腐霉根腐病抗性候选基因GsDYRK2研究及大豆DYRK基因生物信息学分析

Study on Pythium Root Rot Resistance Candidate Gene GsDYRK2 and Bioinformatics Analysis of Soybean DYRK Genes

为挖掘大豆腐霉根腐病抗性基因,对野生大豆腐霉根腐病抗性候选基因—双特异性酪氨酸磷酸化调节激酶基因GsDYRK2进行研究,在大豆全基因组信息中比对该基因的同源基因家族并进行初步生物信息学分析,进一步对18份野生大豆材料接种大豆腐霉根腐病病菌并进行鉴定和基因实时表达检测,分析GsDYRK2表达量与野生大豆资源腐霉根腐病抗性的关系。结果表明:GsDYRK2与GmDYRK2序列相似度最高为98.21%,由722个氨基酸残基组成,分子质量为82 573.92 Da,等电点为4.63,脂肪系数为75.18,不稳定系数为49.01,为不稳定蛋白,且为亲水性蛋白。大豆DYRK家族成员编码的蛋白质序列长度为409~1 179 aa,分子量为46 356.53~132 314.75 Da,理论等电点为4.63~8.66,不稳定系数为34.88~51.26,脂肪系数为70.90~87.01。GsDYRK2蛋白序列仅含PKc_DYRK_like一个一级结构域。二级结构类型及含量为:无规卷曲(48.06%)>α螺旋(36.7%)>延伸链(11.91%)>β转角(3.32%)。大豆DYRK家族可分为4个亚家族,分别为Yat、DYRK1、DYRK2和DYRKP。GsDYRK2和GmDYRK2均定位于细胞核,GmDYRK7定位于细胞质,其它大豆DYRK蛋白均定位于细胞核。大豆DYRK基因启动子区域主要有18种顺式作用元件,可能与激素信号调控及非生物胁迫相关。经腐霉根腐病抗性鉴定,18份野生大豆中,1级抗病材料1份,2级抗病材料12份,3级抗病材料5份;qRT-PCR检测结果显示,GsDYRK2基因的表达量与野生大豆腐霉根腐病抗性有明显的正相关性。研究结果说明GsDYRK2基因的表达量可作为抗腐霉根腐病野生大豆资源辅助筛选的手段之一。

In order to explore the resistance gene to soybean pythium root rot, we studied the resistance candidate gene GsDYRK2 of wild soybean, compared and preliminary analyzed the homologous gene family of the gene in the whole genome with bioinformatics of soybean. We further inoculated 18 wild materials with soybean pythium root rot pathogen and identified, and detected the gene expression with real time PCR, and then analyzed the relationship between GsDYRK2 relative expression level and the resistance to pythium root rot of wild soybean. The results showed that, the sequence similarity of GsDYRK2 and GmDYRK2 was 98.21%, composed of 722 amino acid residues, molecular weight was 82 573.92 Da, isoelectric point was 4.63, fat coefficient was 75.18, instability coefficient was 49.01, and it was a hydrophilic protein. The protein sequences length of DYRK family members were 409-1 179 aa, the molecular weight was 46 356.53-132 314.75 Da, the theoretical isoelectric point was 4.63-8.66, the instability coefficient was 34.88-51.26 and the fat coefficient was 70.90-87.01. The GsDYRK2 sequence contains only one PKc_DYRK_like domain. The secondary structure and content was random curl(48.06%)>α-helix(36.7%)>extension chain(11.91%)>β-angle(3.32%). Soybean DYRK family can be divided into four subfamilies including Yat, DYRK1, DYRK2 and DYRKP. Subcellular localization showed that both GsDYRK2 and GmDYRK2 were localized in the nucleus, GmDYRK7 was localized in cytoplasm and other soybean DYRK proteins were localized in nucleus. The promoter region of the soybean DYRK gene consisted of 18 cis-regulatory element, and soybean DYRK genes may be related to hormone signal regulation and abiotic stress. The resistance of 18 wild soybeans to pythium root rot was identified. Among them, a material was with resistance grade 1, twelve materials were with resistance grade 2, five materials were with resistance grade 3. And the qRT-PCR result showed that there was a positive correlation between the expression of GsDYRK2 gene and the resistance of wild soybean to pythium root rot. This study showed that the expression of GsDYRK2 gene could be used as an assistant screening method for wild soybean resistant to pythium root rot.