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Transient Transformation of Artemisia annua
青蒿素的瞬时转化   

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Abstract

Transient transformation of Artemisia annua does not depend on chromosomal integration of heterologous DNA, and recombinant DNA can be introduced into plant cells via Agrobacterium aided by vacuum. The leaves of 7th and 8th internode from 4-week-old seedlings were chosen as explants, a vacuum system was applied to facilitate agrobacteria into plant cells, the co-cultivation was in the dark at 25 °C for 36-72 h, then GUS or GFP maker genes were used for testing the efficiency of the transformation. The method is used for quick transferring of genes into Artemisia annua (A. annua) by transient transformation.

Keywords: Artemisia annua(阿尔泰米夏黄花), Agrobacterium tumefaciens(农杆菌介导法), Transient transformation(瞬时转化)

Materials and Reagents

  1. Artemisia annua leaves from 4-week-old aseptic seedlings
  2. Agrobacterium tumefaciens (A. tumefaciens) strain EHA105 (or LBA4404)
  3. Lysogeny broth (LB) medium (pH 7.0, containing 50 mg/L rifampicin and 50 mg/L kanamycin)
  4. Murashige and Skoog medium (MS) liquid medium (Sigma-Aldrich, catalog number: M5524 )
  5. 100 μM acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  6. 10 mM MgCl2
  7. 0.005% Silwet L-77 (Agri-Turf Supplies, catalog number: VIS-01 )
  8. Glycerol
  9. Filter paper (Lab Depot, catalog number: TLDCFP1-032 )
  10. Yeast extract broth (YEB) medium (see Recipes)

Equipment

  1. Vacuum pump (Barnant, model: 400-1903 )
  2. Desiccator (Ted Pella, catalog number: 2246 )
  3. Erlenmeyer flasks (VWR International, catalog number: 89095-266 )

Procedure

  1. Glycerol stocks of A. tumefaciens strain EHA105 (or LBA4404) containing plant expression vector construct were thawed and then streaked onto solid LB medium for 48 h (pH 7.0, containing 50 mg/L rifampicin and vector-corresponding antibiotics such as 50 mg/L kanamycin), then individual single colonies were inoculated into 5 ml of liquid YEB medium containing 50 mg/L rifampicin and 50 mg/L kanamycin.
  2. These cultures were grown overnight with shaking at 28 °C/200 rpm. The subsequent 100 ml cultures were inoculated with 1 ml of the preceding 5 ml overnight (10-12 h) cultures, and again incubated with shaking at 28 °C/200 rpm until OD600 = 0.5.
  3. Bacterial cells were harvested by centrifugation at 6,000 x g at 4 °C for 5 min and washed once with 10 ml of washing solution containing 10 mM MgCl2 and 100 μM acetosyringone. After centrifugation at 6,000 x g at 4 °C for another 5 min, the pellet of bacterial cells was resuspended in 50 ml of liquid MS medium.
  4. Seed sterilization, germination, and growth. Artemisia annua seeds were treated for 1 min in 1 ml of 70% ethanol in a sterile Eppendorf tube. The tube was vortexed and ethanol was then removed. Seeds were subsequently washed four times using aseptic H2O. These surface-sterilized seeds were then treated for 10 min using 10% clorox in a sterile Eppendorf tube, during which the tube was vortexed at least 5 times. Then clorox was removed, seeds were washed four times using aseptic H2O. Sterilized seeds were placed on phytoagar-solidified MS medium, then placed in an incubator with necessary photoperiod and temperature.
  5. Artemisia annua aseptic seedlings were sub-cultured, after 4-week-growth (Figure 1), the leaves of 7th and 8th internode were chosen and petioles of leaves far from stem were cut before soaked with 50 ml of MS liquid medium containing A. tumefaciens cells supplemented with 0.005% (v/v; i.e. 50 μl/L) surfactant Silwet L-77 in 100 ml sterilized Erlenmeyer flasks, then the Erlenmeyer flasks, covered with a filter to prevent contamination, were placed in the desiccator and a vacuum was applied at 30 inHg for 10 min under the control of a vacuum pump. More than 30 leaves were cut and vacuumed at the same time. The vacuum pump was then switched off and air was slowly let into the desiccator, so as to minimize the damage to the tissues.


