橡胶树CRISPR/Cas9编辑植株出现预期表型的突变阈值

Mutant Threshold Capable of Inducing Expected Phenotype in Rubber Tree CRISPR/Cas9 Edited Seedlings

本课题组前期在橡胶树原生质体中分别实现了基于CRISPR/Cas9-RNP及质粒介导的瞬时转化基因编辑,并以HbPDS基因为靶标,在橡胶树愈伤组织中实现了农杆菌介导的稳定转化基因编辑,并获得了出现白化表型的愈伤组织,但由于橡胶树愈伤诱导体胚的技术还不稳定,导致未能获得基因编辑植株。为了获得橡胶树基因编辑幼苗,本研究继续以HbPDS基因为靶标,改用橡胶树体胚为侵染受体,开展农杆菌介导的遗传转化,经潮霉素抗性筛选获得增殖的T_0代抗性体胚,通过Cas9基因分子检测共挑选出116个阳性转化体胚,通过对靶点处的测序检测发现有5个胚块发生了基因编辑,编辑效率为4.3%。通过植株再生程序,获得了2株再生植株,表型观测均是嵌合体,表现为同一编辑植株上同时存在绿色及白化叶片。同时取白化叶片和绿色叶片进行高通量测序,发现二者均已发生了基因编辑,除了1个白化叶片发生了纯合双等位突变之外,其余叶片均为嵌合突变。其中,白化叶片编辑细胞的占比介于86%~100%之间,而绿色部位编辑细胞所占比例介于66%~69%之间,说明橡胶树CRISPR/Cas9编辑植株出现预期表型的突变阈值高于69%,介于70%~85%之间,为今后创制有表型的橡胶树基因编辑幼苗提供理论指导。同时,本研究证明了T_0代转化体胚及其获得的再生植株嵌合比例太高,不宜作为植株再生的材料,为今后通过对T_0代阳性体胚进行继代获得T_1代纯合体胚,并以T_1代体胚为转化受体获得纯合基因编辑植株提供思路。本研究首次公开报道获得了橡胶树基因编辑植株,尽管是嵌合植株,但也增进了对CRISPR/Cas9在橡胶树中作用的理解,为下一步在橡胶树中改进并应用基因编辑技术奠定基础。

Previously,we achieved gene editing using CRISPR/Cas9-RNP and plasmid in the PEG-mediated protoplasts transient transformation of rubber tree,and by targeting the HbPDS gene,callus with albino phenotype were obtained,but no edited plants were regenerated because the technology of embryogenesis from callus is not yet mature.In order to obtain gene edited seedlings,we used the same HbPDS target as previous in the callus editing,but used somatic em-bryos as the transformation receptor instead of callus.After hygromycin resistance screening,116 positive T0 generation embryos were selected through Cas9 gene PCR detection,following by next generation sequencing,five embryos were found to be edited at the target,accounting for 4.3%of PCR positive embryos.At last,two regenerated plants were ob-tained,both were chimeric because only partial albino leaves appeared in the plantlet.Sequencing of both albino and green parts revealed that gene editing had occurred in all samples,besides a homozygous biallelic mutation in one al-bino leaf,all other leaves exhibited chimeric mutations,with mutant sequences in albino parts accounting for 86%to 100%ratio,while green parts accounting for 66%to 69%ratio.This indicates that the mutation threshold inducing the expected phenotype in rubber tree CRISPR/Cas9 editing plants is higher than 69%,ranging from 70%to 85%,providing theoretical guidance for obtaining gene editing seedlings with expected phenotype in the future.Meanwhile,it is proven that nearly all the regenerated plantlets obtained from T0 generation somatic embryos are chimeric,thus T0 generation embryos are not suitable as regenerated materials,but also providing insights to improve the regeneration procedure by using T1 embryo to get homozygous seedlings in the future.This is the first report about gene editing plants in rubber tree,although they are chimeric,it still enhances the understanding of the function of CRISPR/Cas9 in rubber tree,lay-ing the foundation for improving and applying gene editing technology in rubber tree.