王雪 龙健梅 董甜甜 郑丹菁 张立定 彭昌操

摘 要：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。

关键词：黄梁木，激光显微切割（LMD），石蜡切片，维管组织，RNA完整性

中图分类号：Q944.5

文献标识码：A

文章编号：1000-3142（2021）08-1226-11

Abstract： 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.

Key words： Neolamarckia cadamba， laser microdissection （LMD）， paraffin section， vascular tissue， RNA integrity

维管组织由木质部和韧皮部组成，它们在初生生长期间从原形成层产生，在次生生长期间从形成层中不断产生（Campbell & Turner，2016）。维管系统延伸到整个植物体，提供机械支撑和资源运输（Zhu et al.，2019）。增殖的形成层细胞是分化木质部（木材）和韧皮部细胞的来源，形成层活性导致茎的增粗和生物量的增加（Elo et al.，2009）。因为大多数植物生物量由形成层产生，所以对形成层的获得和研究可望进一步了解林木树种的发育机制并进行生物量遗传改良。……