基于CRISPR/Cas9的毛果杨bHLH106转录因子的功能研究  

A functional study of bHLH106 transcription factor based on CRISPR/Cas9 in Populus trichocarpa

作　　者：孙佳彤 国艳娇 李爽[1] 周晨光[1] 姜立泉 李伟[1] SUN Jiatong;GUO Yanjiao;LI Shuang;ZHOU Chenguang;CHIANG Vincent;LI Wei(State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin 150040,China)

机构地区：[1]林木遗传育种国家重点实验室(东北林业大学),黑龙江哈尔滨150040

出　　处：《南京林业大学学报（自然科学版）》2021年第6期15-23,共9页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：国家自然科学基金青年科学基金项目(32001332);中央高校基本科研业务费专项资金项目(2572018CL01)。

摘　　要：【目的】采用CRISPR/Cas9基因编辑系统创制毛果杨(Populus trichocarpa)bHLH106(Basic Helix-Loop-Helix 106)基因的突变体,分析植株的表型特征,初步揭示PtrbHLH106基因在毛果杨木材形成过程中的功能。【方法】基于前期对毛果杨野生型(WT)茎干的不同细胞类型(形成层、木质部和韧皮部细胞)RNA-seq数据,克隆得到一个在形成层及木质部较高表达的bHLH基因PtrbHLH106。采用CRISPR/Cas9基因编辑技术创制毛果杨PtrbHLH106的功能缺失突变体。对生长60、90、120 d的毛果杨ptrbhlh106突变体和WT植株进行表型观察;对生长120 d的植株各茎节进行石蜡切片,利用甲苯胺蓝染色观察并进行细胞统计分析。【结果】获得毛果杨ptrbhlh106突变体;与WT相比,突变体植株的株高、地径无明显差异;在整个测量的生长周期中,第8茎节长度有缩短的趋势,茎节数量有增加的趋势;形成层细胞层数有增加的趋势但差异不显著,导管细胞孔径显著增大,纤维细胞数量显著减少。【结论】ptrbhlh106突变体与WT植株在导管孔径和纤维细胞数量上存在差异,初步证明PtrbHLH106基因参与了调控毛果杨次生木质部的发育。【Objective】 We generated CRISPR-based mutants of PtrbHLH106 to explore its function in the wood formation of Populus trichocarpa. 【Method】 Based on the previous RNA-seq data of various cell types(cambium, xylem and phloem cells) of P. trichocarpa, we cloned a bHLH transcription factor, PtrbHLH106, which is highly expressed in the cambium and xylem. The CRISPR/Cas9 gene editing system was used to generate ptrbhlh106 mutants. Phenotype observations of ptrbhlh106 and wild-type(WT) plants grown for 60, 90 and 120 d were carried out. Paraffin sections of stem internodes of ptrbhlh106 and WT plants grown for 120 days were created, and toluidine blue staining was used for observation and statistical analyses of different wood cells. 【Result】 Phenotype observation showed that there were no significant differences in plant height and ground diameter. Compared to that in WT plants, the internodes length of 8 th stem reduced, total number of stem internodes increased, and the layer of cambium cells increased in mutants. However, the differences in the above indexes were not significant. In addition, the lumen area of per vessel increased, and the number of fiber cells decreased significantly in ptrbhlh106. 【Conclusion】 The phenotypic differences between ptrbhlh106 mutants and WT plants showed that PtrbHLH106 is involved in the regulation of secondary xylem development in P. trichocarpa.

关 键 词：毛果杨 次生木质部发育 CRISPR/Cas9基因编辑 PtrbHLH106转录因子 

分 类 号：S792.11[农业科学—林木遗传育种] Q943.2[农业科学—林学]