超声波辅助农杆菌转化OT百合‘罗宾娜’的研究  

Sonication-assisted Agrobacterium-mediated transformation of OT (Lilium tenuifolium oriental×tumpet) lily ‘Robina’

作　　者：王岚[1] 刘雅莉[1] 娄倩[2] 田菲菲[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100 [2]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2013年第6期161-166,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目"百合花色形成关键基因(CHS)特异启动子功能与应用研究"(30871734)

摘　　要：【目的】通过超声波处理辅助农杆菌转化OT百合小鳞片,提高农杆菌介导法转化百合的效率。【方法】以OT百合‘罗宾娜’(Lilium tenuifoliumoriental×tumpet‘Robina’)的再生小鳞片为外植体,分别以80W功率的超声波处理1,2,3,4,5min,通过超声波处理辅助根癌农杆菌进行外源基因的转化,同时以未进行超声波处理为对照;利用GUS组织化学法,分别检测转化脱菌后筛选培养7d的外植体和筛选培养60d后形成的潮霉素抗性再生植株叶片、小鳞片、根系的GUS瞬时表达和稳定表达,提取潮霉素抗性植株基因组DNA进行GUS和HPT基因的PCR扩增分析。【结果】超声波处理的农杆菌介导转化的百合外植体经GUS组织化学染色,多数可见蓝色斑点,且以3min超声波处理的蓝色最深;3min超声波处理的外植体对潮霉素的抗性率最高达48.85%,未经超声波处理的仅为15.56%,二者差异达显著水平。从53个潮霉素抗性再生植株的DNA样品中扩增得到了GUS基因和HPT基因的扩增片段,其中超声波处理3min的PCR阳性植株转化率最高,为28.30%,而未进行超声波处理的PCR阳性植株转化率仅为5.66%,初步证明外源基因可能整合到百合基因组中。【结论】20℃条件下80W功率超声波处理3min,可以有效提高农杆菌介导法转化OT百合的效率。【Objective】 The aim of our study was to enhance the Agrobacterium-mediated transformation efficiency of OT lily ＇Robina＇ and to produce large populations of transformants.【Method】 In this study,the regenerated small scales of OT lily ＇Robina＇ were used for Agrobacterium-mediated transformation 1,2,3,4 and 5 min of sonication treatments were compared with conventional Agrobacterium-mediated inoculation method.The GUS histochemical assay was performed to estimate the transient expression of the GUS gene in the transformed explants and the stable expression in the transformed plantlets.PCR analysis of GUS gene and HPT gene were tested for stable transformation efficiency.【Result】 For the histochemical GUS assay,blue foci were found on most of the transformed bulb scale explants treated with sonication before Agrobacterium infection.Moreover,explants treated with 3 min sonication displayed a substantial number of blue foci on the explant surface.The highest hygromycin-resistant frequency 48.85% was obtained from 3 min sonication treatment and the lowest 15.56%was transformed explants without sonication.In total,the expected GUS and HPT gene fragments of 557 and 476 bp,respectively,were amplified from DNA samples of fifty-three transgenic plantlets and the highest transfromation efficiency of PCR positive plants,28.30%,was obtained from 3 min sonication treatment and the lowest,5.66%,was obtained without sonication.It preliminarily proved the integration of the foreign genes to lily genome.【Conclusion】 It could highly enhance the Agrobacterium-mediated transformation efficiency of OT lily ＇Robina＇ when the explants were treated with 3 min sonication at 20 ℃ and 80 W.

关 键 词：超声波 百合 农杆菌 转基因 

分 类 号：S682.265.032[农业科学—观赏园艺] Q785[农业科学—园艺学]