海岛棉体细胞胚发育特异性基因GbLEC2启动子的克隆和活性检测  

Cloning and Activity Assay of Somatic Embryogenesis-specific Gene GbLEC2 Promoter in Gossypium barbadense

作　　者：赵雪 柳涛 黄明婧 张佳伟 姑扎丽努尔·吐鲁洪 张霞[1] ZHAO Xue;LIU Tao;HUANG Ming-Jing;ZHANG Jia-Wei;GUZHALINUER•Tu-Lu-Hong;ZHANG Xia(College of Life Sciences,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学生命科学学院,乌鲁木齐830052

出　　处：《农业生物技术学报》2023年第4期695-703,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32060044)。

摘　　要：在发育调控因子介导的植株再生过程中,发育调控因子的过量表达会引起植株畸形、花粉败育等多重效应。寻找合适的启动子来驱动发育调控因子在植物中的恰当表达是获得发育正常的再生植株的方法之一。B3转录因子LEAFY COTYLEDON2(LEC2)在胚胎发育过程中特异表达,在种子成熟过程中发挥着重要的调控作用。本研究通过半定量PCR初步检测到海岛棉(Gossypium barbadense)GbLEC2在根、茎、叶和胚性愈伤组织中不表达或表达量微弱,而在球形胚、鱼雷胚和子叶胚中的表达量较高。生物信息学分析结果表明,GbLEC2启动子序列中含有控制种子特异性表达的Skn-1和RY repeat顺式作用元件。为了进一步研究GbLEC2启动子的转录特性,本研究借助瞬时转染法将ProGbLEC2-1(2000 bp)、ProGbLEC2-2(1500 bp)、ProGbLEC2-3(1000 bp)、ProGbLEC2-4(500 bp)等4种不同长度的GbLEC2启动子片段转入海岛棉不同的组织细胞中。GUS染色分析结果显示,ProGbLEC2-3(1000 bp)和ProGbLEC2-4(500 bp)启动子片段仅在体细胞胚胎发生时期有转录活性,这初步符合Lowe等对驱动“发育调控因子”表达的启动子要求。本研究克隆获得的GbLEC2启动子片段有望作为一种新型候选启动子精准调控发育调控因子的表达,从而应用于发育调控因子促植株再生体系介导的新型海岛棉分子育种技术平台。In the process of plant regeneration mediated by developmental regulators,overexpression of developmental regulators could lead to pleiotropic effects such as developmental malformation and pollen abortion.Therefore,finding a suitable promoter to control the suitable expression of developmental regulators in plants may be one of the methods to obtain normal regenerated plants.LEAFY COTYLEDON2(LEC2),a specific B3 transcription factor in plant embryogenesis,plays a crucial regulatory role in seed maturation.In the study,the results of semi-quantitative PCR showed that GbLEC2 was not or weakly expressed in roots,stems,leaves or embryogenic callus in Gossypium barbadense,but significantly highly expressed in globular embryo,torpedo embryo and cotyledon embryo.Bioinformatics analysis displayed that there were 2 seedspecific cis-elements Skn-1 element and RY repeat element in GbLEC2 promoter sequence.To further identify the transcriptional characters of GbLEC2 promoter,4 different GbLEC2 promoter fragments including ProGbLEC2-1(2000 bp),ProGbLEC2-2(1500 bp),ProGbLEC2-3(1000 bp)and ProGbLEC2-4(500 bp)were constructed and then transformed into the different cotton tissues and cells by Agrobacterium-mediated transient transformation method.And the GUS activity assay demonstrated that ProGbLEC2-3(1000 bp)and ProGbLEC2-4(500 bp)promoter fragments displayed a specific transcriptional activity in somatic embryogenesis,which preliminarily met the Lowe’s requirements for the promoters to drive suitable expression of developmental regulators in plant.Therefore,the GbLEC2 promoter fragment cloned in the study is expected to be a new candidate promoter to precisely regulate the expression of developmental regulators,and thus be applied to a novel molecular breeding technolody platform mediated by developmental regulators-promoting plant regenesis in G.barbadense.

关 键 词：海岛棉 GbLEC2启动子 体细胞胚发生 GUS活性 

分 类 号：S5[农业科学—作物学]