过表达酵母PAC1增强转基因大豆对大豆花叶病毒的抗性

doi:10.3864/j.issn.0578-1752.2018.02.002

摘要/Abstract

摘要： 【目的】大豆花叶病毒（soybean mosaic virus，SMV)病是中国大豆产区最主要的病害之一，严重影响大豆产量和籽粒品质。核糖核酸酶PAC1能够识别和降解植物RNA病毒或类病毒复制过程中产生的dsRNAs，从而有效抑制病毒在寄主中的复制与积累。PAC1的这一特点为广谱抗RNA病毒及类病毒转基因作物的创制和培育提供了有效的靶标基因。本研究利用转基因技术，将来源于粟酒裂殖酵母菌(Schizosaccharomyces pombe)的PAC1导入栽培大豆，研究过表达PAC1对大豆SMV抗性的影响，为抗SMV转基因大豆新品种选育提供依据。【方法】采用酶切连接技术，将PAC1连接到双元表达载体pCAMBIA3300中，构建植物表达载体pCAMBIA3300-PAC1。目的基因启动子为组成型强启动子CaMV 35S，终止子为NOS，筛选标记为草铵膦抗性基因BAR。采用农杆菌介导转化法，将PAC1导入栽培大豆品种Williams82。在利用PAT/BAR试纸、PCR及除草剂（500 mg·L^-1Basta）喷施检测基础上，通过Southern杂交技术进一步分析外源基因在转基因大豆中的整合情况和拷贝数。采用人工摩擦接种法，对T₂和T₃代转基因大豆株系进行田间抗SMV鉴定农艺性状调查，分析转基因大豆对SMV抗性及遗传稳定性。并利用qRT-PCR技术分析接种SMV 28 d后转基因大豆中SMV积累水平。【结果】共转化2 600多个外植体，获得耐草铵膦（5 mg·L^-1）大豆再生植株76株。PCR检测结果表明，其中65株能够扩增出目的条带，大豆遗传转化效率为2.48%。对T₁—T₃代转基因大豆株系喷施除草剂表明，在500 mg·L^-1 Basta处理7 d后，转基因植株表型没有明显变化，而对照（非转基因大豆）植株叶片则黄化枯死。Southern杂交结果表明，外源基因以低拷贝的方式(1—2个)整合至大豆基因组中。摩擦接种SMV SC-3鉴定表明，在接种35 d后，对照出现严重花叶、皱缩等典型SMV发病症状，而转基因大豆仅部分叶片表现出轻微的花叶症状，其病情指数降低至11.11—22.22，较对照（病情指数36.81—46.24）显著降低，且SMV抗性在转基因大豆不同代际间能够稳定遗传。qRT-PCR分析表明，在接种SMV SC-3株系28 d后，转基因大豆中SMV CP表达水平较对照极显著下降。农艺性状调查表明，在未接种SMV条件下，转基因大豆在叶形、花色、种皮色、种脐色、株高、节数、结荚高度、生育期及百粒重等方面与对照没有显著差异。【结论】PAC1过表达显著抑制了SMV的积累及症状发展，增强了转基因大豆对SMV的抗性水平。

关键词: 大豆, 大豆花叶病毒, PAC1, 农杆菌介导转化, 抗性

Abstract: 【Objective】Soybean mosaic virus (SMV) is one of the most prevalent viral diseases in major soybean growing areas of China and cause significant yield losses and quality deterioration of soybean seeds. Ribonuclease PAC1 was known to bind and degrade the dsRNA molecules generated during plant RNA viruses or viroids replication, thus effectively inhibited replication and accumulation of viruses or viroids in host plants. This feature of PAC1 provides an effective target gene for the creation and cultivation of the broad-spectrum anti RNA virus and viroid transgenic crops. The objective of this study is to introduce a PAC1 derived from Schizosaccharomyces pombe into soybean by transgenic technique, research the effects of over-expression of PAC1 onSMV resistance of soybean, and to provide a basis for the development of SMV-resistant soybean cultivars. 【Method】 The PAC1 was subcloned into the binary expression vector pCAMBIA3300 by restriction enzyme digestion and ligation to generate recombinant construct pCAMBIA3300-PAC1. In the resulting construct, the PAC1 was located between the constitutive promoter CaMV 35S and the terminator NOS. The construct also contained a BAR which confers resistance to the herbicide phosphinothricin (PPT). The soybean cultivar Williams82 was used for Agrobacterium-mediated transformation. Based on the detection of LibertyLink^® strip, PCR and herbicide spray (500 mg·L^-1Basta), the integration and copy numbers of the exogenous gene in transgenic lines were further analyzed by Southern blot hybridization. Mechanical rub inoculation was performed to evaluate the resistance of the T₂ and T₃transgenic lines and agronomic traits were analyzed under field conditions. Furthermore, the accumulation of SMV in the transgenic lines 28 days after SMV inoculation was analyzed using qRT-PCR. 【Result】A total of 76 PPT-tolerant plants were generated from 2 600 explants after Agrobacterium-mediated transformation. Among them, 65 regenerated plants were confirmed positive as shown by PCR and LibertyLink^® strip analysis, and the transformation efficiency was 2.48%. The transgenic plants in subsequent generations (T₁to T₃) were further screened by spraying 500 mg·L^-1Basta. The transgenic plants showed no visible phenotype change 7 days after treatment, whereas the non-transgenic (NT) plants exhibited symptoms such as chlorosis or necrosis. Southern blot analysis showed that the exogenous gene was integrated into soybean genome with 1-2 copies of T-DNA insertions in the selected transgenic plants. Resistance evaluation by rub inoculation with SMV showed that the NT plants displayed serious mosaic patterns and curling leaves, while only mild mosaic symptom was observed on some of the leaves of the transgenic plants (35 days after inoculation). Moreover, the transgenic lines had significantly lower average disease indices (11.11-22.22) than the non-transformed (NT) control plants (36.81-46.24) and showed enhanced and stable resistance to SMV. The qRT-PCR result further confirmed that the SMV CP expression in the transgenic plants was significantly decreased than that of the NT control 28 days after SMV inoculation. The investigation of agronomic traits showed that there was no significant difference in the leaf shape, flower color, seed color, hilum color, plant height, node number, podding height, maturity period and weight per 100 seeds between the transgenic lines and NT control plants without inoculation SMV. 【Conclusion】The over-expression of PAC1 significantly inhibited the accumulation of SMV and the development of the symptoms, thus enhancing the resistance level to SMV in transgenic soybean plants.

Key words: soybean, Soybean mosaic virus (SMV), PAC1, Agrobacterium-mediated transformation, resistance

牛陆，赵倩倩，杨静，邢国杰，张伟，贺红利，杨向东. 过表达酵母PAC1增强转基因大豆对大豆花叶病毒的抗性[J]. 中国农业科学, 2018, 51(2): 217-225.

NIU Lu, ZHAO QianQian, YANG Jing, XING GuoJie, ZHANG Wei, HE HongLi, YANG XiangDong. Over-Expression of Yeast PAC1 Confers Enhanced Resistance to Soybean mosaic virus in Transgenic Soybean[J]. Scientia Agricultura Sinica, 2018, 51(2): 217-225.

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