Rd29A启动子驱动AmDHN基因转化紫花苜蓿

Transformation of AmDHN Gene Drived by Rd29A Promoter into Alfalfa(Medicago sativa L.)

为获得抗逆性强、生长正常的转基因紫花苜蓿植株,本研究以野生型拟南芥(Arabidopsis thaliana)和蒙古沙冬青(Ammppiptanthus mongolicus)为材料,利用PCR和DNA重组技术,克隆了拟南芥Rd29A基因ATG上游共1520 bp的启动子区域;克隆了蒙古沙冬青脱水素基因(AmDHN)共572 bp的开放阅读框,构建了Rd29A启动子驱动AmDHN基因表达的植物表达载体pCHF-RD-DHN。以紫花苜蓿(Medicago satiua)品种‘敖汉苜蓿’的下胚轴和子叶为受体材料,利用农杆菌介导法,将构建的pCHF-RD-DHN表达载体转入‘敖汉苜蓿’中,经筛选获得了抗性再生植株。通过PCR检测显示,Rd29A启动子驱动的AmDHN基因已转入‘敖汉苜蓿’的基因组中。利用20%PEG模拟干旱胁迫后,经RT-PCR分析证实,当用20%的PEG胁迫转基因苜蓿植株时,转基因苜蓿株系中AmDHN基因表达量明显增强,而在水处理的条件下,转基因植株中AmDHN基因微量表达。本研究结果将为逆境诱导型启动子驱动抗逆基因在牧草中的表达研究及其遗传改良提供依据。

In order to ob tain normal growth and resistant drought transgenic alfalfa(Medicago satiua)using Arabidopsis thaliana and Ammppiptanthus mongolicus as material,DNA sequence of AtRd29 A gene ATG upstream 1520 bp is cloned with PCR and recombinant DNA technologies.Open reading frame of 572 bp of AmDHN gene was cloned by PCR.We construct the plant expression vector of AmDHN gene drived by Rd29 A promoter.The cotyledon and hypocotyl of alfalfa variety’Aohanmuxu’was acted as the receptor material and the vector pCHF-RD-DHN was transformed into alfalfa by Agrobacterium-mediated method.After callus selection and regeneration,regenerated plants with resistance were obtained successfully.The PCR analysis proved that the AmDHN gene has been transformed into the genome of the alfalfa.After drought stress using 20%PEG,RT-PCR analysis proved that the alfalfa plants with 20%PEG stress,AmDHN gene expression in transgenic alfalfa strain quantity obviously increased,and under the condition of water treatment,AmDHN gene was microexpressed in transgenic plants.This study will provide a basis for the expression of resistant genes driven by stress-inducible promoters and genetic improvement for forage grass.