CAJ | 학술논문

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%.将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HindⅢ与Eco RⅠ之间,从而获得高效植物表达载体.将RokⅡ-GDH质粒转移到根癌土壤杆菌(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105中,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PPT的MS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;在MS培养基中用0.5 μg/mL 的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.
용RT-PCR방법종소구조(Chlorella sorokiniana)중극륭료안유도표체적이보매Ⅱ위보기적곡안산탈경매(NADP-GDH)기인적cDNA편단,DNA측서분석표명여이보도적해기인c DNA서렬동원성위94%.장NADP-GDH기인선삽입도SPDK621질립적2CaMV35S계동자화Ω증강서렬지후,연후장2CaMV35S-Ω-GDH-NOS표체단원구건도RokⅡ질립적HindⅢ여Eco RⅠ지간,종이획득고효식물표체재체.장RokⅡ-GDH질립전이도근암토양간균(Agro bacterium tumefaciens (Smith et Townsend) Conn) EHA105중,대연초(Nico tiana tabacum L.)진행전화병득도양성전화후대.대전기인연초분석표명,재저담배양기혹재저담질석중기생장속도화협편수명현고우대조;안독성실험표명,무론재저안혹고안조건하,접충재MS고화배양기상적전기인록협원편존활시간장,협록소함량고.저사결과설명외원NADP-GDH증강료식물대담소적흡수화이용.령외,전화후대환표현료대제초제련화맥황동(PPT)구유교강적항성;배양재함유불동농도PPT적MS고화배양기상적전기인록협원편,기유상화정도명현고우대조;재MS배양기중용0.5 μg/mL 적PPT가이대체잡나매소대전화후대진행사선,저암시 NADP-GDH기인가이작위일충신적선택표기용우식물기인공정적연구.
A full-length cDNA has been cloned encoding nicotinamide adenine dinucleotide ph osphate-specific glutamate dehydrogenase (NADP-GDH) from Chlorella sorokiniana with the RT-PCR method. The complete nucleotide sequence of NADP-GDH gen e had 94% homology to the previously reported one . The NADP-GDH gene was co nst ructed into a vector highly expressed in plants. The specific activity of NADP-G DH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transfor med seedlings grown in lower concentration of nitrogen vermiculite had higher gr owth rate and more leaves than the control plants. Transformed leaf discs cultur ed on MS medium containing different nitrogen concentrations had more chlorophyl l contents compared to the controls. These results suggested that exogenous NADP -GDH may enhance the absorption and utilization to ammonium in plants. The incre ase d weight of transformed leaf discs cultured on medium supplemented with differen t 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 be used as a new selecting gene in the future research of plant gene engineering.