低能离子注入与~（60）Coγ辐照小麦成熟胚诱发反转录转座子WIS2-1A变化的研究
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摘要
目的:低能N~+和~(60)Coγ射线在诱变育种应用上已经取得了较好的成效,但对于诱变效应的机理研究相对滞后。本实验以小麦中反转录转座子WIS2-1A为研究对象,将用于诱变育种的物理诱变源低能N~+和~(60)Coγ射线应用到反转录转座子的研究上。从DNA及cDNA水平初步探讨了低能离子辐射及~(60)Coγ射线对WIS2-1A反转录酶的基因及表达的影响。然后以其中WIS2-1A表达大幅度提高的样本为研究对象,用基因芯片的方法研究其整个转录本的表达变化情况,并据此推断这些表达变化与反转录转座子WIS2-1A表达之间的相关关系,推测辐射引起生物学效应的可能原因及反转录转座子WIS2-1A在其中的作用,为辐射诱变生物学效应的机理研究奠定基础。
结论:
1.本研究以各种剂量低能N~+和~(60)Coγ射线辐照小麦种子,发现经过处理的种子培养30 h、45 h和60 h后,胚中的反转录转座子WIS2-1A反转录酶的保守序列在DNA水平没有太大变化。与Genebank中标准序列相比,低能N~+注入的小麦种胚中WIS2-1A反转录酶的保守序列变化相对较大。
2.1.0×10~(17)N~+/cm~2注入的小麦种子培养45 h时,种胚中WIS2-1A反转录酶保守序列克隆中,发现一个克隆序列在187-195bp处缺失了9个碱基(AGTGAGTGG)。将其翻译成氨基酸序列,发现其中有6个终止子。这种缺失突变和终止密码子突变可能是造成反转录转座子异质性的主要原因。
3.实时定量PCR结果表明,不同剂量低能N~+注入和~(60)Coγ射线处理的种胚中,都发现WIS2-1A有持续低水平的表达,且WIS2-1A表达活性随着种子培养时间延长而下降。~(60)Coγ辐照能够小幅度地刺激或抑制WIS2-IA表达,影响相对较小且没有规律性;低能N~+注入对WIS2-1A表达影响较大,且WIS2-1A表达变化与低能N~+注入剂量之间有一定规律性。种子培养30 h时,这种剂量依赖关系表现尤其明显:随着注入剂量增大,WIS2-1A表达呈现先增加后降低的趋势,在0.5×10~(17)N~+/cm~2表达达到最大值,即相对表达量是对照的138倍。
4.从基因芯片结果看,受到0.5×10~(17)N~+/cm~2低能N~+注入刺激的小麦种子,在培养30 h时,呼吸作用增强,碳水化合物和蛋白质的水解加快而合成减慢,低温及高温应激蛋白、抗病及抗逆蛋白明显增多。据此推断,0.5×10~(17)N~+/cm~2低能N~+注入对小麦种胚细胞造成了某些伤害,经过30 h培养,细胞正处于主动的抗逆状态。
5.小麦种子受到0.5×10~(17)N~+/cm~2低能N~+注入刺激后,与对照相比,细胞内抗逆基因和反转录转座子WIS2-1A的表达都显著提高。推断胁迫条件下,反转录转座子激活与防卫基因激活之间可能存在相似的分子机理。
Differential expression of retrotransposon WIS 2-1 A in wheatresponse to low-energy N~+ implantation and ~(60)Coγ-ray irradiation
Purpose: This paper tried to investigate the variation of retrotransposon WIS 2-1Aactivity in wheat (Triticum aestivum L.) embryos at three different developmentalstages (30 h, 45 h and 60 h) after treating with 0.5×10~(17),1.0×10~(17),1.5×10~(17) N~+/cm~2implantation and 100Gy, 300Gy, 500Gy ~(60)Coγ-ray irradiation respectively. Wediscussed the relationship between the expression of WIS2-1A and the variable dose ofN~+ and ~(60)Coγ-ray from the level of DNA and cDNA. What's more, we tried to find theinfluence of the high expression of WIS2-1A to the whole other transcripts using thewheat genome array. This paper could promote the theory of radiant aberrance.
Results:
1 .The DNA composition changed a little of WIS2-1A reverse transcriptase aftersufferring N~+ implantation and ~(60)Coγ-ray irradiation.
2. There is a vacancy of 9bp in the sequence of W1S2-1A reverse transcriptase aftersufferring 1.0×10~(17) N~+/cm~2 implantation. It's translation product has 6 terminators andthat probably result the heterogeneity.
3. WIS 2-1A always had the transcription activity in the first 60 h of incubation and areverse relationship between the time of embryo development and the sensitivity ofretrotransposon WIS 2-1A. For N~+ the expression showed a regular fluctuation withthe gradual doses. The most positive stimulation occurred at the dose of 1.5×10~(17)N~+/cm~2 at 30 h. For ~(60)Coγ-ray the expression showed a irregular fluctuation and theexpression changed a little.
