雌雄青杨组织培养和PcXTH22基因过表达及敲除  

Tissue regeneration of male and female Populus cathayana and overexpression and knockdown of PcXTH22 gene

作　　者：陈遥 李婧 赖诗宜 张雪驰 陈凡 张胜 CHEN Yao;LI Jing;LAI Shi-Yi;ZHANG Xue-Chi;CHEN Fan;ZHANG Sheng(Laboratory of Plant Physiology and Ecology,College of Life Sciences,Sichuan University,Chengdu 610065,China;Power China Guiyang Engineering Corporation Limited,Guiyang 550009,China)

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065 [2]中国电建集团贵阳勘测设计研究院有限公司,贵阳550009

出　　处：《四川大学学报(自然科学版)》2025年第2期259-270,共12页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(32271830);青藏高原寒旱区大规格乔木移植关键技术研究及应用项目(2846G202400001)。

摘　　要：为能通过基因工程手段对青杨(Populus cathayana Rehd.)进行遗传改良,本研究以雌雄青杨为材料建立了适用于不同性别的组培再生体系,确定了青杨雌株分化、增殖和生根阶段最佳培养基分别为:MS+30 g/L蔗糖+6 g/L琼脂+0.1 mg/L TDZ+0.3 mg/L IBA,MS+30 g/L蔗糖+7 g/L琼脂+0.1 mg/L 6-BA+0.5 mg/L IBA,WPM+20 g/L蔗糖+7 g/L琼脂+0.1 mg/L IBA+0.2 mg/L NAA;青杨雄株分化、增殖和生根阶段的最优培养基分别为:WPM+30 g/L蔗糖+6 g/L琼脂+0.1 mg/L TDZ+0.3 mg/L NAA,MS+30 g/L蔗糖+7g/L琼脂+1.0 mg/L 6-BA+0.1 mg/L NAA,WPM+20 g/L蔗糖+7 g/L琼脂+0.2 mg/L IBA+0.02 mg/L NAA.随后使用继代培养1 mo的青杨雌株叶片作为遗传转化受体,根癌农杆菌菌液侵染10~15 min后,共培养48 h,然后转入含10 mg/L潮霉素和200 mg/L特美汀的各阶段培养基中进行选择培养.定量PCR发现过表达株系中PcXTH22的转录表达水平提高了40~824倍,转化率为100%.在CRISPR-Cas9转化成功的5个株系中发现1个株系的两个等位基因同时发生移码突变而蛋白翻译提前终止(即纯合株系),编辑效率为20%.因此,该研究为使用我国特有乡土树种进行遗传改良提供了优秀的范例.To achieve genetic improvement of Populus cathayana Rehd through genetic engineering methods,male and female poplar were used as materials to establish a systematic tissue culture regeneration.The optimal mediums for the differentiation,proliferation and rooting of female P.cathayana were MS+30 g/L sucrose+6 g/L agar+0.1 mg/L TDZ+0.3 mg/L IBA,MS+30 g/L sucrose+7 g/L agar+0.1 mg/L 6-BA+0.5 mg/L IBA,WPM+20 g/L sucrose+7 g/L agar+0.1 mg/L IBA+0.2 mg/L NAA;those for male P.cathayana were:WPM+30 g/L sucrose+6 g/L agar+0.1 mg/L TDZ+0.3 mg/L NAA,MS+30 g/L sucrose+7 g/L agar+1.0 mg/L 6-BA+0.1 mg/L NAA,WPM+20 g/L sucrose+7 g/L agar+0.2 mg/L IBA+0.02 mg/L NAA.Subsequently,the leaves of female P.cathayana after 1 month of sub‑culture were used as genetic transformation receptor material for overexpression and knockout of the PcXTH22 gene.After infecting with Agrobacterium solution for 10~15 minutes,the leaf discs were cocultured with Agrobacterium for 48 h and then transferred into mediacontaining 10 mg/L hygromycin and 200 mg/L temetine for selective culture.The obtained resistant plants were detected by PCR,and the transformation rate was almost 100%.Quantitative PCR showed that the transcriptional expression level of PcXTH22 in overexpressed strains increased by 40-824 times.Among the 5 CRISPR-Cas9-transformed lines,1 line was found to have frameshift mutation on both alleles and the protein translation was terminated prematurely,and the editing efficiency was 20%.Therefore,the study provides an excellent example of genetic improvement using our endemic native tree species.

关 键 词：青杨 雌雄植株 组织再生 遗传转化体系 PcXTH22 CRISPR-Cas9基因编辑 

分 类 号：Q94[生物学—植物学]