Zhou Lian, He Hongli, Liu Ruifang, Han Qiang, Shou Huixia, Liu Bao
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, P, R, China.
BMC Plant Biol. 2014 Jun 3;14:154. doi: 10.1186/1471-2229-14-154.
Soybean mosaic virus (SMV) is the most prevalent viral disease in many soybean production areas. Due to a large number of SMV resistant loci and alleles, SMV strains and the rapid evolution in avirulence/effector genes, traditional breeding for SMV resistance is complex. Genetic engineering is an effective alternative method for improving SMV resistance in soybean. Potassium (K+) is the most abundant inorganic solute in plant cells, and is involved in plant responses to abiotic and biotic stresses. Studies have shown that altering the level of K+ status can reduce the spread of the viral diseases. Thus K+ transporters are putative candidates to target for soybean virus resistance.
The addition of K+ fertilizer significantly reduced SMV incidence. Analysis of K+ channel gene expression indicated that GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, was significantly induced by SMV inoculation in the SMV highly-resistant genotype Rsmv1, but not in the susceptible genotype Ssmv1. Transgenic soybean plants overexpressing GmAKT2 were produced and verified by Southern blot and RT-PCR analysis. Analysis of K+ concentrations on different leaves of both the transgenic and the wildtype (Williams 82) plants revealed that overexpression of GmAKT2 significantly increased K+ concentrations in young leaves of plants. In contrast, K+ concentrations in the old leaves of the GmAKT2-Oe plants were significantly lower than those in WT plants. These results indicated that GmAKT2 acted as a K+ transporter and affected the distribution of K+ in soybean plants. Starting from 14 days after inoculation (DAI) of SMV G7, severe mosaic symptoms were observed on the WT leaves. In contrast, the GmAKT2-Oe plants showed no symptom of SMV infection. At 14 and 28 DAI, the amount of SMV RNA in WT plants increased 200- and 260- fold relative to GmAKT2-Oe plants at each time point. Thus, SMV development was significantly retarded in GmAKT2-overexpressing transgenic soybean plants.
Overexpression of GmAKT2 significantly enhanced SMV resistance in transgenic soybean. Thus, alteration of K+ transporter expression is a novel molecular approach for enhancing SMV resistance in soybean.
大豆花叶病毒(SMV)是许多大豆产区最普遍的病毒性病害。由于存在大量SMV抗性位点和等位基因、SMV株系以及无毒/效应子基因的快速进化,传统的SMV抗性育种很复杂。基因工程是提高大豆对SMV抗性的一种有效替代方法。钾(K+)是植物细胞中最丰富的无机溶质,参与植物对非生物和生物胁迫的反应。研究表明,改变K+状态水平可以减少病毒性病害的传播。因此,K+转运蛋白是大豆抗病毒的潜在靶点。
施用钾肥显著降低了SMV发病率。对K+通道基因表达的分析表明,拟南芥K+弱通道编码基因AKT2的直系同源基因GmAKT2在SMV高抗基因型Rsmv1中受SMV接种显著诱导,但在感病基因型Ssmv1中未被诱导。构建了过表达GmAKT2的转基因大豆植株,并通过Southern杂交和RT-PCR分析进行了验证。对转基因植株和野生型(Williams 82)植株不同叶片的K+浓度分析表明,GmAKT2过表达显著提高了植株幼叶中的K+浓度。相反,GmAKT2过表达植株老叶中的K+浓度显著低于野生型植株。这些结果表明,GmAKT2作为一种K+转运蛋白,影响了K+在大豆植株中的分布。从接种SMV G7后14天开始,野生型叶片上出现严重的花叶症状。相比之下,GmAKT2过表达植株未表现出SMV感染症状。在接种后14天和28天,野生型植株中SMV RNA的量在每个时间点相对于GmAKT2过表达植株增加了200倍和260倍。因此,在过表达GmAKT2的转基因大豆植株中,SMV的发展显著延迟。
GmAKT2过表达显著增强了转基因大豆对SMV的抗性。因此,改变K+转运蛋白的表达是增强大豆对SMV抗性的一种新的分子方法。