CsAHL25通过影响CsHB1、LEC1/B3基因表达调控柑橘体细胞胚发生  

CsAHL25 regulates citrus somatic embryogenesis by affecting the expression of CsHB1 and LEC1/B3 genes

作　　者：叶长宁 徐梦梦 刘兰兰 付玉洁 葛晓霞 YE Changning;XU Mengmeng;LIU Lanan;FU Yujie;GE Xiaoxia(College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,Hubei,China;Journal Center of Academy of Science and Technology Development,Huazhong Agricultural University,Wuhan 430070,Hubei,China;Center of Applied Boitechnology,Wuhan Institute of Bioengineering,Wuhan 430415,Hubei,China)

机构地区：[1]华中农业大学园艺林学学院,武汉430070 [2]华中农业大学科学技术发展研究院期刊中心,武汉430070 [3]武汉生物工程学院应用生物技术研究中心,武汉430415

出　　处：《果树学报》2024年第4期579-589,共11页Journal of Fruit Science

基　　金：国家自然科学基金项目(31872084);湖北省高等学校优秀中青年科技创新团队计划项目(T2021039)。

摘　　要：【目的】基于柑橘体细胞胚发生相关基因CsHB1的启动子筛选其上游转录因子,以期为柑橘体细胞胚发生分子机制研究提供可靠的候选基因。【方法】利用CsHB1启动子(-1018~-558 bp)进行酵母单杂筛库实验,筛选出CsHB1上游转录因子CsAHL25;利用亚细胞定位实验,确定CsAHL25在细胞中的位置;通过酵母单杂点对点、双荧光素酶实验验证CsAHL25对CsHB1表达的影响;利用qRT-PCR探究CsAHL25基因在柑橘体细胞胚诱导过程中的表达模式;在柑橘愈伤组织中瞬时表达该基因,并检测体细胞胚发生相关基因的表达变化。【结果】CsAHL25在柑橘体细胞胚诱导过程中呈现先上升后下降的表达模式,该蛋白定位在细胞核中,能与CsHB1启动子结合并下调CsHB1的表达。瞬时表达CsAHL25会导致CsHB1表达量下调,及CsABI3、CsFUS3、CsLEC1、CsL1L等促进体细胞发生的LEC1/B3基因表达量上调。【结论】CsAHL25能直接下调CsHB1的表达,并使LEC1/B3基因表达量上升。CsAHL25可能通过调整CsHB1、LEC1/B3基因的表达促进体细胞胚发生。【Objective】Somatic embryogenesis(SE)is widely used in the conservation and utilization of plant germplasm resources.However,there is significant variation in the somatic embryogenesis(SE)capacity of calls derived from different citrus varieties.Furthermore,their SE capacity gradually dimin-ishes during culture,posing a significant hindrance to the conservation and utilization of citrus germ-plasm resources.CsHB1,an HD-ZIP II gene associated with enhancing SE,was isolated from a citrus variety exhibiting robust SE capabilities.In this study,we harnessed the promoter of CsHB1(pCsHB1)to search for upstream transcription factors to provide reliable candidate genes for the study on plant so-matic embryogenesis.【Methods】To identify the upstream transcription factors of CsHB1,we cloned pCsHB1(-1018 to-558 bp)into pAbAi and utilized a yeast one-hybrid(Y1H)assay to obtain the candi-date transcription factor CsAHL25 from a yeast library.Using SMART,candidate genes were analyzed for domains and named based on annotations in the Citrus Pangenome Breeding Database.The expres-sion pattern of this gene was measured by qRT-PCR in various somatic embryo developmental stages of citrus,aiming to deduce the function of CsAHL25.The gene was cloned and inserted into pRI121,trans-ferred into GV3101 and Marker mixed annotated Nicotiana benthamiana.After 2 d,the localization in the cells was observed using the laser scanning confocal microscopy.CsAHL25 was cloned and inserted into pGADT7 and transfected into Y1HGold with pCsHB1-AbAi for the Y1H assay.A Y1H assay was performed to determine whether the two were complementary or not based on the growth of the yeast cells in the screening medium.The gene was cloned and inserted into the overexpression vector pC-MBAI1300-35S,and pCsHB1(-2377-0 bp)was cloned and inserted into pGreenII 0800-LUC.The two vectors were then separately transferred into GV3101 and mixed to transiently transform N.benthami-ana,with empty vector used as a control.After 2 d,the fluorescence of LUC was observed u

关 键 词：柑橘:体细胞胚发生 HD-ZIP AT-HOOK 

分 类 号：S666[农业科学—果树学]