基于CRISPR/Cas9的毛果杨PtrHBI1基因功能解析

doi:10.12302/j.issn.1000-2006.202107030

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (6): 31-39.doi: 10.12302/j.issn.1000-2006.202107030

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PtrHBI1基因功能解析

王竹雯(), 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:2021-08-19 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项目(32001332);中央高校基本科研业务费专项资金项目(2572018CL01)

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

摘要/Abstract

摘要： 目的利用CRISPR/Cas9系统创制毛果杨PtrHBI1功能缺失突变体,初步解析该转录因子在木材形成过程中的功能,为利用基因工程手段培育制浆造纸优良性状的林木新品种提供新思路。方法以毛果杨(Populus trichocarpa)为研究材料,根据激光显微切割技术捕获的野生型毛果杨不同细胞类型(形成层、木质部和韧皮部)RNA-seq数据,筛选得到1个bHLH家族转录因子PtrHBI1,利用毛果杨木质部原生质体系统对该基因进行亚细胞定位分析,采用原位杂交技术分析PtrHBI1组织特异性表达,采用CRISPR/Cas9技术创制毛果杨ptrhbi1突变体。对ptrhbi1突变体进行生长表型分析,利用石蜡切片对茎段横切面的各细胞类型进行形态分析,利用Klason酸水解法检测突变体木质素含量,使用高效液相色谱测定突变体纤维素和半纤维素含量。结果亚细胞定位显示PtrHBI1基因定位于细胞核,原位杂交结果表明PtrHBI1主要在形成层和木质部表达。通过CRISPR/Cas9创制的毛果杨ptrhbi1突变体株高显著增加,茎节数和地径在一定时期显著大于野生型。此外,突变体的导管孔径显著增大,纤维细胞数量显著减少。导管细胞的数量和形成层细胞层数与野生型无差异。木材组分分析表明,与野生型植株相比,ptrhbi1植株的纤维素含量增加,木质素总量无显著变化,紫丁香基木质素与愈创木基木质素之质量比(S/G)显著降低。结论 PtrHBI1参与调控毛果杨的生长及次生木质部发育,可能在木材形成过程起着较为重要的调控作用。

关键词: 毛果杨, 木材形成调控, CRISPR/Cas9, PtrHBI1转录因子

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

中图分类号:

王竹雯,国艳娇,李爽,等. 基于CRISPR/Cas9的毛果杨PtrHBI1基因功能解析[J]. 南京林业大学学报（自然科学版）, 2021, 45(6): 31-39.

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 (Natural Science Edition), 2021, 45(6): 31-39.DOI: 10.12302/j.issn.1000-2006.202107030.

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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^**

基于CRISPR/Cas9的毛果杨PtrHBI1基因功能解析

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

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