基于CRISPR/Cas9的毛果杨bHLH106转录因子的功能研究

doi:10.12302/j.issn.1000-2006.202107031

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (6): 15-23.doi: 10.12302/j.issn.1000-2006.202107031

所属专题：专题报道；林木 CRISPR/Cas基因编辑专题

• 专题报道（执行主编施季森尹佟明陈金慧） • 上一篇下一篇

基于CRISPR/Cas9的毛果杨bHLH106转录因子的功能研究

孙佳彤(), 国艳娇, 李爽, 周晨光^*(), 姜立泉, 李伟^*()

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

收稿日期:2021-07-20 接受日期:2021-08-25 出版日期:2021-11-30 发布日期:2021-12-02
通讯作者: 周晨光,李伟
基金资助:
国家自然科学基金青年科学基金项目(32001331);中央高校基本科研业务费专项资金项目(2572018CL02)

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

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

Received:2021-07-20 Accepted:2021-08-25 Online:2021-11-30 Published:2021-12-02
Contact: ZHOU Chenguang,LI Wei

摘要/Abstract

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。

关键词: 毛果杨, 次生木质部发育, CRISPR/Cas9基因编辑, PtrbHLH106转录因子

Abstract:

【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 8th 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.

Key words: Populus trichocarpa, secondary xylem development, CRISPR/Cas9 gene editing system, PtrbHLH106 transcription factor

中图分类号:

孙佳彤,国艳娇,李爽,等. 基于CRISPR/Cas9的毛果杨bHLH106转录因子的功能研究[J]. 南京林业大学学报（自然科学版）, 2021, 45(6): 15-23.

SUN Jiatong, GUO Yanjiao, LI Shuang, ZHOU Chenguang, CHIANG Vincent, LI Wei. A functional study of bHLH106 transcription factor based on CRISPR/Cas9 in Populus trichocarpa[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(6): 15-23.DOI: 10.12302/j.issn.1000-2006.202107031.

图/表 7

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引物名称 primer name	引物序列 sequences	用途 application
F	5'-CACCATGCAGCCTGAAAACTGTCAGGAG-3'	PtrbHLH106基因克隆 cloning of PtrbHLH106 gene
R	5'-CATAGCCATTCAACGTCCAAAGAT-3'	PtrbHLH106基因克隆 cloning of PtrbHLH106 gene
T7-bHLH106-stem-loop	5'-TAATACGACTCACTATAGGTTCCATTCTCGGTAAGAAAC GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGG-3'	体外检测的gRNA转录模板 gRNA transcription template for in vitro detection
pUC19-F	5'-CTAGGGATCCCTTCACTTGCGGGTCATCTC-3'	体外检测中的模板DNA the template DNA for in vitro detection
pUC19-R	5'-CTAGGTCGACGACTTGTTTGTGTAGATCCAAG-3'	体外检测中的模板DNA the template DNA for in vitro detection
gRNA-F	5'-GATTGTTCCATTCTCGGTAAGAAAC-3'	pEgP237载体构建,R端引物亦用于转基因植株鉴定 vector construction of pEgP237, gRNA-R also for transgenic identification
gRNA-R	5'-GTTTCTTACCGAGAATGGAACCAAA-3'
M13F	5'-GTAAAACGACGGCCAG-3'	转基因鉴定 transgenic identification
PtrbHLH106-F	5'-ACTTGCGGGTCATCTCCCTT-3'	靶位点编辑情况的鉴定 identification of gene editing
PtrbHLH106-R	5'-GCCTGCAACACCTAAAAATATT-3'	靶位点编辑情况的鉴定 identification of gene editing

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

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