农杆菌介导苹果PPO基因dsRNAi转化‘丰水’梨的研究
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摘要
砂梨果实在加工和切割的过程中普遍存在的褐变问题,给广大果农和果商带来巨大的经济损失。虽然通过多种手段获得了比较满意的抑制褐变的效果,但是随之带来的负面效果也很多。培育抗褐变的品种是解决这一问题的根本途径之一。在已建立‘丰水'叶片不定芽再生体系的基础上,我们以‘丰水'梨为材料,以苹果多酚氧化酶双链RNA为目的基因,通过农杆菌介导方法转化‘丰水'梨,以期获得抗褐变的转基因砂梨。实验内容和研究结果如下:
1、我们研究继代培养基对本实验室保存的‘丰水'梨组培苗生长发育的影响;再以不同继代培养基上培养的叶片为材料,研究叶片生理状态以及氨苄青霉素(Amp)、羧苄青霉素(Carb)、头孢霉素(Cef)和卡那霉素(Km)4种抗生素对不定芽再生的影响。结果表明,以AS为基本培养基,附加BA 0.5mg/L+NAA0.1 mg/L适合‘丰水'梨继代培养,以该配方继代培养21d～32d的叶片不定芽再生率达92.21%～95.38%。在农杆菌介导的‘丰水'梨遗传转化中,单纯考虑杀菌剂对‘丰水'梨叶片再分化的影响,可使用100～300mg/L的Amp、或100mg/L Carb、或100～300 mg/L的Cef,10mg//L为Km筛选的临界浓度。
2、我们以‘丰水'梨组培苗叶片为外植体,通过试验初步确定‘丰水'梨遗传转化体系为:农杆菌菌液浓度OD_(600)=0.4～0.5,以含75μmol/L乙酰丁香酮(AS)的NN_(69)O,pH5.5为悬浮液活化菌株,叶块浸染8min后吸干,置于含75μmol/L乙酰丁香酮(AS)的再生培养基上黑暗共培养4天,综合考虑3种抗生素对叶片再分化的影响和对农杆菌抑制效果,我们认为200mg/L～300 mg/L Amp或Cef是理想的杀菌剂,附加杀菌剂延后培养3d,再转入附加杀菌剂和10mg/LKm的选择培养基上黑暗培养21d,再光照培养至不定芽长出。从转化的2019个外植体中获得了16个不定芽,平均不定芽再生率为0.79%。
3、‘丰水'梨组培苗叶片按照第三章的体系进行转化试验,延迟培养时间由3d延长至21d,再转入附加10mg/LKm的选择培养基上光照培养至不定芽长出,抗性芽率为4.91%。对获得的8株Km抗性芽进行GUS组织化学染色,结果显示AG180-2和AG180-4为GUS阳性植株;PCR检测初步证实AG180-1、AG180-2、AG180-3、AG180-4等4个株系基因组中已整合APPO基因dsRNAi;RT-PCR检测,证实株系AG180-2中的APPO基因dsRNAj已在转录水平表达。多酚氧化酶(PPO)活性测定结果表明,株系AG180-2的多酚氧化酶(PPO)活性比对照极显著降低了49.45%。可见,将苹果PPO基因双链RNA导入苹果中和导入梨中的效果有差异,这可能是由于导入了异源的PPO基因,导致双链RNA的干扰效果变差。
When sand pear are processed and incised,there are ubiquitous browning which bring huge economic loss for a great number of fruit growers andtraders.Although relative satisfactory effects are got by many means,which brign negative impacts with.Cultivating anti-browning varieties is one basic way to solve the problem.In this paper,on the ground of developing shoots regeneration from leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro,we chose 'Hosui' as plant material,dsRNAi of APPO gene as target gene in Agrobacterium-mediated transformation of 'Hosui'.We look forward to getting anti-browning varieties.Experiment contents and research results as following:
1.We studied effects of different subculture media on growing of 'Hosui' shoots perservated in our laboratory;Afterward,chose leaves in vitro on different subculture medium as explants,we studied effects of physiological state of leaves and antibiotics of Amp,Carb,Cef,Km on shoots regeneration frenqucy.The results indicated:'Hosui' shoots had a good state in AS~·medium with BA 0.5mg/L,NAA 0.1mg/L.
