Fan Sujie, Jiang Liangyu, Wu Junjiang, Dong Lidong, Cheng Qun, Xu Pengfei, Zhang Shuzhen
Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, 150030, Heilongjiang, People's Republic of China.
Soybean Research Institute, Heilongjiang Academy of Agricultural Sciences, Collaborative Innovation Center of Grain Production Capacity Improvement in Heilongjiang Province, Harbin, 150086, Heilongjiang, People's Republic of China.
PLoS One. 2015 Oct 16;10(10):e0140364. doi: 10.1371/journal.pone.0140364. eCollection 2015.
Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean 'Suinong 10' infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homology of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved 'P-loop' (phosphate-binding loop) motif at residues 47-55 aa and a Bet v 1 domain at residues 87-120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible 'Dongnong 50' soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.
由大豆疫霉（Phytophthora sojae）引起的大豆疫霉根腐病是全球许多大豆种植区的一种毁灭性病害。在之前的一项研究中，在感染大豆疫霉的高抗大豆品种‘绥农10’中，发现了杏（Prunus armeniaca）主要过敏原Pru ar 1的一个表达序列标签（EST）同源物上调表达。在此，利用cDNA末端快速扩增（RACE）技术分离出该EST的全长。与已报道的8个大豆过敏原家族比较后发现，其与Gly m 4的同源性最高，为53.46%，并将其命名为Gly m 4样蛋白（Gly m 4l，GenBank登录号：HQ913577.1）。Gly m 4l的cDNA全长为707 bp，包含一个474 bp的开放阅读框，编码一个由157个氨基酸组成的多肽。序列分析表明，Gly m 4l在第47 - 55位氨基酸残基处含有一个保守的“P环”（磷酸结合环）基序，在第87 - 120位氨基酸残基处含有一个Bet v 1结构域。Gly m 4l的转录丰度受到大豆疫霉、水杨酸（SA）、NaCl的显著诱导，并且对茉莉酸甲酯（MeJA）和乙烯（ET）也有响应。重组Gly m 4l蛋白具有核糖核酸酶活性，并表现出直接的抗菌活性，能够抑制大豆疫霉的菌丝生长并减少游动孢子的释放。进一步分析表明，在玉米素存在的情况下，重组蛋白降解tRNA的核糖核酸酶活性受到显著影响。在感病的‘东农50’大豆中过表达Gly m 4l，对大豆疫霉的抗性增强。这些结果表明，Gly m 4l蛋白在大豆抵御大豆疫霉感染的防御过程中发挥了重要作用。
