苹果潜隐性病毒的检测与脱除技术研究
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摘要
本研究用热处理+茎尖培养、热处理+茎尖培养+板蓝根、超低温+茎尖培养三种方法对苹果病毒进行了脱除，通过分析各方法的主要影响因子，确立了苹果脱毒的优化体系；建立了一种简便有效的苹果总RNA提取新方法；用RT-PCR对苹果潜隐性病毒进行了检测，并对RT-PCR体系进行了优化。结果如下：
1．用斑点酶联免疫吸附法检测了苹果褪绿叶斑病毒，该法操作简单，可有效地减轻单宁等物质对苹果褪绿叶斑病毒的钝化作用，提高检测的灵敏度。
2．研究了苹果总RNA提取方法，设计了一种新的RNA提取方法，用酸酚及140mM的NaCl来去除DNA，水不溶性PVP、β-巯基乙醇去除酚类物质，15％无水乙醇、1／5体积的3M KAC(pH4.8)来去除多糖，得到高质量的总RNA，该法操作简单，只需要3-4个小时。
3．用RT-PCR法对苹果潜隐性病毒ACLSV、ASGV、ASPV进行了检测。约0.02μg的苹果总RNA依然能扩增出特异性带。
4．用热处理+茎尖培养脱除病毒，转接后试管苗的培养时间以7—15天为宜；采用32℃8h和37℃8h交替处理60天的变温热处理法，试管苗热处理的成活率提高了11％，脱毒率提高了7.5％；茎尖分化培养基是影响茎尖分化的重要因素，不同的品种最佳培养基激素浓度配比不同，茎尖分化培养基BA与NAA配比应适当高于该品种继代培养基BA、NAA的配比，长富二号、金矮生适宜的分化培养基为MS+BA1.0mg／L+NAA0.1mg／L，王林、美富、新乔纳金适宜的分化培养基为MS+BA2.0mg／L+NAA0.1mg／L。
5．在热处理+茎尖培养法的基础上，茎尖分化培养基中添加2.0g／L的板蓝根，可达到100％的脱毒率。但药害现象严重，茎尖的分化率只有16％，生长量大大降低。
6．用超低温+茎尖培养法脱除了苹果病毒，脱毒率可达70％。研究了3种玻璃化保护剂对成活率的影响，玻璃化保护剂PVS2、PVS3的效果最好，相差不大，其组成为PVS2：35％甘油+15％EG+35％蔗糖MS培养基；PVS3：40％甘油+10％EG+45％蔗糖+10％DMSO；PVS一步装载处理90min茎尖再生率最高，为45.5％；采用逐步装载，即先用装载液(15％甘油+15％蔗糖)处理20min，60％PVS3处理20min，PVS3处理80min，效果好于一步装载，茎尖再生率可达57.3％。
The virus elimination in apple were achieved by the ways of shoot tip culture + heat treatment, shoot tip culture + heat treatment + Radix Isatidis and cryopreservation +shoot tip culture. Factors affecting elimination of virus were studied, the efficient elimination methods of apple virus were developed. New method of extracting total RNA from apple was posed; and the apple latent virus(ACLSV, ASGV, ASPV)were detection by reverse transcription polymerase chain reaction, and the RT-PCR detection system was optimized. Results are following:
1. The ACLSV was detected by Dot-ELISA, The method of Dot-ELISA is simple and save time, The effect of passivation substance on ACLSV is milder than other method of serology.
2. A rapid and simple procedure was posed for diffidently extracting total RNA from apple. The RNA can be used for RT-PCR. Acid phenol and 140mM NaCl were used to removal of DNA,insoluble PVP and 2-mercapto-ethanol were used to removal phenolic compounds, 15% ethanol and 1/5 volume of 3M KAc were used to removal polysaccharides. The procedure can be completed in less than four hours.
3. The ACLSV, ASGV and ASPV were detected by reverse transcription polymerase chain reaction.. The RT-PCR method is sensitivity and save tune.
4. The virus elimination in apple were achieved by the way of combining shoot tip culture with heat treatment. Before being heat treated, the shoots were cultured on new medium for 7-15 days, a higher survival rate was gained. The rate of survival is 11 % higher and the rate of elimination is 7.5% higher treamented by variable temperature than by invariable heat treament The regrowth rate changed when the shoot tips were cultured on mediums containing different concentration of hormone. Cultured on regeneration medium contain higher propotion by concentration of BA and NAA than stock medium, the regrowth tate of shoot tips is higher. The Nagafu-2 and Gold Spur Deli, would regenerate normally in medurm MS+BA1.0mg/L+NAA 0.1 mg/L, and New Jonagold, Orin would in medium MS+BA2.0mg/L+NAAO. 1 mg/L.
5. Based on the method of shoot tip culture+heat treatment, the antivirus Radix Isatidis was applied into medium of tip culture, the results shows that the elimination
rate can attained 100%, but Radix Isatidis have strong inhibiting effect on apple tip growth.
6. The apple virus can be successfully eliminated by cryopreservation of in vitro-grow shoot tips, the rate of elimination is 70%. Three compound cryopretectants were designed and the effects were investigated. The PVS2(35%glycerol+15% EG+35%sucrose MS liquid medium) and PVS3(40 % glycerol + 10% EG+ 45% sucrose +10 %DMSO) was more effective. Higher survival rate was gained by dehydration hi PVS3 for 90 min directly. The survival rate would higher by loading step by step in load solution (15%glycerol+15%sucrose)and diluted PVS3.