'Hosui' leaves cultured for 21d～32d on the subculture medium had 92.21%～95.38% regeneration frequency.In Agrobacterium-mediated transformation of 'Hosui',only considered the effects of bactericides on redifferentiation,we could use 100～300mg /L Amp and Cef,100 mg/L Carb,10mg/L was critical concentration of Km slection. 2.We chose leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro as explants.The system for Agrobacterium-mediated genetic transformation of 'Hosui' was primarily developed via experiments:The concentration of A.tumefacien was OD_(600) = 0.4～0.5. A.tumefacien strain was been activated by NN_(69)0 suspensions with 75μmol/L AS and adjusted to pH5.5.Leaves were inoculated with Agrobacterium solution for 8 min and co-cultivated on regeneration medium with 75μmol/L AS in the dark for 4 days after absorbed drying.Comprehensively considered effects of antibiotics on redifferentiati- on and inhibition of A.tumefaciens,200 mg/L～300 mg/L Amp or Cef were ideal bactericides. Delayed cultivation for 3 days with bactericide.The cultures were kept in the dark in the first 3 weeks and then cultured in the light till adventitious buds were regenerated on slective medium with 10mg/L Km and bactericide.We got 16 buds from 2019 explants,average shoot regeneration was 0.79%.
3.According to system for Agrobacterium-mediated genetic transformation of 'Hosui' in chapter 3,leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro were kept in the dark in the first 3d adjusted to 3 weeks on delayed cultivation medium,and then cultured in the light till adventitious buds were regenerated on slective medium with 10mg/L Km. Adventitious buds regeneration frequency was enhanced to 4.91%.Histochemical GUS assay showed AG180-2,AG180-4 were GUS positive.PCR and RT-PCR analysis with primers designed to amplify fragments of dsRNAi of APPO gene.The results primarily confirmed dsRNAi of APPO gene was been transferred into transgene lines AG180-1,AG180-2,AG180-3,AG180-4,and transgene lines AG180-2 with DSAP -PO gene expression in the level of transcription.The result of PPO activity test indicated:The PPO activity of transgene lines AG180-2 significantly decreased by 49.45%,compeared with control.It is obvious that there was difference between transfering dsRNAi of APPO gene into apple and into pear.As a result of that heterogenous PPO gene was transferred into 'Hosui',the effects of dsRNAi got worse.
1、我们研究继代培养基对本实验室保存的‘丰水'梨组培苗生长发育的影响;再以不同继代培养基上培养的叶片为材料,研究叶片生理状态以及氨苄青霉素(Amp)、羧苄青霉素(Carb)、头孢霉素(Cef)和卡那霉素(Km)4种抗生素对不定芽再生的影响。结果表明,以AS为基本培养基,附加BA 0.5mg/L+NAA0.1 mg/L适合‘丰水'梨继代培养,以该配方继代培养21d～32d的叶片不定芽再生率达92.21%～95.38%。在农杆菌介导的‘丰水'梨遗传转化中,单纯考虑杀菌剂对‘丰水'梨叶片再分化的影响,可使用100～300mg/L的Amp、或100mg/L Carb、或100～300 mg/L的Cef,10mg//L为Km筛选的临界浓度。
2、我们以‘丰水'梨组培苗叶片为外植体,通过试验初步确定‘丰水'梨遗传转化体系为:农杆菌菌液浓度OD_(600)=0.4～0.5,以含75μmol/L乙酰丁香酮(AS)的NN_(69)O,pH5.5为悬浮液活化菌株,叶块浸染8min后吸干,置于含75μmol/L乙酰丁香酮(AS)的再生培养基上黑暗共培养4天,综合考虑3种抗生素对叶片再分化的影响和对农杆菌抑制效果,我们认为200mg/L～300 mg/L Amp或Cef是理想的杀菌剂,附加杀菌剂延后培养3d,再转入附加杀菌剂和10mg/LKm的选择培养基上黑暗培养21d,再光照培养至不定芽长出。从转化的2019个外植体中获得了16个不定芽,平均不定芽再生率为0.79%。
3、‘丰水'梨组培苗叶片按照第三章的体系进行转化试验,延迟培养时间由3d延长至21d,再转入附加10mg/LKm的选择培养基上光照培养至不定芽长出,抗性芽率为4.91%。对获得的8株Km抗性芽进行GUS组织化学染色,结果显示AG180-2和AG180-4为GUS阳性植株;PCR检测初步证实AG180-1、AG180-2、AG180-3、AG180-4等4个株系基因组中已整合APPO基因dsRNAi;RT-PCR检测,证实株系AG180-2中的APPO基因dsRNAj已在转录水平表达。多酚氧化酶(PPO)活性测定结果表明,株系AG180-2的多酚氧化酶(PPO)活性比对照极显著降低了49.45%。可见,将苹果PPO基因双链RNA导入苹果中和导入梨中的效果有差异,这可能是由于导入了异源的PPO基因,导致双链RNA的干扰效果变差。
