Functional analyses of PtrHBI 1 gene in Populus trichocarpa based on CRISPR/Cas9

doi:10.12302/j.issn.1000-2006.202107030

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (6): 31-39.doi: 10.12302/j.issn.1000-2006.202107030

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

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Functional analyses of PtrHBI 1 gene in Populus trichocarpa based on CRISPR/Cas9

WANG Zhuwen(), 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:2021-08-19 Online:2021-11-30 Published:2021-12-02
Contact: ZHOU Chenguang,LI Wei E-mail:wzw981012@163.com;zhouchenguang@nefu.edu.cn;weili2015@nefu.edu.cn

Abstract

Abstract:

【Objective】 We used the CRISPR/Cas9 system to generate PtrHBI1 loss of function mutants of Populus trichocarpa in order to investigate the function of the PtrHBI1 transcription factor in wood formation. This investigation provides new clues for creating superior varieties of trees suitable for pulp and papermaking. 【Method】 Based on RNA-seq data of different cell types (cambium, xylem and phloem) of wild-type P. trichocarpa by laser capture microdissection, the bHLH transcription factor PtrHBI1 was identified. Subcellular localization of PtrHBI1 was analyzed using the stem-differentiating xylem (SDX) protoplast system of P. trichocarpa, and tissue-specific expression of PtrHBI1 was analyzed by in situ hybridization. We then used the CRISPR/Cas9 system to generate ptrhbi1 mutants of P. trichocarpa and analyzed the growth traits of ptrhbi1. Meanwhile, we performed a morphological analysis using paraffin sections and analyzed the wood composition of the mutant using the Klason acid hydrolysis method and high-performance liquid chromatography. 【Result】 According to the results, we found that PtrHBI1 was localized in the nucleus of P. trichocarpa SDX cells. The results of in situ hybridization showed that PtrHBI1 was mainly expressed in the cambium and xylem regions. The height of ptrhbi1 mutants created by CRISPR/Cas9 was significantly increased, and the internode number and stem basal diameter were significantly larger than those of the wild type during a certain period. In addition, the ptrhbi1 mutant showed a significant increase in the lumen area of per vessel cells and a significant decrease in the number of fiber cells. However, the number of vessel cells and layers of cambium cells in the ptrhbi1 mutant were similar to those in the wild type. The wood composition analysis showed that the glucose content of the ptrhbi1 mutant increased. Compared with the wild type, although the total lignin content was not significantly changed, the mass ratio of syringly to guaiacy (S/G) ratio of the mutant was significantly decreased. 【Conclusion】 PtrHBI1 was involved in regulating the growth and secondary xylem development of P. trichocarpa, which may play an important role in wood formation.

Key words: Populus trichocarpa, wood formation regulation, CRISPR/Cas9, PtrHBI1 transcription factor

CLC Number:

WANG Zhuwen, GUO Yanjiao, LI Shuang, ZHOU Chenguang, CHIANG Vincent, LI Wei. Functional analyses of PtrHBI 1 gene in Populus trichocarpa based on CRISPR/Cas9[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(6): 31-39.

Figures/Tables 9

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引物名称 primer name	序列 sequences	用途 application
PtrHBI1-1F	5'-CACCATGCTGTCTCAAAAACACATCTTT-3'	PtrHBI1基因克隆 cloning of PtrHBI1 gene
PtrHBI1-1R	5'-TCATTCAGGACCATTACTGTAATA-3'	PtrHBI1基因克隆 cloning of PtrHBI1 gene
PtrHBI1-2F	5'-CTAGGGATCCATGCTGTCTCAAAAACACATCT-3'	pUC19-35S-PtrHBI1-GFP瞬时表达载体构建 construction of pUC19-35S-ptrHBI1-GFP transient expression vector
PtrHBI1-2R	5'-CTAGGTCGACATGGAATCCCAAATTTTGAAGG-3'
PtrHBI1-3F	5'-AGCTGATTAGCCAATTCTATCA-3'	PtrHBI1基因探针 PtrHBI1 gene probe
PtrHBI1-3R	5'-GGTTTACGTAGTTGTTCTCCT-3'
PtrHBI1-4F	5'GAATTGTAATACGACTCACTATAGGGAGCTGATT AGCCAATTCTATCA-3'
PtrHBI1-4R	5'GAATTGTAATACGACTCACTATAGGGGGTTTACGT AGTTGTTCTCCT-3'
PtrHBI1-g-F PtrHBI1-g-R	5'-GATTGAGAAGCAGAGGTAACGGCA-3' 5'-TGCCGTTACCTCTGCTTCTCCAAA-3'	pEgP237载体构建,R端引物亦用于转基因植株鉴定 vector construction of pEgP237,PtrHBI1-g-R also for transgenic identification
M13F	5'-GTAAAACGACGGCCAG-3'	转基因鉴定 transgenic identification
PtrHBI1-5F PtrHBI1-5R	5'-GAGATAGGATTGATTGGAAGG-3' 5'-AGTCTTCTGATTTTCCTTCGA-3'	靶位点编辑情况的鉴定 indetification of gene editing

样本 sample	碳水化合物占比carbohydrates rate					木质素占比lignin rate
样本 sample	葡萄糖 glucose	木糖 xylose	半乳糖 galactose	阿拉伯糖 the Arab sugar	总计 total	酸不可溶性木质素 acid insoluble lignin	酸可溶性木质素 acid soluble lignin	总计 total
野生型 WT	53.70±0.75	12.25±0.31	1.25±0.03	2.36±0.05	69.55±1.10	19.40±0.58	3.63±0.05	23.03±0.62
突变体 ptrhbi1	57.61±0.23^**	11.64±0.08	1.04±0.02^**	2.5±0.01^*	72.79±0.17^*	17.39±0.33^*	3.84±0.05^*	21.22±0.28

样本 sample	占比/% rate			S/G
样本 sample	S型木质素 S-lignin	G型木质素 G-lignin	H型木质素 H-lignin	S/G
野生型 WT	61.55±0.30	28.18±0.19	10.27±0.18	2.18±0.07
突变体 ptrhbi1	60.46±0.23^*	33.76±0.17^**	5.77±0.14^**	1.79±0.01^**

Functional analyses of PtrHBI 1 gene in Populus trichocarpa based on CRISPR/Cas9

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