1．用斑点酶联免疫吸附法检测了苹果褪绿叶斑病毒，该法操作简单，可有效地减轻单宁等物质对苹果褪绿叶斑病毒的钝化作用，提高检测的灵敏度。
2．研究了苹果总RNA提取方法，设计了一种新的RNA提取方法，用酸酚及140mM的NaCl来去除DNA，水不溶性PVP、β-巯基乙醇去除酚类物质，15％无水乙醇、1／5体积的3M KAC(pH4.8)来去除多糖，得到高质量的总RNA，该法操作简单，只需要3-4个小时。
3．用RT-PCR法对苹果潜隐性病毒ACLSV、ASGV、ASPV进行了检测。约0.02μg的苹果总RNA依然能扩增出特异性带。
4．用热处理+茎尖培养脱除病毒，转接后试管苗的培养时间以7—15天为宜；采用32℃8h和37℃8h交替处理60天的变温热处理法，试管苗热处理的成活率提高了11％，脱毒率提高了7.5％；茎尖分化培养基是影响茎尖分化的重要因素，不同的品种最佳培养基激素浓度配比不同，茎尖分化培养基BA与NAA配比应适当高于该品种继代培养基BA、NAA的配比，长富二号、金矮生适宜的分化培养基为MS+BA1.0mg／L+NAA0.1mg／L，王林、美富、新乔纳金适宜的分化培养基为MS+BA2.0mg／L+NAA0.1mg／L。
5．在热处理+茎尖培养法的基础上，茎尖分化培养基中添加2.0g／L的板蓝根，可达到100％的脱毒率。但药害现象严重，茎尖的分化率只有16％，生长量大大降低。
6．用超低温+茎尖培养法脱除了苹果病毒，脱毒率可达70％。研究了3种玻璃化保护剂对成活率的影响，玻璃化保护剂PVS2、PVS3的效果最好，相差不大，其组成为PVS2：35％甘油+15％EG+35％蔗糖MS培养基；PVS3：40％甘油+10％EG+45％蔗糖+10％DMSO；PVS一步装载处理90min茎尖再生率最高，为45.5％；采用逐步装载，即先用装载液(15％甘油+15％蔗糖)处理20min，60％PVS3处理20min，PVS3处理80min，效果好于一步装载，茎尖再生率可达57.3％。
The virus elimination in apple were achieved by the ways of shoot tip culture + heat treatment, shoot tip culture + heat treatment + Radix Isatidis and cryopreservation +shoot tip culture. Factors affecting elimination of virus were studied, the efficient elimination methods of apple virus were developed. New method of extracting total RNA from apple was posed; and the apple latent virus(ACLSV, ASGV, ASPV)were detection by reverse transcription polymerase chain reaction, and the RT-PCR detection system was optimized. Results are following:
1. The ACLSV was detected by Dot-ELISA, The method of Dot-ELISA is simple and save time, The effect of passivation substance on ACLSV is milder than other method of serology.
2. A rapid and simple procedure was posed for diffidently extracting total RNA from apple. The RNA can be used for RT-PCR. Acid phenol and 140mM NaCl were used to removal of DNA,insoluble PVP and 2-mercapto-ethanol were used to removal phenolic compounds, 15% ethanol and 1/5 volume of 3M KAc were used to removal polysaccharides. The procedure can be completed in less than four hours.
3. The ACLSV, ASGV and ASPV were detected by reverse transcription polymerase chain reaction.. The RT-PCR method is sensitivity and save tune.
4. The virus elimination in apple were achieved by the way of combining shoot tip culture with heat treatment. Before being heat treated, the shoots were cultured on new medium for 7-15 days, a higher survival rate was gained. The rate of survival is 11 % higher and the rate of elimination is 7.5% higher treamented by variable temperature than by invariable heat treament The regrowth rate changed when the shoot tips were cultured on mediums containing different concentration of hormone. Cultured on regeneration medium contain higher propotion by concentration of BA and NAA than stock medium, the regrowth tate of shoot tips is higher. The Nagafu-2 and Gold Spur Deli, would regenerate normally in medurm MS+BA1.0mg/L+NAA 0.1 mg/L, and New Jonagold, Orin would in medium MS+BA2.0mg/L+NAAO. 1 mg/L.
5. Based on the method of shoot tip culture+heat treatment, the antivirus Radix Isatidis was applied into medium of tip culture, the results shows that the elimination
rate can attained 100%, but Radix Isatidis have strong inhibiting effect on apple tip growth.
6. The apple virus can be successfully eliminated by cryopreservation of in vitro-grow shoot tips, the rate of elimination is 70%. Three compound cryopretectants were designed and the effects were investigated. The PVS2(35%glycerol+15% EG+35%sucrose MS liquid medium) and PVS3(40 % glycerol + 10% EG+ 45% sucrose +10 %DMSO) was more effective. Higher survival rate was gained by dehydration hi PVS3 for 90 min directly. The survival rate would higher by loading step by step in load solution (15%glycerol+15%sucrose)and diluted PVS3.
引文
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