التفاصيل البيبلوغرافية
| العنوان: |
菠萝叶基原生质体分离与瞬时转化体系的建立. (Chinese) |
| Alternate Title: |
Establishment of an isolation and transient transformation system for leaf base protoplast in pineapple (Ananas comosus). (English) |
| المؤلفون: |
贺涵, 阳习鹏, 赵婉欣, 邵雪花, 杨凤玺, 刘传和 |
| المصدر: |
Journal of Northwest A & F University - Natural Science Edition; 2024, Vol. 52 Issue 7, p81-98, 9p |
| Abstract (English): |
[Objective] This study investigated efficient methods for separation and transient transformation of pineapple leaf base protoplasts to provide technical support for pineapple gene function analysis. [Method] Young leaf bases from 'Comtede Paris' cultivar were used for a three-factor and three-level orthogonal test to analyze the effects of cellulase concentration (6,12 and 20 g/L), macerozyme concentration (3,6 and 9 g/L) and mannitol concentration (0.4,0.5 and 0.6 mol/L) on protoplast yields and vitalities. A single-factor test of digestion time (2,4 and 6 h) was also used to determine the optimal digestion time for protoplast isolation. Protoplasts were prepared under optimal conditions and transformed with plasmids using the polyethylene glycol (PEG) mediated transformation method to establish an efficient protoplast transformation system in pineapple. [Result] The orthogonal test revealed that the protoplast yield of 'Comtede Paris' leaf bases ranged from 1.01×106 g-1 to 1.93×106 g-1 under different treatments, and cellulase concentration was the primary factor influencing protoplast yield. Protoplast vitality ranged from 65.55% to 87.00%, and mannitol concentration was the most influential factor. Optimal conditions for pineapple leaf base protoplast isolation were determined as 12 g/L cellulase, 6 g/L macerozyme and 0.6 mol/L mannitol, resulting in 1.96×106 g-1 yield of protoplasts with 85.54% vitality after 4 hours of digestion in dark. The obtained protoplasts were successfully transformed with the pAN580-WRKY75 plasmid, and green fluorescence signal was observed in the nucleus of transformed protoplasts under a confocal microscope. [Conclusion] This study determined the optimal conditions for isolating and transforming pineapple leaf base protoplasts and established a transient expression system for pineapple gene function analysis. [ABSTRACT FROM AUTHOR] |
| Abstract (Chinese): |
[目的] 研究菠萝叶基原生质体的高效分离方法,并对制备的原生质体进行瞬时转化,为利用菠萝原 生质体进行菠萝基因功能研究提供技术支持。[方法] 以'巴厘'菠萝(Ananas comosus,'Comtede Paris')幼嫩叶片的 叶基为材料,采用3因素3水平正交试验L9(33)分析纤维素酶质量浓度(6,12,20 g/L)、离析酶质量浓度(3,6,9 g/L)和甘露醇浓度(0.4,0.5,0.6 mol/L)对分离原生质体产量与活力的影响;同时,利用单因素试验(设置酶解时间 2,4,6 h)筛选制备原生质体的最适酶解时间,确定分离菠萝叶基原生质体的最适条件;在最适条件下制备原生质体, 采用聚乙二醇(PEG)介导法转化质粒,建立菠萝原生质体瞬时转化体系。[结果] L9(33)正交试验结果显示,不同处 理分离的原生质体产量为1.01×106~1.93×106 g-1,纤维素酶质量浓度是决定原生质体产量的关键因素;原生质体 活力为65.55%~87.00%,甘露醇浓度对原生质体活力的贡献最大。最适酶解条件为:菠萝叶基在含12 g/L纤维素酶、 6 g/L离析酶、0.6 mol/L甘露醇的酶液中避光酶解4 h,产量可达1.96×106 g-1,活力为85.54%。菠萝叶基原生质体 通过PEG介导法转化pAN580-WRKY75质粒后,可在激光共聚焦显微镜下观察到核内的绿色荧光信号。[结论] 确定 了菠萝叶基原生质体的最适分离条件与瞬时转化方法,建立了适用于菠萝基因功能分析的原生质体瞬时表达体系。. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
Complementary Index |