高级检索+

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

  • 摘要:
    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。

     

    Abstract:
    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.

     

/

返回文章
返回