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

doi:10.12302/j.issn.1000-2006.202107031

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (6): 15-23.doi: 10.12302/j.issn.1000-2006.202107031

Special Issue: 专题报道；林木 CRISPR/Cas基因编辑专题

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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 E-mail:sunjtt89@163.com;zhouchenguang@nefu.edu.cn;weili2015@nefu.edu.cn

Abstract

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

CLC Number:

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, 2021, 45(6): 15-23.

Figures/Tables 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

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

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