When sand pear are processed and incised,there are ubiquitous browning which bring huge economic loss for a great number of fruit growers andtraders.Although relative satisfactory effects are got by many means,which brign negative impacts with.Cultivating anti-browning varieties is one basic way to solve the problem.In this paper,on the ground of developing shoots regeneration from leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro,we chose 'Hosui' as plant material,dsRNAi of APPO gene as target gene in Agrobacterium-mediated transformation of 'Hosui'.We look forward to getting anti-browning varieties.Experiment contents and research results as following:
1.We studied effects of different subculture media on growing of 'Hosui' shoots perservated in our laboratory;Afterward,chose leaves in vitro on different subculture medium as explants,we studied effects of physiological state of leaves and antibiotics of Amp,Carb,Cef,Km on shoots regeneration frenqucy.The results indicated:'Hosui' shoots had a good state in AS~·medium with BA 0.5mg/L,NAA 0.1mg/L.
'Hosui' leaves cultured for 21d～32d on the subculture medium had 92.21%～95.38% regeneration frequency.In Agrobacterium-mediated transformation of 'Hosui',only considered the effects of bactericides on redifferentiation,we could use 100～300mg /L Amp and Cef,100 mg/L Carb,10mg/L was critical concentration of Km slection. 2.We chose leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro as explants.The system for Agrobacterium-mediated genetic transformation of 'Hosui' was primarily developed via experiments:The concentration of A.tumefacien was OD_(600) = 0.4～0.5. A.tumefacien strain was been activated by NN_(69)0 suspensions with 75μmol/L AS and adjusted to pH5.5.Leaves were inoculated with Agrobacterium solution for 8 min and co-cultivated on regeneration medium with 75μmol/L AS in the dark for 4 days after absorbed drying.Comprehensively considered effects of antibiotics on redifferentiati- on and inhibition of A.tumefaciens,200 mg/L～300 mg/L Amp or Cef were ideal bactericides. Delayed cultivation for 3 days with bactericide.The cultures were kept in the dark in the first 3 weeks and then cultured in the light till adventitious buds were regenerated on slective medium with 10mg/L Km and bactericide.We got 16 buds from 2019 explants,average shoot regeneration was 0.79%.
3.According to system for Agrobacterium-mediated genetic transformation of 'Hosui' in chapter 3,leaves of Pyrus pyrifolia Nakai 'Hosui' in vitro were kept in the dark in the first 3d adjusted to 3 weeks on delayed cultivation medium,and then cultured in the light till adventitious buds were regenerated on slective medium with 10mg/L Km. Adventitious buds regeneration frequency was enhanced to 4.91%.Histochemical GUS assay showed AG180-2,AG180-4 were GUS positive.PCR and RT-PCR analysis with primers designed to amplify fragments of dsRNAi of APPO gene.The results primarily confirmed dsRNAi of APPO gene was been transferred into transgene lines AG180-1,AG180-2,AG180-3,AG180-4,and transgene lines AG180-2 with DSAP -PO gene expression in the level of transcription.The result of PPO activity test indicated:The PPO activity of transgene lines AG180-2 significantly decreased by 49.45%,compeared with control.It is obvious that there was difference between transfering dsRNAi of APPO gene into apple and into pear.As a result of that heterogenous PPO gene was transferred into 'Hosui',the effects of dsRNAi got worse.
引文
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