J Integr Plant Biol. ›› 2002, Vol. 44 ›› Issue (3): 318-324.

• Research Articles • Previous Articles     Next Articles

Cloning of Glutamate Dehydrogenase cDNA from Chlorella sorokiniana and Analysis of Transgenic Tobacco Plants

HUANG Guo-Cun   

Abstract:

A full length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate specific glutamate dehydrogenase (NADP-GDH) from Chlorella sorokiniana with the RT-PCR method. The complete nucleotide sequence of NADP-GDHgene had 94% homology to the previously reported one . The NADP-GDHgene was constructed into a vector highly expressed in plants. The specific activity of NADP-GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP-GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs.0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP-GDH gene might besed as a new selecting gene in the future research of plant gene engineering.

小球藻NADP-谷氨酸脱氢酶的cDNA克隆及转基因烟草分析

黄国存  孟颂东  王荣  杨怀义  田波*

(中国科学院微生物研究所,北京,100080)

摘要:用RT-PCR方法从小球藻(Chlorella sorokiniana)中克隆了铵诱导表达的以辅酶Ⅱ为辅基的谷氨酸脱氢酶(NADP-GDH)基因的cDNA片段,DNA测序分析表明与已报道的该基因c DNA序列同源性为94%.NADP-GDH基因先插入到SPDK621质粒的2CaMV35S启动子和Ω增强序列之后,然后将2CaMV35S-Ω-GDH-NOS表达单元构建到RokⅡ质粒的HindEco RⅠ之间,从而获得高效植物表达载体.Rok-GDH质粒转移到根癌土壤杆菌(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105,对烟草(Nico tiana tabacum L.)进行转化并得到阳性转化后代.对转基因烟草分析表明,在低氮培养基或在低氮蛭石中其生长速度和叶片数明显高于对照;铵毒性实验表明,无论在低铵或高铵条件下,接种在MS固化培养基上的转基因绿叶圆片存活时间长,叶绿素含量高.这些结果说明外源NADP-GDH增强了植物对氮素的吸收和利用.另外,转化后代还表现了对除草剂膦化麦黄酮(PPT)具有较强的抗性;培养在含有不同浓度PPTMS固化培养基上的转基因绿叶圆片,其愈伤化程度明显高于对照;MS培养基中用0.5 μg/mL PPT可以代替卡那霉素对转化后代进行筛选,这暗示 NADP-GDH基因可以作为一种新的选择标记用于植物基因工程的研究.

关键词 谷氨酸脱氢酶 转基因烟草 氮素吸收 膦化 麦黄酮抗性

*通讯作者

 

Key words: nicotinamide adenine dinucleotide phosphate specific glutamate dehydrogenase(NADP-GDH), transgenic tobacco, ammonium absorption, phosphinothricin resistance

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