摘要
以沙冬青(Ammopiptanthus mongolicus(Maxim.ex Kom.)Cheng f.)幼苗的子叶为材料,对其原生质体的分离、纯化和瞬时表达体系进行了研究。结果表明,子叶原生质体分离的最佳酶解液组成为CPW溶液+3.0%纤维素酶R-10+0.5%离析酶R-10+0.3%半纤维素酶+9.0%甘露醇(p H5.8);最佳酶解条件为室温、避光、40 r/min轻摇14 h。采用W5溶液作为漂洗液将酶解物稀释后进行过滤,将过滤液在4℃、700 r/min条件下离心5 min,所得纯化原生质体的产量约为2.50×106cells/g,活力达到90%;以纯化的原生质体作为受体,利用聚乙二醇(PEG)介导法成功将植物瞬时表达载体p BI-GFP导入其中,转化效率达到50.8%。利用本研究建立的原生质体瞬时表达体系,检测到沙冬青脱水应答转录因子Am DREB1定位于细胞核内。
Using young cotyledons of Ammopiptanthus mongolicus（Maxim. ex Kom.） Cheng f.seedlings as donor material,the technical system for the isolation,purification,and transient expression of the cotyledon protoplasts was studied. Results showed that the optimal enzyme solution for the protoplast isolation was CPW solution ＋ 3.0% cellulase R-10 ＋ 0.5%macerozyme R-10 ＋ 0.3% hemicellulose ＋ 9.0% mannitol（p H5.8）, and the optimal enzymolysis conditions were gentle shaking of the enzyme solution containing cotyledon tissues at 40 r/min in the dark for 14 h at room temperature. Using the W5 solution as a washing solution,the enzymatic hydrolysate was diluted and filtered,with the filtrate then centrifuged at 4℃ and 700 r/min for 5 min. The purified protoplast yield was approximately2.50 × 106 cells/g and the protoplast viability reached 90%. Using the PEG-mediated method,the protoplasts were successfully transformed with plant transient expression vector p BI-GFPand the transformation efficiency was 50.8%. Moreover, using the established protoplast transient expression system,a dehydration-responsive transcription factor of A. mongolicus,namely Am DREB1,was found in the nucleus.
作者
楠迪娜
薛敏
唐宽刚
任美艳
王茅雁
Nan Di-Na;Xue Min;Tang Kuan-Gang;Ren Mei-Yan;Wang Mao-Yan(College of Life Sciences,Inner Mongolia Agricultural University,Hohhot 010018,China)
出处
《植物科学学报》
CAS
CSCD
北大核心
2018年第4期562-568,共7页
Plant Science Journal
基金
国家自然科学基金项目(31260256
31560299)
内蒙古自然科学基金重大项目(2012ZD02)~~
关键词
沙冬青
原生质体
瞬时表达
DREB转录因子
亚细胞定位
Ammopiptanthus mongolicus
Protoplast
Transient expression
DREB transcriptionfactor
Subcellular localization
