| 摘要: |
| [目的]了解无机焦磷酸酶对植物钾吸收和相关基因表达的作用.[方法]反转录克隆拟南芥无机焦磷酸酶基因(H+-PPase)AVP2,构建植物表达载体,采用根癌农杆菌介导法将其导人烟草品种K326,经分子鉴定,提取转化烟苗幼根RNA,反转录后,应用荧光定量PCR分析外源基因AVP2和烟草钾通道基因NKT1、钾离子转运体基因NtHAK1、细胞质膜H+-ATPase基因NHA1、液泡膜H+-ATPase基因VAG1和液泡膜H+-PPase基因NVP1的mRNA转录水平,并测定转化烟草叶片的内在化学成分.[结果]获得了GUS染色和AVP2序列PCR扩增呈阳性的卡那霉素抗性转化烟草11株.经Southern杂交鉴定,证实AVP2基因已成功导入烟草,在转化烟草幼根中可以检测到外源基因AVP2的mRNA,证实该基因已整合到烟草基因组并成功表达;与未转化烟草相比,烟草钾离子转运体基因NtHAK1的转录水平显著增加,钾通道基因NKT1的转录稍有增加,而烟草液泡膜H+-ATPase基因VAG1和液泡膜H+-PPase基因NVP1的转录有所降低,烟草细胞质膜H+-ATPase基因NHA1无显著变化;转化烟草材料烟叶K+含量较对照显著增加,而烟碱、还原糖等其他内在化学成分无显著变化.[结论]无机焦磷酸酶基因参与植物的K+吸收. |
| 关键词: 拟南芥AVP2 转基因烟草 吸K+基因 转录 |
| DOI： |
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| 基金项目:国家烟草专卖局科技攻关项目（110200101008） |
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| Transcript analysis of K+ uptake relative genes in AVP2 transgenic tobacco |
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| Abstract: |
| 【Objective】 Nutrition uptake has close relations with energy supply in plant cell.The studly investigated the effects of H+-pyrophosphatase on the expression of plant potassium uptake related genes.【Method】 The vacuolar H+-pyrophosphatase (V-H+-PPase) gene AVP2 which was amplified from Arabidopsis thaliana by RT-PCR was inserted into the plant binary expression vector containing intron kanamycin gene and SAR sequence from tobacco and introduced into tobacco variety K326 by Agro-bacterium mediated transformation.Total RNA was isolated from the roots of AVP2 trans-formants and normal tobacco without genetic modification.The transcript of six genes including tobacco K+ transporter gene NtHAK1,K+ channel gene NKT1,plasma H+-ATPase gene NHA1,vacuole H+-ATPase gene VAG1,vacuole H+-PPase gene NVP1 were analyzed using real-time PCR.【Result】 Eleven AVP2 transgenic tobacco plants were obtained and approved GUS dye and AVP2 sequence PCR test.The mRNA of AVP2 were detected in the trans-formant plant roots which confirmed that AVP2 have been transcript and expressed effectively in the modified tobacco.The results showed that the transcript of K+ transporter gene NtHAK1 and potassium channel NKT1 were significantly stimulated, and that of vacuolar V-H+-ATPase G unit gene VAG1 and V-H+-PPase genes NVP1 were down regulation.There was no significant alteration can be found in the transcript of plasma proton pumping (P-H+-ATPase) gene NHA1.It was found that the potassium contents increased significantly and there were no changes in nicotine,sugar and other chemical contents in the leaves of AVP2 transgenic tobacco T1 generation.【Conclusion】 The results indicate that AVP2 which encoded the vacuolar H+-pyrophosphatase have effects on the potassium uptake in plant. |
| Key words: AVP2 transgenic tobacco K+ uptake transcript |
