小立碗藓PpAux/IAA2基因在原生质体再生过程中的功能分析  

Functional analysis of PpAux/IAA2 in Physcomitrella patens(Hedw.)Mitt.during protoplast regeneration

作　　者：李永红 齐美艳 方圆 付安 王晓琴[1] 何亦騉 Li Yong-Hong;Qi Mei-Yan;Fang Yuan;Fu An;Wang Xiao-Qin;He Yi-Kun(Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing University of Agriculture, Beijing 102206, China;Beijing Bei Nong Enterprise Management Co., Ltd., Beijing 102206, China;College of Life Sciences, Capital Normal University, Beijing 100048, China)

机构地区：[1]北京农学院林木分子设计育种高精尖创新中心,北京102206 [2]北京北农企业管理有限公司,北京102206 [3]首都师范大学生命科学学院,北京100048

出　　处：《植物科学学报》2021年第5期515-525,共11页Plant Science Journal

基　　金：国家自然科学基金项目(31871235,31371243)。

摘　　要：以小立碗藓(Physcomitrella patens(Hedw.)Mitt.)野生型及抗生长素的突变体Ppiaa2-87为实验材料,分析了生长素早期应答基因PpAux/IAA2在原生质体再生过程中的调控机制。分别采用qRT-PCR、FDA染色、DNA倍性分析、DAPI染色、甲基化敏感扩增多态性分析等方法,对原生质体再生过程的相关基因表达、存活率、细胞周期进程、染色体重塑及DNA甲基化进行了分析。结果显示:PpAux/IAA的3个同源基因在野生型0 h原生质体中的表达量较原丝体、48 h和96 h原生质体均明显升高;随着培养时间的延长,Ppiaa2-87原生质体的存活率明显下降;原生质体进入S期受到抑制;部分0 h原生质体的染色质未发生重塑,且染色质重塑复合体SWI/SNF蛋白家族4个基因的表达水平在0 h原生质体中较原丝体下降;Ppiaa2-87的0 h原生质体甲基化程度偏高。研究结果说明,PpAux/IAA2调控的生长素信号通路在小立碗藓原生质体再生过程中具有重要作用,该基因的突变影响了原生质体DNA甲基化及染色质重塑等细胞重新编程过程,导致原生质体不能获得多能性而死亡。We used wild-type(WT)and auxin-resistant mutant Ppiaa2-87 strains of Physcomitrella patens to explore the regulation mechanism of the early auxin-response gene PpAux/IAA2 in protoplast regeneration.Gene expression,survival rate,cell cycle progression,chromosome remodeling,and DNA methylation during protoplast regeneration were analyzed separately using quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR),fluorescein diacetate(FDA)staining,flow cytometry of DNA ploidy,4′,6-diamidino-2-phenylindole(DAPI)staining,and methylation-sensitive amplified polymorphism analysis.Results showed that the expression levels of three PpAux/IAA homologous genes in 0-h protoplasts were significantly higher than that in protonemata,48-h,and 96-h protoplasts;survival rate of PPiaa2-87 protoplasts decreased significantly with culture time;entry into the S phase of PPiaa2-87 protoplasts was inhibited;chromosomes of some Ppiaa2-870-h protoplasts were not remodeled;expression of four genes in the SWI/SNF protein family in the chromatin remodeling complex decreased in 0-h protoplasts compared with that in protonemata;and methylation of Ppiaa2-870-h protoplasts was higher than that of WT protoplasts.This study indicates that the PpAux/IAA2-related auxin signaling pathway plays an important role in protoplast regeneration.Mutation of the PpAux/IAA2 gene affects protoplast DNA methylation and chromatin remodeling,which is involved in cell development reprogramming,leading to protoplast death due to lack of pluripotency.

关 键 词：小立碗藓 生长素 AUX/IAA 原生质体再生 

分 类 号：Q943.2[生物学—植物学]