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
孙佳彤(), 国艳娇, 李爽, 周晨光*(
), 姜立泉, 李伟*(
)
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:
SUN Jiatong(), GUO Yanjiao, LI Shuang, ZHOU Chenguang*(
), CHIANG Vincent, LI Wei*(
)
Received:
2021-07-20
Accepted:
2021-08-25
Online:
2021-11-30
Published:
2021-12-02
Contact:
ZHOU Chenguang,LI Wei
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。
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.
表1
所用引物及序列"
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' | |
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' | |
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' |
图3
毛果杨转基因植株的获得 A. 愈伤组织分化诱导callus induction;B. 抗性芽诱导shoot induction;C. 抗性植株筛选screaning of the resistant plants;D. 转基因植株的PCR鉴定the PCR detection results(M. DNA marker DL 2000。 1. ddH2O为模板的阴性对照negative control with ddH2O as PCR template; 2. pEgP237-U6-PtrbHLH106 gRNA-35S-Cas9质粒为模板的阳性对照positive control with plasmid pEgP237-U6-PtrbHLH106 gRNA-35S-Cas9 as PCR template; 3. 野生型毛果杨叶片DNA为模板的阴性对照negative control with leaf DNA of wild-type plant as PCR template; 4~6. 抗性植株叶片DNA为模板的样品sample with leaf DNA of kanamycin-resistant plant as PCR template)。"
图4
毛果杨PtrbHLH106基因编辑情况 A. 等位基因编辑情况(红色字母表示碱基插入,红色“-”表示碱基缺失,“-16 bp-”表示缺失16个碱基)gene editing of alleles (The red letters represent insertion; the red short lines “-” represent deletion; “-16 bp-” represents 16 nucleotide deletion);B. 被编辑基因的蛋白翻译情况预测(红色块区域代表氨基酸改变) the prediction of amino acid sequence translation (The red short lines represent amino acid changed)。"
图5
毛果杨野生型与ptrbhlh106突变体表型分析 误差线代表由3个生物学重复计算的标准误,*表示通过t检验,突变体与野生型植物各株系之间存在显著差异,*.P<0.05,**.P<0.01)。下同。Statistical analysis of growth index of wild-type(WT) and ptrbhlh106 plants. Error bars represent SE values of three independent experiments. Asterisks indicate significant differences between each line of the mutants and wild-type plants by Student’s t test. The same below."
图6
毛果杨ptrbhh106突变体石蜡切片观察及细胞统计 A. 野生型(WT)和突变体(ptrbhlh106)毛果杨各茎节细胞形态观察(比例尺为200 μm)morphologic observation of stem internodes of WT and ptrbhlh106 (Bars=200 μm);B. 形成层细胞形态观察(红色线表示形成层区域,比例尺为100 μm)morphologic observation of cambium cells (Red line showed cambium areas, Bars=100 μm);C. 单位面积细胞数目统计statistics analysis of number of fiber and vessel cells in per unit cells;D. 导管孔径面积统计statistics analysis of lumen area of per vessel;E. 形成层细胞层数统计statistics analysis of number of cambium cell layer。"
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