4. The membrane permeability, respiration, synthesis of carbohydrates and proteinincreased, but the hydrolysis of carbohydrates and protein slowed down. The proteinof resisting low-temperature, high temperature and stress increased after 1.5×10~(17)N~+/cm~2 implantation at 30 h. Low-energy N~+ implantation caused some injury towheat embryo cells, after 30h culture, the cells are in the active state of stress adaptation.
5.comparing the control,the expression of WIS2-1A and some resistance geneincreased greatly. It seems like that they have the same mechanism to regard the cells.
结论:
1.本研究以各种剂量低能N~+和~(60)Coγ射线辐照小麦种子,发现经过处理的种子培养30 h、45 h和60 h后,胚中的反转录转座子WIS2-1A反转录酶的保守序列在DNA水平没有太大变化。与Genebank中标准序列相比,低能N~+注入的小麦种胚中WIS2-1A反转录酶的保守序列变化相对较大。
2.1.0×10~(17)N~+/cm~2注入的小麦种子培养45 h时,种胚中WIS2-1A反转录酶保守序列克隆中,发现一个克隆序列在187-195bp处缺失了9个碱基(AGTGAGTGG)。将其翻译成氨基酸序列,发现其中有6个终止子。这种缺失突变和终止密码子突变可能是造成反转录转座子异质性的主要原因。
3.实时定量PCR结果表明,不同剂量低能N~+注入和~(60)Coγ射线处理的种胚中,都发现WIS2-1A有持续低水平的表达,且WIS2-1A表达活性随着种子培养时间延长而下降。~(60)Coγ辐照能够小幅度地刺激或抑制WIS2-IA表达,影响相对较小且没有规律性;低能N~+注入对WIS2-1A表达影响较大,且WIS2-1A表达变化与低能N~+注入剂量之间有一定规律性。种子培养30 h时,这种剂量依赖关系表现尤其明显:随着注入剂量增大,WIS2-1A表达呈现先增加后降低的趋势,在0.5×10~(17)N~+/cm~2表达达到最大值,即相对表达量是对照的138倍。
4.从基因芯片结果看,受到0.5×10~(17)N~+/cm~2低能N~+注入刺激的小麦种子,在培养30 h时,呼吸作用增强,碳水化合物和蛋白质的水解加快而合成减慢,低温及高温应激蛋白、抗病及抗逆蛋白明显增多。据此推断,0.5×10~(17)N~+/cm~2低能N~+注入对小麦种胚细胞造成了某些伤害,经过30 h培养,细胞正处于主动的抗逆状态。
5.小麦种子受到0.5×10~(17)N~+/cm~2低能N~+注入刺激后,与对照相比,细胞内抗逆基因和反转录转座子WIS2-1A的表达都显著提高。推断胁迫条件下,反转录转座子激活与防卫基因激活之间可能存在相似的分子机理。
Differential expression of retrotransposon WIS 2-1 A in wheatresponse to low-energy N~+ implantation and ~(60)Coγ-ray irradiation
Purpose: This paper tried to investigate the variation of retrotransposon WIS 2-1Aactivity in wheat (Triticum aestivum L.) embryos at three different developmentalstages (30 h, 45 h and 60 h) after treating with 0.5×10~(17),1.0×10~(17),1.5×10~(17) N~+/cm~2implantation and 100Gy, 300Gy, 500Gy ~(60)Coγ-ray irradiation respectively. Wediscussed the relationship between the expression of WIS2-1A and the variable dose ofN~+ and ~(60)Coγ-ray from the level of DNA and cDNA. What's more, we tried to find theinfluence of the high expression of WIS2-1A to the whole other transcripts using thewheat genome array. This paper could promote the theory of radiant aberrance.
Results:
1 .The DNA composition changed a little of WIS2-1A reverse transcriptase aftersufferring N~+ implantation and ~(60)Coγ-ray irradiation.
2. There is a vacancy of 9bp in the sequence of W1S2-1A reverse transcriptase aftersufferring 1.0×10~(17) N~+/cm~2 implantation. It's translation product has 6 terminators andthat probably result the heterogeneity.
3. WIS 2-1A always had the transcription activity in the first 60 h of incubation and areverse relationship between the time of embryo development and the sensitivity ofretrotransposon WIS 2-1A. For N~+ the expression showed a regular fluctuation withthe gradual doses. The most positive stimulation occurred at the dose of 1.5×10~(17)N~+/cm~2 at 30 h. For ~(60)Coγ-ray the expression showed a irregular fluctuation and theexpression changed a little.
4. The membrane permeability, respiration, synthesis of carbohydrates and proteinincreased, but the hydrolysis of carbohydrates and protein slowed down. The proteinof resisting low-temperature, high temperature and stress increased after 1.5×10~(17)N~+/cm~2 implantation at 30 h. Low-energy N~+ implantation caused some injury towheat embryo cells, after 30h culture, the cells are in the active state of stress adaptation.
5.comparing the control,the expression of WIS2-1A and some resistance geneincreased greatly. It seems like that they have the same mechanism to regard the cells.
引文
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