【作者】 王玉；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 农业硕士（专业学位）， 2018， 硕士

【摘要】 番木瓜环斑病毒(Papaya ringspot virus,PRSV)属于马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)。根据寄主范围的不同,PRSV可分为P和W两个株系,其中W株系(PRSV-W)只侵染葫芦科作物,引起花叶和果实环斑等症状,严重影响作物产量和品质。近年PRSV-W在山东、河南等地区的发生呈上升趋势。本研究制备了PRSV-W抗血清,分析了葫芦科作物品种对PRSV的抗性,测定了弱毒突变体对野生型PRSV的交叉保护效果。主要研究结果如下:1、利用原核表达的衣壳蛋白制备了效价高、特异性强的PRSV抗血清。将PRSV-W衣壳蛋白(CP)基因连接到原核表达载体pEHISTEV上,获得重组质粒,转化大肠杆菌Rosetta。经诱导可表达出约37 kD的蛋白,最适IPTG终浓度0.2 mmol/L、诱导时间4 h。经ELISA检测,抗血清效价为1:8192。Western blotting结果表明,该抗血清只与PRSV-W侵染的西葫芦有特异性反应,而与健康西葫芦、西瓜花叶病毒侵染的西葫芦和马铃薯Y病毒侵染的普通烟均无反应。2、通过室内接种试验鉴定了55个葫芦科作物品种对PRSV的抗性。在鉴定的13个黄瓜品种中,唐山秋美、精选唐山秋瓜、耐热王中王和辽研翠玉4个品种的病情指数分别为32.2、34.4、37.8和38.9,属于中抗品种,其余均为感病或高感品种;七叶早南瓜、瑞丽甜蜜本、日本南瓜和一品甜红栗的病情指数介于31.1与39.4之间,属于中抗品种。长丰肉丝瓜和常丰长香丝瓜的病情指数分别为50.5和54.4,属于感病品种。13个甜瓜品种、11个西瓜品种、11个西葫芦品种和2个瓠瓜品种,病情指数都在60以上,属于高感品种;3、测定了三个弱毒突变体对野生型PRSV的交叉保护效果。在保护间隔期为5天时,三个弱毒突变体均没有表现出交叉保护效果。在保护间隔期为10天时,挑战接种后15天,弱毒突变体N137A预先接种的西葫芦植株均无明显症状,保护效率为100%,与只接种野生型植株相比,预先接种N137A的西葫芦植株中病毒粒子的积累量显著降低;弱毒突变体N346A能够延迟发病15天;弱毒突变体V417A没有表现出交叉保护效果。在保护间隔期为15天时,N137A的保护效率为100%;N346A的保护效率为26.7%,V417A仍然无明显的交叉保护效果。更多还原

【Abstract】 Papaya ringspot virus(PRSV)is a member of the genus Potyvirus belonging to the family Potyviridae.According to the host range,PRSV can be divided into P and W strains.Among them,the W strain(PRSV-W)mainly infects cucurbitaceous crops and causes symptoms of leaf mosaic and fruit ring spots,which seriously affected the yield and quality.In recent years,the occurrence of PRSV-W has been increasing in Shandong,Henan and other provinces.In this study,the PRSV-W antiserum was prepared,the resistance of cucurbitaceous crops to PRSV was analyzed and the cross-protection efficiency of three attenuated mutants on wild-type PRSV was evaluated.The main results are shown as below:1.The PRSV-W antiserum with high titer and strong specificity was prepared using the prokaryotic expressed capsid protein.The PRSV-W capsid protein(CP)gene was ligated into the prokaryotic expression vector pEHISTEV to obtain a recombinant plasmid,which was transformed into E.coli Rosetta.After induction,a protein of about 37 kD was expressed,and the optimal IPTG final concentration was 0.2 mmol/L and the induction time was 4 h.The titer of the resultant antiserum was 1:8192 in ELISA.In Western blotting analysis,the antiserum showed specific positive reaction only with the zucchini plants infected with PRSV,but not with healthy zucchini plants or those infected with watermelon mosaic virus,or Nicotiana tabacum plants infected with potato virus Y.2.The resistance of 55 cucurbitaceous cultivars to PRSV-W was identified by indoor inoculation test.Of the 13 cucumber cultivars identified,the disease indices of Tangshan Qiumei,Tangshan Qiugua,Heat-resistant Wangzhong Wang and Liaoyan Cuiyu were 32.2,34.4,37.8 and 38.9,respectively,indicating that these cultivars had medium resistance;the other cultivars were susceptible or highly susceptible.The disease indices of Qiye Zaonanguan,Ruili Tianmiben,Japanese pumpkin and Yipin Tianhongli were between 31.1 and 39.4,indicating that these cultivars also had medium resistance.The disease indices of Changfeng Rousigua and Changfeng Changxiangsigua were 50.5 and 54.4,respectively,indicating that both cultivars were susceptible cultivars.The disease indices of 13 melon cultivars,11 watermelon cultivars,11 zucchini cultivars and 2 melon cultivars were higher than 60,indicating that all these cultivars were highly susceptible.3.The cross protection effiency of three attenuated mutants on wild-type PRSV was determined.With a protection interval of 5 days,none of the three attenuated mutants showed cross-protective effects.With an interval of 10 days,at 15 days after the challenge,the zucchini plants protected with the attenuated mutant N137 A had no obvious symptoms,and the protection efficiency was 100%.The virus accumulation level in the N137 A pre-inoculated plants was significantly reduced compared to that in plants inoculated with wild-type virus alone.The attenuated mutant N346 A delayed the appearance of PRSV-W symptom for 15 days,while attenuated mutant V417 A showed no cross-protective effect.With the interval of 15 days,the protective efficiencies of N137 A and N346 A were 100% and 26.7%,respectively,while V417 A still had no cross-protection effect.更多还原