    Figure 1. Artemisia annua leaves from 4-week-old aseptic seedlings were used for transient expression, the leaves of 7th and 8th internode were chosen and petiole of leaves far from stem were cut before they were used for transient transformation

  6. Leaves were washed three times by aseptic H2O, then liquid medium was removed using aseptic filter paper, and the explants were plated on MS solid medium for aseptic culture. Usually, when target gene(s) were fused with GUS or GFP maker (Figure 2), the co-cultivation time depends on the target genes expression efficiency, so the co-cultivation was in the dark at 25 °C for 36-72 h.


    Figure 2. GUS staining was done using Artemisia annua leaves through transient transformation

Recipes

  1. Yeast extract broth (YEB) medium
    5 g/L beef extract
    1 g/L yeast extract
    5 g/L peptone
    5 g/L sucrose
    0.5 g/L MgCl2

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 61173098, No. 61379081).

References

  1. Han, J., Wang, H., Ye, H., Liu, Y., Li, Z., Zhang, Y., Zhang, Y., Yan, F. and Li, G. (2005). High efficiency of genetic transformation and regeneration of Artemisia annua L. via Agrobacterium tumefaciens-mediated procedure. Plant Sci 168(1): 73-78.
  2. Ji, Y., Xiao, J., Shen, Y., Ma, D., Li, Z., Pu, G., Li, X., Huang, L., Liu, B., Ye, H. and Wang, H. (2014). Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua. Plant Cell Physiol 55(9): 1592-1604.
  3. Ma, D., Pu, G., Lei, C., Ma, L., Wang, H., Guo, Y., Chen, J., Du, Z., Wang, H., Li, G., Ye, H. and Liu, B. (2009). Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant Cell Physiol 50(12): 2146-2161.

简介

青蒿的瞬时转化不依赖于异源DNA的染色体整合,并且重组DNA可以通过真空辅助土壤杆菌引入植物细胞中。 选择来自4周龄幼苗的第7和第8个节间的叶作为外植体,应用真空系统以将农杆菌促进植物细胞中,共培养在25℃下黑暗中36-72小时,然后 或 GFP 制造商基因用于测试转化的效率。 该方法用于通过瞬时转化将基因快速转移到青蒿(ememansia annua)( A。annua )中。

关键字:阿尔泰米夏黄花, 农杆菌介导法, 瞬时转化

材料和试剂

  1. 青蒿从4周龄无菌幼苗叶子中分离
  2. 根瘤土壤杆菌(根瘤土壤杆菌)菌株EHA105(或LBA4404)
  3. 溶菌酶培养基(LB)培养基(pH7.0,含有50mg/L利福平和50mg/L卡那霉素)
  4. Murashige和Skoog培养基(MS)液体培养基(Sigma-Aldrich,目录号:M5524)
  5. 100μM乙酰丁香酮(Sigma-Aldrich,目录号:D134406)
  6. 10mM MgCl 2/
  7. 0.005%Silwet L-77(Agri-Turf Supplies,目录号:VIS-01)
  8. 甘油
  9. 滤纸(Lab Depot,目录号:TLDCFP1-032)
  10. 酵母提取液(YEB)培养基(见配方)

设备

  1. 真空泵(Barnant,型号:400-1903)
  2. 干燥器(Ted Pella,目录号:2246)
  3. 锥形瓶(VWR International,目录号:89095-266)

