油梨PaWRI1和PaWRI2基因的克隆、序列分析及表达研究
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摘要
以‘哈斯’油梨(PerseaamericanaMill.)为试材,通过RACE及RT-PCR技术分别克隆得到2个AP2/EREBP类转录因子WRI1和WRI2基因的c DNA全长,分别命名为PaWRI1(GenBank登录号:MH367865)和PaWRI2(GenBank登录号:MH367866)。其中,PaWRI1基因全长为1 396 bp,含1 155 bp开放阅读框,编码384个氨基酸;PaWRI2基因全长为1 782 bp,含1 287 bp开放阅读框,编码428个氨基酸。PaWRI1和PaWRI2基因属于AP2/EREBP类转录因子家族,其编码氨基酸序列中分别均存在2个和1个典型的AP2/ERF DNA结合位点。实时荧光定量PCR检测结果表明:在油梨果实发育过程中,果肉中PaWRI1基因的相对表达量始终快速上升,PaWRI2基因的相对表达量先少量下降,而后缓慢上升;PaWRI1基因相对表达量变化与油脂积累的变化较一致,PaWRI2基因相对表达量变化与油脂积累的变化不一致。因此,推测在油梨果实发育过程中,果肉中PaWRI1基因可能参与调控油脂积累过程。
Two AP2/EREBP transcription factor genes, designated as PaWRI1(GenBank accession No. MH367865)and PaWRI2(GenBank accession No. MH367866), were isolated from avocado(Persea americana Mill.) by RACE and reverse transcription PCR technologies. The full-length of cDNA of PaWRI1 was 1 396 bp with an open reading frame of1 155 bp, and encoded a putative protein of 384 amino acids. The full-length of cDNA of PaWRI2 was 1 782 bp with an open reading frame of 1 287 bp, and encoded a putative protein of 428 amino acids. PaWRI1 and PaWRI2 belonged to the AP2/EREBP family of transcription factors, and the amino acid sequences encoded by PaWRI1 and PaWRI2 contained two and one typical AP2/ERF DNA-binding domains, respectively. Real-time quantitative PCR revealed that the relative expression amount of PaWRI1 consistently and rapidly increased during avocado mesocarp development; meanwhile the relative expression amount of PaWRI2 presented a gently descending trend subsequently followed by a small increase. The comprehensive analyses indicated that the change in the relative expression amount of PaWRI1 was consisted with that of oil content, while that of PaWRI2 was not agreement with that of oil content during avocado mesocarp development. Therefore,PaWRI1 might participate in the regulation of oil accumulation during avocado mesocarp development.
Two AP2/EREBP transcription factor genes, designated as PaWRI1(GenBank accession No. MH367865)and PaWRI2(GenBank accession No. MH367866), were isolated from avocado(Persea americana Mill.) by RACE and reverse transcription PCR technologies. The full-length of cDNA of PaWRI1 was 1 396 bp with an open reading frame of1 155 bp, and encoded a putative protein of 384 amino acids. The full-length of cDNA of PaWRI2 was 1 782 bp with an open reading frame of 1 287 bp, and encoded a putative protein of 428 amino acids. PaWRI1 and PaWRI2 belonged to the AP2/EREBP family of transcription factors, and the amino acid sequences encoded by PaWRI1 and PaWRI2 contained two and one typical AP2/ERF DNA-binding domains, respectively. Real-time quantitative PCR revealed that the relative expression amount of PaWRI1 consistently and rapidly increased during avocado mesocarp development; meanwhile the relative expression amount of PaWRI2 presented a gently descending trend subsequently followed by a small increase. The comprehensive analyses indicated that the change in the relative expression amount of PaWRI1 was consisted with that of oil content, while that of PaWRI2 was not agreement with that of oil content during avocado mesocarp development. Therefore,PaWRI1 might participate in the regulation of oil accumulation during avocado mesocarp development.
引文
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[15]Tajima D,Kaneko A,Sakamoto M,et al.Wrinkled 1(WRI1)homologs,AP2-type transcription factors involving master regulation of seed storage oil synthesis in castor bean(Ricinus communis L.).American Journal of Plant Sciences,2013,4:333-339.
[16]Maeo K,Tokuda T,Ayame A,et al.An AP2-type transcription factor,WRINKLED1,of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis.The Plant Journal,2009,60(3):476-487.
[17]Li Q,Shao J H,Tang S H,et al.Wrinkled1 accelerates flowering and regulates lipid homeostasis between oil accumulation and membrane lipid anabolism in Brassica napus.Frontiers in Plant Science,2015,6:1015.
[18]Ruuska S A,Girke T,Benning C,et al.Contrapuntal networks of gene expression during Arabidopsis seed filling.The Plant Cell,2002,14(9):1191-1206.
[19]Baud S,Mendoza M S,To A,et al.WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis.The Plant Journal,2007,50(5):825-838.
