黄梁木维管组织细胞激光显微切割技术体系建立

黄梁木是华南地区重要的速生用材树种,其快速的次生生长主要取决于包括形成层细胞在内的维管组织细胞的发育进程。为了精准获取维管组织形成层细胞与高质量RNA,该研究以黄梁木幼苗为材料,通过石蜡切片与激光显微切割(LMD)结合的方法,成功获得形成层、木质部和韧皮部细胞。结果表明:优化石蜡切片流程,以卡诺/丙酮作为固定剂(丙酮浓度10%~50%),100%正丁醇作为脱水剂和透明剂,之后经过梯度渗蜡、包埋、切片、脱蜡后可获得形态完整的组织切片且保证了组织细胞RNA的完整性。调试LMD参数,放大倍数为10×~20×;激光能量为42~44;切割孔径为4~13;切割速度为16~25;最终获得的这3种细胞的RNA完整性(RIN)均大于5.0,即形成层细胞RIN=5.0,韧皮部细胞RIN=6.6和木质部细胞RIN=7.9,满足后续RNA-seq分析。该研究通过优化石蜡切片和LMD参数,成功建立了黄梁木幼苗茎段维管组织细胞的激光显微切割技术体系,为进一步揭示林木树种维管组织分裂与分化的调控机制奠定基础。

Establishment of vascular tissue cells capture system by laser microdissection in Neolamarckia cadamba

Neolamarckia cadamba is one of the most important fast-growing trees for wood production in South China, and its secondary growth depends on the development process of vascular tissue cells including formative cells. In order to obtain the vascular tissue accurately and high quality RNA of N. cadamba, we presented an optimal method to provide high-quality paraffin section for vascular tissues, in which carnoy/acetone was used as fixative(acetone concentration was 10%-50%)and 100% n-Butanol was used as the dehydrating and infiltration agent, followed by gradient infiltration paraffin, embedding, sectioning and dewaxing. Meanwhile, LMD parameters were set with magnification between 10× and 20×, power between 42 and 44, aperture between 4 and 13 and speed between 16 and 25 for acquisition of vascular cells in N. cadamba. Utilizing this protocol, high quality of RNAs were extracted, with RIN(RNA integrity number)value of 5.0, 6.6 and 7.9 in cambium cells, phloem cells and xylem cells, respectively. These RNAs can be used for subsequent RNA-seq analysis. Accordingly, the vascular tissue cells capture system by LMD was established in N. cadamba, which would be of value for revealing the regulatory mechanism of vascular tissue division and differentiation in N. cadamba, and provide a reference for capturing vascular cell in other forest tree species.