利用绿色荧光蛋白优化辣椒遗传转化体系

Optimization of Genetic Transformation System of Peppers by Green Fluorescent Protein

【目的】农杆菌介导的辣椒遗传转化较为困难,至今仍未建立高效转化体系。绿色荧光蛋白(GFP)基因是植物遗传转化中常用的报告基因,以GFP为报告基因,优化农杆菌介导的辣椒遗传转化体系。【方法】利用GFP表达系统,统计3个辣椒品种(‘HP’‘8214’和‘L55’)中3种不同外植体(子叶、下胚轴和Flamingo-bill外植体)的不定芽分化率、不定根分化率与荧光阳性率,探究农杆菌侵染浓度、侵染时间、预培养时间和共培养时间等因素对不定芽、不定根分化率及荧光阳性率的影响。【结果】3个辣椒品种的Flamingo-bill外植体不定芽分化率均显著高于下胚轴与子叶外植体,其中‘L55’Flamingo-bill外植体的不定芽分化率最高、达77.59%,故选用‘L55’Flamingo-bill外植体进行后续研究。4种农杆菌不同侵染浓度和时间组合下,‘L55’Flamingo-bill外植体均可产生不定芽、不定根及表达GFP的愈伤组织,当农杆菌侵染浓度为OD_(600)=0.05,侵染时间为30 min时,不定芽分化率和荧光阳性率最高,分别达48.39%、4.84%。在6种不同预培养与共培养时间组合处理下,Flamingo-bill外植体也均产生不定芽和不定根,预培养1 d、共培养1~2 d处理下的不定芽分化率和荧光阳性率最高,分别达48.44%、12.50%。最后对表达GFP荧光的不定根与愈伤组织进行PCR检测,结果显示GFP、Kan和Cas9基因在荧光阳性的组织中均被检测到,表明农杆菌介导的T-DNA插入成功且转化稳定。【结论】辣椒的外植体类型、农杆菌侵染浓度、侵染时间、预培养和共培养时间均对辣椒遗传转化效率有影响。以GFP为报告基因,筛选合适的转化条件可提高农杆菌介导的辣椒遗传转化效率。

【Objective】The genetic transformation of pepper mediated by Agrobacterium was a very difficult process,and no efficient transformation system had been established so far.Green fluorescent protein(GFP) gene is a common reporter gene in plant genetic transformation.In the study,GFP was used as the reporter gene to optimize the Agrobacterium-mediated genetic transformation system of pepper.【Method】By using GFP expression system,three types of explants(cotyledons,hypocotyls and Flamingo-bill explants) from 3 pepper varieties('HP' '8214' and 'L55') were counted according to the adventitious bud differentiation rate,adventitious root differentiation rate and fluorescence positive rate.The effects of factors including infection concentration and infection time,pre-culture time and co-culture time on adventitious bud differentiation rate,adventitious root differentiation rate and fluorescence positive rate were explored.【Result】The adventitious bud differentiation rate of Flamingo-bill explants was significantly higher than that of the hypocotyl and cotyledon explants,among which the 'L55' Flamingo-bill explant had the highest adventitious bud differentiation rate of 77.59%.Therefore,the 'L55'Flamingo-bill explant was selected for subsequent research.Under 4 different combinations of Agrobacterium infection concentration and infection time,all of them could produce adventitious buds,adventitious roots and GFP-expressing calluses.When the infection concentration of Agrobacterium was OD_(600)=0.05 and the infection time was 30 min,the adventitious bud differentiation rate and fluorescence positive rate of Flamingo-bill explants were the highest,reaching 48.39% and 4.84%,respectively.Under 6 different pre-culture and co-culture time combinations,the Flamingo-bill explants also produced adventitious buds and adventitious roots,and the highest adventitious bud differentiation rate and fluorescence positive rate were 48.44% and 12.50%,respectively,under the treatment of pre-culture for 1 d and co-culture for 1-2 d.Finally,PCR detection of adventitious roots and calluses with GFP fluorescence expression showed that the 3 target genes GFP,Kan and Cas9 all presented signal in the fluorescence positive tissues,indicating that the T-DNA insertion mediated by Agrobacterium was successful and the transformation was stable.【Conclusion】The explant type of pepper,Agrobacterium infection concentration,infection time,pre-culture time and co-culture time all have effects on the genetic transformation efficiency of pepper.Screening appropriate transformation conditions with GFP as a reporter gene could improve the efficiency of Agrobacterium-mediated genetic transformation of pepper.