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AP2亚族在植物的生长发育过程中起着至关重要的作用。ANT是一类重要的植物转录因子,与植物器官的发育过程密切相关,属于AP2/EREBP转录因子超家族中花卉同源基因AP2亚族,于1996年由Elliott等人从拟南芥中首次发现[1]。有研究表明,ANT基因的突变会影响拟南芥花瓣的数量,萼片的融合及花器官的位置和生长[2];ANT基因受到生长素信号的诱导影响花器官中营养物质的积累[3];ANT基因的缺失会导致植物叶片器官发育的矮小[4];ANT基因超表达会导致拟南芥各种营养器官,如叶、茎、花梗、萼片、花瓣、雄蕊、雌蕊、胚珠、果实等形态大小的增加[5];ANT基因突变会导致胚珠不正常发育以及雌配子消失[6];ANT基因促进了开花植物叶尖侧器官的生长及极性发育[7];ANT基因参与番茄花器官发育、果实初期颜色和果实大小的发育[8],参与葡萄的浆果大小[9],以及油菜、大白菜叶球的大小的发育[10]。ANT家族在众多植物生长发育中的功能已经得到证实,但在荔枝中的研究尚未见报道。荔枝(Litchi chinensis Soon.)为华南地区产量最大的热带果树之一,在经济、文化和食用价值上都具有不可忽视的地位。在荔枝实际生产过程中常因雄、雌花周期相遇短造成授粉困难,导致坐果不佳,严重影响荔枝果实产量,从而制约了荔枝产业的发展。‘妃子笑’荔枝为偏早熟品种,以肉多、味美、品相佳,产量好等优点在华南地区广泛种植,因雄花比例大,雌花比例小,易受天气影响等问题导致产量不稳定,有研究表明‘妃子笑’胚珠的发育与花型的形成密切相关[11]。随着生物技术的发展,转基因技术不断成熟,已经成为改良作物的主要手段之一[12],挖掘荔枝开花,营养器官发育相关基因,将有助于利用分子育种手段改良荔枝开花及发育过程,从而改良荔枝果实品质。本研究拟从筛选并克隆荔枝花器官相关基因ANT基因入手,通过生物信息学分析手段初步了解荔枝ANT(LcANT)的生物学特征,构建系统进化树,分析起源分化,进行组织特异性表达分析探究LcANT基因在‘妃子笑’荔枝中的表达模式,从而有助于推测ANT类转录因子在‘妃子笑’荔枝发育过程的机理,为调控‘妃子笑’荔枝品种开花结果提供分子水平上的理论基础。
Cloning and Expression Profiling of an AP2 Homologous Gene of Litchi ‘Feizixiao’
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摘要: 根据笔者课题组荔枝基因组数据库,从‘妃子笑’荔枝中克隆了1个AP2同源基因LcANT,其cDNA全长2 087 bp,编码695个氨基酸,含有2个保守的AP2/EREBP结构域。生物信息学分析结果表明,LcANT含有7个内含子,亚细胞定位预测于细胞核,LcANT蛋白为疏水性蛋白,二级结构多为无规则卷曲。通过BLAST比对发现,LcANT与许多植物的ANT转录因子具有高度同源性,与开心果相似度最高,高达82%。利用q−PCR,对LcANT在‘妃子笑’荔枝中的表达模式进行了表征,发现其在春梢、秋梢和花序中表达量较高,暗示其可能参与调控花器官及其他植物营养器官的发育。LcANT基因的获得为后续研究其对荔枝营养器官生长发育的调控作用奠定了基础。Abstract: An AP2 homologous gene LcANT was cloned from Litchi ‘Feizixiao’ (Litchi chinensis Sonn. Feizixiao) based on the litchi whole genome database available from the Environment and Plant Protection Institute. The gene LcANT was 2 087 bp in full-length cDNA, encoded 695 amino acids and contained two AP2/EREBP conserved domains. Bioinformatics analysis showed that the LcANT contained 7 introns, and that the subcellular localization was predicted in the nuclear. The LcANT protein was hydrophobic and its secondary structure wass mostly random coils. BLAST sequence alignment analysis showed that the LcANT had a high homology in amino acid sequences with the ANT transcription factors from other plants, and had a maximum identity of up to 82% with the ANT from pistachio (Pistacia vera). The q-PCR was used to characterize the expression pattern of the LcANT in the tissues of Litchi 'Feizixiao', and the results showed that the LcANT had higher expression levels in spring shoots, ovary, inflorescence, and flower buds, implying that this gene may regulate the growth and development of floral organs and other plant vegetative organs. The acquisition of the LcANT gene provides a molecular basis for further research focused on its function on the growth and development of vegetative organs of Litchi.
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Key words:
- Litchi chinensis Sonn. /
- ANT transcription factor /
- expression analysis
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图 7 LcANT与其他物种ANT蛋白的系统进化关系
Hu:哥伦比亚锦葵;Tc:可可;Dz:榴莲;Pv:开心果;Jr:胡桃;Vv:葡萄;Os:水稻;At:拟南芥;Bn:甘蓝型油菜;Nt:美花烟草;Ns:红花烟草;Mt:蒺藜苜蓿 。
Fig. 7 Phylogenetic relationship of LcANT with ANT of other plant species
Hu: Herrania umbratica; Tc: Theobroma cacao; Dz: Durio zibethinus; Pv: Pistacia vera; Jr: Juglans regia; Vv: Vitis vinifera; Os: Oryza sativa; At: Arabidopsis thaliana; Bn: Brassica napus; Nt: Nicotiana tabacum; Ns: Nicotiana sylvestris; Mt: Medicago truncatula.
图 8 1个蛋白的氨基酸序列比对
黑色区域表示100%的匹配,红色区域表示75%的匹配,蓝色区域表示50%的匹配。
Fig. 8 Multiple alignments between LcANT sequences and other ANT sequences
The black background denotes the conserved amino acid residues (100%); the red background denotes the conserved amino acid residues (>75%); the blue background denotes the conserved amino acid residues (>50%).
图 9 荔枝LcANT基因在不同器官组织中的相对表达量
1. 根;2. 茎;3. 春梢;4. 秋梢;5. 花序;6. 花药;7. 果皮;8. 果肉;9. 种子。
Fig. 9 Relative expression and transcript abundances of LcANT in different organs of litchi
1. Root; 2. Stem; 3. Leaves of spring shoot; 4. Leaves of autumn shoot; 5. Inflorescence; 6. Anther; 7. Pericarp; 8. Pulp; 9. Seed.
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