陆地棉BABY BOOM基因的克隆及特征分析  

Cloning and Characterization Analysis of the BABY BOOM Gene in Gossypium hirsutum

作　　者：束立哲 宫则宇 雷忠萍[3] 唐保善[1] 卢碧霞[1] 宋银 贺道华[1] SHU Li-Zhe;GONG Ze-Yu;LEI Zhong-Ping;TANG Bao-Shan;LU Bi-Xia;SONG Yin;HE Dao-Hua(College of Agronomy,Northwest A&F University,Yangling 712100,China;College of Innovation and Experiment,Northwest A&F University,Yangling 712100,China;College of Life Sciences,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学农学院,杨凌712100 [2]西北农林科技大学创新实验学院,杨凌712100 [3]西北农林科技大学生命科学学院,杨凌712100

出　　处：《农业生物技术学报》2021年第5期885-899,共15页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFD0100300);陕西省自然科学基金(2019JQ645);现代农业产业技术体系建设专项资金(CARS-15-44);全省种质资源保护利用(20171010000004)。

摘　　要：BABY BOOM(BBM)是植物特有的AP2/ERF(APETALA2/ethylene-responsive factor)家族转录因子,在体细胞胚胎发生过程中发挥重要的调控作用。本研究利用同源克隆技术从陆地棉(Gossypium hirsutum)品种’豫早1号’(YZ-1)中克隆到GhBBMa(GenBank No.MW836956)和GhBBMd(GenBank No.MW836957)两个基因,结合3个自然群体的重测序数据和表型数据,以及中棉所24号(ZM24)的基因组数据,通过软件分析BBM基因的DNA序列及其编码的蛋白等特征;通过RNA-seq数据和qRT-PCR实验分析BBM基因表达特征。结果表明,GhBBMa/d分别位于A08和D08染色体上,均由9个外显子组成,长度分别为2028 bp、2025 bp,分别编码675、674个氨基酸。GhBBMa/d氨基酸序列含有2个保守的AP2结构域,无信号肽和跨膜结构;亚细胞定位结果显示其位于细胞质中。系统发育树显示,GhBBMa/d与同属锦葵目的可可树(Theobroma cacao)亲缘关系最近。自然群体的重测序数据表明,GhBBMa和GhBBMd的序列多态性分别为1.222和1.422 SNP/kb,连锁不平衡(linkage disequilibrium,LD)衰减距离为5.54、12.76 kb,说明GhBBMd比GhBBMa受到更强烈的选择压;关联作图显示,GhBBMa/d对马克隆值等性状有显著的表型效应。RNA-seq数据表明GhBBMa/d均在根和胚珠中表达量较高,在其他时空条件下不表达或表达很低;与非胚性愈伤相比,GhBBMd在胚性愈伤中显著高量表达。qRT-PCR实验显示GhBBMa/d均在30 d的胚性愈伤和开花前三天(-3 days post anthesis,DPA)的胚珠中表达量最高。综上所述,本研究克隆的GhBBMa和GhBBMd具有表型效应,在胚胎发生过程中的不同时期表达量不同,可能在体细胞胚胎发生及胚珠发育过程中行使重要的功能,为进一步探究GhBBM参与体细胞胚胎发生过程的分子机制提供参考。BABY BOOM(BBM)proteins are plant-specific transcription factors belonging to APETALA2/ethylene-responsive factor(AP2/ERF)family that may play an important regulatory role in somatic embryogenesis.By means of homology-based cloning,GhBBMa(GenBank No.MW836956)and GhBBMd(GenBank No.MW836957)were obtained from upland cotton(Gossypium hirsutum)cultivar YZ-1.The resequencing and phenotypic data of three natural populations,and the genome sequence of cultivar ZM24 were collected to characterize BBM genes and corresponding proteins.Especially,the expression profiles were analyzed by RNA-seq dataset and qRT-PCR assay.GhBBMa and GhBBMd were located on chromosomes A08 and D08.The opening reading frame(ORF)of GhBBMa/d were both composed of 9 exons,correspondingly with 2028 bp and 2025 bp in coding sequence(CDS),encoding 675 and 674 amino acids,respectively.The GhBBMa/d proteins contained two AP2 domains and excluded signal peptide or transmembrane domains.The bioinformatics subcellular location indicated that GhBBM appeared to accumulate in the cytoplasm.The phylogenetic tree showed that the GhBBMs were closely related to that from Theobroma cacao,which both belonged to the Malvales.The resequencing data of the natural populations showed that the sequence polymorphisms of GhBBMa and GhBBMd were 1.222 and 1.422 SNP/kb,respectively.The linkage disequilibrium(LD)decay distances were over 5.54 kb and 12.76 kb for GhBBMa and GhBBMd,respectively,indicating that GhBBMd was subjected to more evolutional selective pressure than GhBBMa.Association mapping showed that GhBBMa and GhBBMd had significant phenotypic effects on traits such as micronaire values and so on.RNA-seq dataset showed that the expression level of GhBBMa and GhBBMd genes was high in roots and ovules,but was very low in other tissues.GhBBMd was more vigorously transcripted in embryogenic callus than that in non-embryogenic callus.qRT-PCR assays showed that GhBBMa and GhBBMd had the highest expression accumulation in 30 d embryogenic callus and-3 days post anthesis(DPA

关 键 词：陆地棉 BABY BOOM(BBM) 体细胞胚胎发生 基因克隆 QRT-PCR 

分 类 号：S512.3[农业科学—作物学]