOX2 and GmMIOX4 protein were closely related to Arabidopsis thaliana At MIOX1 and At MIOX4 respectively.