程序

  1. 甘油储备液。 将含有植物表达载体构建体的根瘤土壤杆菌菌株EHA105(或LBA4404)解冻,然后在固体LB培养基上划线48小时(pH 7.0,含有50mg/L利福平和载体相应的抗生素例如50mg/L卡那霉素 ),然后将单个单菌落接种到5ml含有50mg/L利福平和50mg/L卡那霉素的液体YEB培养基中。
  2. 将这些培养物在28℃/200rpm振荡下生长过夜。随后的100ml培养物用1ml前面的5ml过夜(10-12h)培养物接种,并再次在28℃/200rpm下摇动孵育直到OD 600 = 0.5。
  3. 通过在4℃下以6000xg离心5分钟收获细菌细胞,并用10ml含有10mM MgCl 2和100μM乙酰丁香酮的洗涤溶液洗涤一次。在4℃下以6,000×g离心另外5分钟后,将细菌细胞沉淀重悬浮于50ml液体MS培养基中。
  4. 种子灭菌,发芽和生长。将青蒿种子在无菌Eppendorf管中在1ml 70%乙醇中处理1分钟。涡旋该管,然后除去乙醇。随后将种子用无菌H 2 O洗涤四次。然后将这些表面灭菌的种子在无菌Eppendorf管中使用10%clorox处理10分钟,在此期间将管涡旋至少5次。然后除去Clorox,用无菌H 2 O洗涤种子四次。将灭菌的种子放在植物琼脂固化的MS培养基上,然后置于具有必要光周期和温度的培养箱中。
  5. 在培养4周后(图1),选择第7和第8个节间的叶,并且将远离茎的叶的叶柄切开,然后用50ml的枸杞子浸泡的含有A的MS液体介质。在100ml无菌锥形瓶中用补充有0.005%(v/v;即50μl/L)表面活性剂Silwet L-77的根瘤细胞接种锥形瓶,然后用锥形瓶防止污染,放置在干燥器中,并在真空泵的控制下在30英寸Hg下真空10分钟。同时切下和抽真空30多片叶子。然后关闭真空泵,并且将空气缓慢地放入干燥器中,以使对组织的损害最小化。


    图1. 艾蒿青蒿 4周龄无菌幼苗的叶片用于瞬时表达,7号和8号选择节间,并且在它们用于瞬时转化
    之前切割远离茎的叶的叶柄
  6. 将叶子用无菌H 2 O洗涤三次,然后使用无菌滤纸除去液体培养基,将外植体铺在MS固体培养基上用于无菌培养。通常,当靶基因与GUS 或 GFP 标签融合时(图2),共培养时间取决于靶基因表达效率, - 在25℃黑暗中培养36-72小时。


    图2.通过瞬时转化使用青蒿叶进行GUS染色

食谱

  1. 酵母提取物肉汤(YEB)培养基
    5g/L牛肉提取物
    1g/L酵母提取物
    5 g/L蛋白胨
    5g/L蔗糖 0.5g/L MgCl 2·h/v

致谢

这项工作得到了中国国家自然科学基金(61173098,No. 61379081)的支持。

参考文献

  1. Han,J.,Wang,H.,Ye,H.,Liu,Y.,Li,Z.,Zhang,Y.,Zhang,Y.,Yan,F.and Li, 遗传效率高 通过根癌农杆菌介导的方法转化和再生青蒿。植物科学168(1):73-78 。
  2. Ji,Y.,Xiao,J.,Shen,Y.,Ma,D.,Li,Z.,Pu,G.,Li,X.,Huang,L.,Liu, Wang,H。(2014)。 克隆和表征AabHLH1,一种正调节青蒿中青蒿素生物合成的bHLH转录因子。植物细胞生理学 55(9):1592-1604。
  3. Ma,D.,Pu,G.,Lei,C.,Ma,L.,Wang,H.,Guo,Y.,Chen,J.,Du,Z.,Wang,H.,Li, Ye,H。和Liu,B。(2009)。 AaWRKY1的隔离和表征,AaWRKY1是一种调节青蒿素的转录因子紫穗槐-4,11-二烯合酶基因,青蒿素生物合成的关键基因。植物细胞生理学50(12):2146-2161。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ma, D. and Wang, H. (2015). Transient Transformation of Artemisia annua. Bio-protocol 5(10): e1476. DOI: 10.21769/BioProtoc.1476.
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