CMO启动子驱动CMO基因在烟草中的表达
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摘要
甜菜碱是一种非毒性的小分子渗透调节剂,多种高等植物在盐碱或干旱的环境下在细胞中积累甜菜碱,以维持细胞正常的膨压。在植物中甜菜碱是由胆碱经两步反应生成的,催化第一步反应的酶是胆碱单加氧酶(choline monooxygenase,CMO)。本实验室的前期工作中分离了辽宁碱蓬CMO基因、CMO启动子,发现CMO启动子(pC:-267～+1bp)是盐诱导启动子,在400 mmol/L NaCl条件下处理时,其驱动的GUS基因活性是未经NaCl诱导时的5倍。本文构建了pC启动子驱动CMO基因的植物表达载体,转化烟草,分析pC启动子驱动CMO基因在转基因烟草中的表达及转基因烟草的甜菜碱含量,为胁迫诱导型启动子驱动外源基因的应用奠定了基础。具体工作及结果如下:
利用PCR技术从克隆载体pUC19-CMO中扩增出约1.3kb的CMO基因编码区,经过酶切、连接,替代表达载体pCAMBIA1301-pC-GUS中的GUS基因获得新的表达载体pCAMBIA1301-pC-CMO。
冻融法将pCAMBIA1301-pC-CMO导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,获得工程菌LBA4404- pCAMBIA1301-pC-CMO。
采用农杆菌介导的叶盘转化法将pCAMBIA1301-pC-CMO、pBI121-CMO (CaMV 35S启动子驱动CMO基因)转入烟草, PCR检测获得阳性植株。半定量RT-PCR检测表明,35S-CMO转基因烟草CMO基因的表达量高于pC-CMO转基因烟草;甜菜碱含量检测表明,pC-CMO转基因烟草甜菜碱含量与野生烟草的甜菜碱含量相近或略高于野生烟草的甜菜碱含量,35S-CMO转基因烟草中的甜菜碱含量高于pC-CMO转基因烟草。
100 mmol/L NaCl胁迫处理转基因烟草和野生烟草24 h后,pC-CMO转基因烟草中CMO基因表达量高于35S-CMO转基因烟草,与此相对应,pC-CMO转基因烟草中甜菜碱含量均提高,而野生型烟草和35S-CMO转基因烟草中的甜菜碱含量基本没有变化,且pC-CMO转基因烟草中甜菜碱含量均高于野生烟草、35S-CMO转基因烟草。说明pC可以驱动CMO基因在转基因烟草中适时表达,从而相应提高甜菜碱含量。
Betaine is a non-toxic osmopretectant which protects the cell from salt or drought stress by maintaining an osmotic balance with the environment. In plants,glycine betaine is synthesized via the two pathway from choline, and the first step is catalyzed by choline monooxygenase (CMO).Our preliminary work showed that CMO gene promoter (pC: -267 ~ +1bp) is salt-induced promoter. Compared to noninductive leaves, the GUS enzyme activity driven by CMO promoter was enhanced 5-fold in transgenic tobacco leaves in the presence of 400mmol/L NaCl. In this article, the pCAMBIA1301-pC-CMO was constructed and transferred into tobacco. The expression of CMO gene in transgenic plants was analysed.
The 1.3 kb coding region of CMO gene was amplified from cloning vector pUC19-CMO by PCR. CMO gene was inserted into pCAMBIA1301-pC-GUS by replacing GUS gene, and the new expression vector, pCAMBIA1301-pC-CMO was obtained. Then pCAMBIA1301- pC-CMO was transferred into Agrobacterium tumefaciens strain LBA4404.
pCAMBIA1301-pC-CMO, pBI121-CMO(35S-CMO) were transferred into tobaccos (Nictiana tabacum L. cv. 89) via Agrobacterium mediation. PCR positive tobaccos were obtained. Semi-Quantitative RT-PCR detection showed that CMO gene has expressed in two types transgenic tobaccos. The expression of CMO gene in pC-CMO transgenic plants is lower than that in 35S-CMO transgenic plants. Betaine content analysis in the transgenic tobacco leaves showed that the betaine content in pC-CMO transgenic plants is less than that in 35S-CMO transgenic plants.
