RNAi介导的山核桃嫁接砧穗基因沉默信号的表达机理研究

The genetically modified fruits safety is an important event in the society. RNA interference (RNAi), is also called RNA mediated gene silencing or post transcriptional gene silencing (PTGS) or co-suppression, is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules and is potently initiated by double-stranded RNA (dsRNA). The current study shows that the silencing signal which mediated by RNAi can be transferred from grafting rootstock to scion and improve the agronomic traits of wild type scions. This can reduce the risk of genetically modified woody fruit dramatically. Studies showed gene signal silencing can be transferred from rootstock to scion in Arabidopsis successfully. Gibberellins (GAs) constitute a large group of natural tetracyclic diterpenoids. Biologically active GAs have a profound effect on plant growth and development. The gibberellin 20-oxidase played an important role in the biosynthesis of gibberellic acid as one of the key oxidase enzymes regulating directly the generation of biological GAs. GA20ox silencing signal can be mediated by RNA interference in some crops. However, whether GA20ox gene silencing signal which mediated by RNAi in the rootstocks of hickory (Carya cathayensis Sarg.) can be transferred from rootstock to scion and lead to the target gene silencing or not is not sure now. This project will base on the somatic embryogenesis and genetic transformation system of hickory to study GA20ox gene signal silencing between rootstocks and scions in hickory which mediated by RNA interference. The purposes are as follows: ① To optimize the hickory genetic transformation system; ② To construct a stable hickory GA20ox RNA interference system; ③ To analysis the silence effect of hickory GA20ox RNA interference transformants; ④ To clarify the expression of silencing signal mediated by RNA interference between hickory RNAi rootstock and wild type scion. These results will illustrate GA20ox gene silencing mechanism between hickory rootstock and scion which mediated by RNA interference. We seek to explore a hemi-dwarf RNAi rootstock and hemi-dwarf non transgenic scion grafting system in order to solve the huge problem between high quantity and nontransgenic dwarf woody fruit trees.

转基因果品安全是当今民众关心的重要事件。RNA干扰，也称作RNA介导的基因沉默，是一种由双链RNA信号引发同源mRNA降解的基因转录后沉默现象，被认为是生物自身调节基因表达和自我保护的一种有效手段。研究表明，RNA干扰介导的沉默信号能够有效地从拟南芥等模式植物的RNAi型嫁接砧木传递至野生型接穗并定向改良接穗的重要性状。然而，RNAi介导的山核桃嫁接砧木GA20ox基因沉默信号是否能长距离传递并导致野生型非转基因接穗的目的基因沉默？本课题在近年研究的基础上，探讨RNAi介导的山核桃嫁接砧穗基因沉默信号的长距离传递与表达，包括①优化山核桃遗传转化体系；② 构建稳定的GA20ox RNAi干扰转化体系；③分析山核桃GA20ox RNAi干扰转化子的沉默效应；④ 明晰RNAi 介导下嫁接砧穗间基因沉默信号表达。从而阐明GA20ox RNAi型山核桃嫁接砧木对野生型接穗GA20ox基因沉默效应的调控机制，以期获得山核桃半矮化RNAi型砧木及半矮化型非转基因接穗的嫁接培育体系，为解决山核桃等木本果树高产和非转基因矮化的矛盾提供良好的理论依据和技术支持。

转基因果品的安全性已逐渐成为当今社会民众十分关心的重要事件。研究表明， RNA干扰产生的砧木目的基因转录后沉默信号可以跨越嫁接面在植物嫁接体内进行长距离快速传递，从而有效剪切野生型非转基因接穗中目的基因的mRNA而产生系统获得性沉默。本研究推测山核桃GA20ox基因转录后沉默信号很可能从RNAi型砧木长距离运输至野生型接穗，可导致接穗GA20ox基因的沉默，并获得半矮化的野生型接穗，避免直接对山核桃接穗进行转基因。因此，本项目在近年研究的基础上，探讨了RNAi介导的山核桃嫁接砧穗基因沉默信号的长距离传递与表达，包括：构建稳定的GA20ox RNAi干扰转化体系；分析山核桃GA20ox RNAi干扰转化子的沉默效应并阐明RNAi 介导下嫁接砧穗间基因沉默信号表达，从而解析GA20ox RNAi型山核桃嫁接砧木对野生型接穗GA20ox基因沉默效应的调控机制。具体研究结果如下：通过生长调节物质等主导因子筛选，优化了山核桃体胚发生体系，并在此基础开展了山核桃遗传转化，构建了良好的遗传转化体系；克隆获得了山核桃GA20ox基因保守片段（226 bp），BLAST比对结果表明，该保守片段与核桃（Juglans regia）的同源性为96%，并成功构建了pTCK303-GA20ox RNA干扰载体；克隆获得了山核桃GA20ox基因ORF全长（1140bp），采用同源重组法成功构建了pCAMBIA1300-GA20ox过表达载体，并对山核桃pCAMBIA1300-GA20ox载体进行烟草叶表皮亚细胞定位发现CcGA20ox蛋白定位在细胞核与细胞膜；通过pTCK303-GA20ox干扰载体与pCAMBIA1300-GA20ox过表达载体的遗传转化，验证了山核桃GA20ox基因功能，发现其与山核桃植株株高正相关；开展了山核桃pTCK303-GA20ox与野生型植株互为砧穗进行嫁接，采用RT-PCR及Northern blot检测了砧穗mRNA的变化趋势，发现pTCK303-GA20ox RNA干扰可导致砧木或接穗中GA20ox mRNA不同程度的降低。进一步验证发现，该种传递是双向的，即既可从砧木传递至接穗，也可双接穗传递至砧木。本研究结果为山核桃等木本果树矮化/半矮化新品种的创制与规模化生产提供理论依据和技术支持，极大程度地降低了山核桃等木本果树树种转基因的风险。