丹波黑大豆GmDREPP基因的克隆与耐铝性

Cloning of GmDREPP gene in Tamba Black soybean and its effect to aluminum resistance

发育调节质膜多肽(developmentally-regulated plasma membrane polypeptide,DREPP)是一类植物特异性蛋白,参与植物对生物和非生物胁迫的响应与调控。本研究以耐铝丹波黑大豆(Glycine max cv.Tamba)为试验材料,克隆获得编码丹波黑大豆GmDREPP蛋白基因的CDS序列,构建组成型过表达载体pCXSN-GmDREPP,成功遗传转化烟草(Nicotiana tabacum),并运用qRT-PCR定量技术研究GmDREPP基因在丹波黑大豆中的表达情况。结果表明,铝胁迫下GmDREPP在丹波黑大豆的根、茎、叶、子叶中均有表达,根中又以根尖的表达量最高;GmDREPP的根尖表达水平随Al3+浓度增加而上升,Al3+浓度高于50μmol·L-1后其表达量无显著变化。对转基因烟草阳性植株进行RT-PCR分析,选取3个表达水平较高的转基因株系(GmDREPP-2、GmDREPP-4、GmDREPP-5)进行耐铝性分析,结果表明:(1)50μmol·L-1 Al3+处理24 h后转基因烟草根的相对伸长量、NtALS3和NtMATE的表达量与柠檬酸分泌量均极显著(P<0.01)高于野生型;(2)与野生型相比,转基因植株根系过氧化物酶(peroxidase,POD)和超氧化物歧化酶(superoxide dismutase,SOD)活性极显著(P<0.01)升高,而丙二醛(malonaldehyde,MDA)含量较野生型极显著(P<0.01)降低;(3)伊文思蓝和苏木精染色结果显示,转基因烟草根尖颜色较野生型浅,说明受铝毒害减轻。通过在烟草中过表达GmDREPP基因的研究,证明该基因具有增强耐铝性的功能,可以为培育耐铝新品种提供基因资源。

Developmentally-regulated plasma membrane polypeptide(DREPP),acting as a kind of plant-specific protein,is involved in the response and regulation of plants to biologic and abiotic stresses.CDS sequence encoding the GmDREPP protein was cloned from an aluminum-resistance cultivar Glycine max cv.Tamba,and pCXSN-GmDREPP,the overexpression vector,was constructed and transformed into wild type tobaccos via transformation mediated by agrobacterium to obtain transgenic tobaccos.The expression of GmDREPP in Tamba Black soybean was analyzed by qRT-PCR,and the results showed that GmDREPP expressed in roots,stems,leaves and cotyledons,while the expression level in root tips was the highest in root system.Besides,the mRNA level of GmDREPP increased with the increase of the Al3+concentration,and had no significant changes when Al3+concentration was higher than 50μmol·L-1.To investigate the aluminum resistance,the transgenic tobacco lines(GmDREPP-2,GmDREPP-4,GmDREPP-5)selected by RT-PCR were challenged by Al(pH 4.5,0.5 mmol·L-1 CaCl2,50μmol·L-1 AlCl3)compared with wild type(WT).The results were as following:(1)Root elongation,citrate content and the expression of NtALS3 and NtMATE were significantly(P<0.01)increased in transgenic tobacco lines compared with WT.(B)By contrast,POD and SOD activities significantly(P<0.01)increased and MDA content significantly(P<0.01)decreased in roots of transgenic tobacco lines.(C)Root tips stains using Evans blue and hematoxylin manifested transgenic tobaccos were lighter than WT,indicating a reduction in aluminum toxicity.This research verified that over-expression of GmDREPP could enhance the resistance of the transgenic tobaccos to aluminum and provide new genetic resources to breed new varieties with aluminum resistance.