Under 100 mmol/L NaCl stress, Semi-Quantitative RT-PCR detection showed that the expression of CMO gene in plants was different. The expression of CMO gene in pC-CMO transgenic plants is higher than that in 35S-CMO transgenic plants. Further detection showed that the betaine content in pC-CMO transgenic plants is more than wild type and35S-CMO transgenic plants. The results showed that under salt stress the expression of CMO gene driven by CMO promoter increased in transgenic plants..
利用PCR技术从克隆载体pUC19-CMO中扩增出约1.3kb的CMO基因编码区,经过酶切、连接,替代表达载体pCAMBIA1301-pC-GUS中的GUS基因获得新的表达载体pCAMBIA1301-pC-CMO。
冻融法将pCAMBIA1301-pC-CMO导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,获得工程菌LBA4404- pCAMBIA1301-pC-CMO。
采用农杆菌介导的叶盘转化法将pCAMBIA1301-pC-CMO、pBI121-CMO (CaMV 35S启动子驱动CMO基因)转入烟草, PCR检测获得阳性植株。半定量RT-PCR检测表明,35S-CMO转基因烟草CMO基因的表达量高于pC-CMO转基因烟草;甜菜碱含量检测表明,pC-CMO转基因烟草甜菜碱含量与野生烟草的甜菜碱含量相近或略高于野生烟草的甜菜碱含量,35S-CMO转基因烟草中的甜菜碱含量高于pC-CMO转基因烟草。
100 mmol/L NaCl胁迫处理转基因烟草和野生烟草24 h后,pC-CMO转基因烟草中CMO基因表达量高于35S-CMO转基因烟草,与此相对应,pC-CMO转基因烟草中甜菜碱含量均提高,而野生型烟草和35S-CMO转基因烟草中的甜菜碱含量基本没有变化,且pC-CMO转基因烟草中甜菜碱含量均高于野生烟草、35S-CMO转基因烟草。说明pC可以驱动CMO基因在转基因烟草中适时表达,从而相应提高甜菜碱含量。
Betaine is a non-toxic osmopretectant which protects the cell from salt or drought stress by maintaining an osmotic balance with the environment. In plants,glycine betaine is synthesized via the two pathway from choline, and the first step is catalyzed by choline monooxygenase (CMO).Our preliminary work showed that CMO gene promoter (pC: -267 ~ +1bp) is salt-induced promoter. Compared to noninductive leaves, the GUS enzyme activity driven by CMO promoter was enhanced 5-fold in transgenic tobacco leaves in the presence of 400mmol/L NaCl. In this article, the pCAMBIA1301-pC-CMO was constructed and transferred into tobacco. The expression of CMO gene in transgenic plants was analysed.
The 1.3 kb coding region of CMO gene was amplified from cloning vector pUC19-CMO by PCR. CMO gene was inserted into pCAMBIA1301-pC-GUS by replacing GUS gene, and the new expression vector, pCAMBIA1301-pC-CMO was obtained. Then pCAMBIA1301- pC-CMO was transferred into Agrobacterium tumefaciens strain LBA4404.
pCAMBIA1301-pC-CMO, pBI121-CMO(35S-CMO) were transferred into tobaccos (Nictiana tabacum L. cv. 89) via Agrobacterium mediation. PCR positive tobaccos were obtained. Semi-Quantitative RT-PCR detection showed that CMO gene has expressed in two types transgenic tobaccos. The expression of CMO gene in pC-CMO transgenic plants is lower than that in 35S-CMO transgenic plants. Betaine content analysis in the transgenic tobacco leaves showed that the betaine content in pC-CMO transgenic plants is less than that in 35S-CMO transgenic plants.
Under 100 mmol/L NaCl stress, Semi-Quantitative RT-PCR detection showed that the expression of CMO gene in plants was different. The expression of CMO gene in pC-CMO transgenic plants is higher than that in 35S-CMO transgenic plants. Further detection showed that the betaine content in pC-CMO transgenic plants is more than wild type and35S-CMO transgenic plants. The results showed that under salt stress the expression of CMO gene driven by CMO promoter increased in transgenic plants..
引文
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[4] Zhu Q, Dabi T, Lamb C. TATA box and initiator functions in the accurate transcription of a plant minimal promoter invitro. Plat Cell, 1995 ,7 (10):16-19.
[5]李杰,张福城,王文泉,等.高等植物启动子的研究进展.生物技术通讯, 2006,17 (4) : 658-661.
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