基于sfGFP系统的嫁接体接口处的细胞质融合检测

doi:10.7668/hbnxb.20192980

摘要/Abstract

摘要：

为了探究接穗和砧木间的细胞如何相互作用而形成功能性嫁接体,利用自组装拆分的超折叠绿色荧光蛋白(sfGFP)系统来监测嫁接体接口处的细胞质融合发生事件。通过花侵染转化得到在细胞质中单独表达sfGFP1-10和mCherry-11的转基因拟南芥植株以及通过叶盘法转化得到在细胞质中单独表达sfGFP1-10和mCherry-11的转基因烟草植株。T1转基因拟南芥和T1转基因烟草在含相应抗生素的培养基中生长,培养8 d后选取生长健壮的幼苗用于嫁接,构建远缘嫁接体(At-1-10/Nb-11)、自体嫁接体(At-1-10/At-11、Nb-1-10/Nb-11)以及对照嫁接体(At-11/Nb-11、At-11/At-11、Nb-11/Nb-11)。嫁接后第4天,利用激光共聚焦显微镜观察接口处GFP信号并统计嫁接体接口处的GFP荧光强度。结果显示:在嫁接后第4天,远缘嫁接体和自体嫁接体接口处均监测到GFP信号,且远缘嫁接体的GFP荧光强度显著高于自体嫁接。在细胞质中单独表达sfGFP1-10和mCherry-11的转基因拟南芥未嫁接幼苗、细胞质中单独表达sfGFP1-10和mCherry-11的转基因烟草未嫁接幼苗以及对照嫁接体中均未检测到GFP信号。结果表明:嫁接体接口处细胞质在嫁接后第4天已经发生融合,拆分的sfGFP片段在砧穗的连接下重组并发出荧光,定位于细胞质中,且远缘嫁接体和自体嫁接体在细胞质融合上存在差异;自组装拆分的sfGFP系统可用于检测嫁接体接口处细胞质的融合,为研究嫁接体接口处细胞学事件发生提供了新的视角。

关键词: 拟南芥, 本生烟草, 远缘嫁接体, 愈合机制, sfGFP系统

Abstract:

In order to explore how cells from different plant individuals reestablish the connection between scion and stock to form a functional graft,self-assembling split super-folder greenfluorescent protein(sfGFP)system was used to detect the cytoplasm fusion at the graft junction.Transgenic Arabidopsis thaliana and Nicotiana benthamiana plants expressing sfGFP1-10 and mCherry-11 in cytoplasm were obtained by floral dip transformation and leaf disc transformation respectively,and 8-day old T1 transgenic plant were used for grafting.Interfamilial grafts(At-1-10/Nb-11),autografts(At-1-10/At-11,Nb-1-10/Nb-11)and control grafts(At-11/Nb-11,At-11/At-11,Nb-11/Nb-11)were constructed.On the fourth day after grafting,the sfGFP signal at the junction was observed under confocal microscope,and the fluorescence intensity of the grafts was calculated.The results showed that the interfamilial grafts and antografts displayed GFP signal in junction.GFP fluorescence intensity of interfamilial At/Nb grafts was significantly higher than that of autografts.GFP signal was not observed in cells of transgenic Arabidopsis thaliana and Nicotiana benthamiana expressing sfGFP1-10 or mCherry-11 alone and control grafts.These results indicated that at the fourth day,the fusion of cytoplasm at junction readily occurred.The sfGFP fragments were reassembled at the junction through the contact between the stock and the scion targeting in cytoplasm.Moreover,the reassembled fluorescence intensity in the interfamilial At/Nb heterografts was stronger than autografts,suggesting an extensive cellular fusion for At/Nb distant graft.This study provides a new perspective for the cellular events occurring at the graft junction.

Key words: Arabidopsis thaliana, Nicotiana benthamiana, Interfamilial grafting, Healing mechanism, sfGFP system

李冬怡, 邓竹英, 梁大成. 基于sfGFP系统的嫁接体接口处的细胞质融合检测[J]. 华北农学报, 2022, 37(4): 151-157. doi: 10.7668/hbnxb.20192980.

LI Dongyi, DENG Zhuying, LIANG Dacheng. Cytoplasmic Fusion Detection at the Grafting Junction Based on Split sfGFP System[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 151-157. doi: 10.7668/hbnxb.20192980.

http://www.hbnxb.net/CN/Y2022/V37/I4/151

图/表 5

图1 转基因拟南芥与烟草及构建的嫁接体

Fig.1 Transgenic Arabidopsis thaliana and Nicotiana benthamiana plants and constructed grafts

图2 转基因拟南芥单株及拟南芥/拟南芥嫁接体GFP信号观察

Fig.2 GFP signal observation of transgenic Arabidopsis thaliana plants and At/At grafts

图3 转基因烟草单株及烟草/烟草嫁接体GFP信号观察

Fig.3 GFP signal observation of transgenic Nicotiana benthamiana plants and Nb/Nb grafts

图4 拟南芥/烟草嫁接体GFP信号观察

Fig.4 GFP signal observation of At/Nb grafts

图5 远缘嫁接与自体嫁接嫁接体GFP荧光强度不同小写字母表示数据之间存在显著差异(P<0.05)。

Fig.5 GFP fluorescence intensity of heterograft and autografts Different lowercase letters indicated significant differences between the data(P<0.05).

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