AtMET1基因在84K杨中的遗传转化及诱导表达分析

Genetic Transformation of AtMET1 in Populus alba×P.glandulosa‘84K’and Its Chemical-inducible Expression Analysis

[目的]研究拟南芥甲基化转移酶基因AtMET 1在银腺杨84K(Populus alba×P. glandulosa ‘84K’)中的诱导表达特性,为建立杨树甲基化诱导变异体系、进而实现杨树品种改良等奠定基础。[方法]以白杨派优良品种84K杨的无菌苗叶片为受体材料,采用农杆菌介导法将化学诱导启动子与AtMET1基因导入84K杨基因组中;经潮霉素筛选获得抗性植株,通过常规PCR检测、DNA测序等方法对抗性植株进行鉴定。通过化学诱导剂17-β-雌二醇对随机挑选的1个转基因株系离体叶片进行诱导处理,处理时间为0、3、6、12、24、48、96、144 h,采用实时荧光定量PCR(qRT-PCR)检测外源基因AtMET1表达量变化。[结果]本研究共获得了潮霉素抗性芽648个,经生根筛选获得18株抗性植株,经分子检测证实全部为转基因植株,分别标号为AM-1~18#。qRT-PCR结果显示:化学诱导剂17-β-雌二醇处理3 h时,目的基因AtMET1的表达量即达到最大值,随后在处理6 h时表达量下降,处理12 h时有所回升,处理24 h后表达量降低至不足12 h时的一半。[结论]化学诱导剂17-β-雌二醇能迅速有效地调控转基因杨树中AtMET1基因的表达,为进一步研究MET1基因在杨树基因组甲基化调控方面的作用机制奠定基础,也为今后杨树化学诱导表达研究及品种改良提供新思路、新方法。

[Objective]To study the chemically-induced expression characteristics of Arabidopsis thaliana methyltransferase gene AtMET1 in Populus alba×P.glandulosa'84K',to lay a foundation for the establishment of the poplar methylation-induced variation system and genetic improvement of poplars.[Method]The chemically-induced promoter and AtMET1 were transformed into genome of P.alba×P.glandulosa'84K'using Agrobacterium-mediated transformation.The hygromycin resistant plants were obtained and transgenic plants were identified as by traditional PCR and DNA sequencing.The chemical inducer 17-β-estradiol was used to induce expression of AtMET1 in leaves of in vitro leaves of a transgenic line for 0,3,6,12,24,48,96 and 144 h,and the expression of AtMET 1 gene was detected by Quantitative Real-time PCR(qRT-PCR).[Result]A total of 648 hygromycin resistant buds were obtained,among them 18 hygromycin resistant plants were screened,and all of them were identified to be transgenic plants by molecular detection method.qRT-PCR showed that the expression of AtMET1 reached its highest at 3 h by17-β-estradiol treatment,then decreased at 6 h,increased at 12 h and decreased again to less than half of the expression at 12 h.[Conclusion]The chemical inducer can efficiently and rapidly induce the expression of AtMET1 in transgenic poplar plant,that laid a solid foundation for the regulation mechanism of MET1 in poplars and provided new idea and method for chemical induction of genes and genetic improvement of poplars.