[20]Baud S,Wuilleme S,To A,et al.Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis.The Plant Journal,2009,60(6):933-947.
[21]Wu X L,Liu Z H,Hu Z H,et al.Bn WRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.Journal of Integrative Plant Biology,2014,56(6):582-593.
[22]Kilaru A,Cao X,Dabbs P B,et al.Oil biosynthesis in a basal angiosperm:transcriptome analysis of Persea Americana mesocarp.BMC Plant Biology,2015,15:203.
[23]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25(4):402-408.
[24]Shen B,Allen W B,Zheng P Z,et al.Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize.Plant Physiology,2010,154(2):980-987.
[25]Adhikari N D,Bates P D,Browse J.WRINKLED1 rescues feedback inhibition of fatty acid synthesis in hydroxylase-expressing seeds.Plant Physiology,2016,171(1):179-191.
[26]Qu J,Ye J,Geng Y F,et al.Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing.Plant Physiology,2012,160:738-748.
[2]Vinha A F,Moreira J,Barreira S V P.Physicochemical parameters,phytochemical composition and antioxidant activity of the Algarvian avocado(Persea americana Mill.).Journal of Agricultural Science,2013,5(12):100-109.
[3]Galv?o M D S,Narain N,Nigam N.Influence of different cultivars on oil quality and chemical characteristics of avocado fruit.Food Science Technology,Campinas,2014,34(3):539-546.
[4]Ge Y,Si X Y,Cao J Q,et al.Morphological characteristics,nutritional quality,and bioactive constituents in fruits of two avocado(Persea americana)varieties from Hainan province,China.Journal of Agricultural Science,2017,9(2):8-17.
[5]黄胜和,丁永红,李东明,等.植物含油量相关转录因子WRINKLED1的研究进展.生命科学,2012,24(3):262-265.
[6]丁霄,杨淑巧,许琦,等.转录因子WRI1在主要作物中的研究进展.分子植物育种,2015,13(3):697-701.
[7]张琳.椰子胚乳中WRI1-like类转录因子Co WRI1基因的克隆和功能初步分析.海口:海南大学,2011.
[8]Dussert S,Guerin C,Andersson M,et al.Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition.Plant Physiology,2013,162:1337-1358.
[9]Focks N,Benning C.Wrinkled1:a novel,low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism.Plant Physiology,1998,118:91-101.
[10]To A,Joubès J,Barthole G,et al.WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis.The Plant Cell,2012,24(12):5007-5023.
[11]Cernac A,Benning C.WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis.The Plant Journal,2004,40(4):575-585.
[12]Pouvreau B,Baud S,Vernoud V,et al.Duplicate maize Wrinkled1transcription factors activate target genes involved in seed oil biosynthesis.Plant Physiology,2011,156:674-686.
[13]李鑫,王正明,薛伟,等.棉花中一个新型转录因子Gh WRI1的表达特征与功能分析.生物技术通报,2013(6):80-86.
[14]施春霖,刘聪,肖旦望,等.甘蓝型油菜WRI1基因cDNA的克隆与序列分析.湖南农业大学学报(自然科学版),2013,39(3):247-252.
[15]Tajima D,Kaneko A,Sakamoto M,et al.Wrinkled 1(WRI1)homologs,AP2-type transcription factors involving master regulation of seed storage oil synthesis in castor bean(Ricinus communis L.).American Journal of Plant Sciences,2013,4:333-339.
[16]Maeo K,Tokuda T,Ayame A,et al.An AP2-type transcription factor,WRINKLED1,of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis.The Plant Journal,2009,60(3):476-487.
[17]Li Q,Shao J H,Tang S H,et al.Wrinkled1 accelerates flowering and regulates lipid homeostasis between oil accumulation and membrane lipid anabolism in Brassica napus.Frontiers in Plant Science,2015,6:1015.
[18]Ruuska S A,Girke T,Benning C,et al.Contrapuntal networks of gene expression during Arabidopsis seed filling.The Plant Cell,2002,14(9):1191-1206.
[19]Baud S,Mendoza M S,To A,et al.WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis.The Plant Journal,2007,50(5):825-838.
[20]Baud S,Wuilleme S,To A,et al.Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis.The Plant Journal,2009,60(6):933-947.
[21]Wu X L,Liu Z H,Hu Z H,et al.Bn WRI1 coordinates fatty acid biosynthesis and photosynthesis pathways during oil accumulation in rapeseed.Journal of Integrative Plant Biology,2014,56(6):582-593.
[22]Kilaru A,Cao X,Dabbs P B,et al.Oil biosynthesis in a basal angiosperm:transcriptome analysis of Persea Americana mesocarp.BMC Plant Biology,2015,15:203.
[23]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25(4):402-408.
[24]Shen B,Allen W B,Zheng P Z,et al.Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize.Plant Physiology,2010,154(2):980-987.
[25]Adhikari N D,Bates P D,Browse J.WRINKLED1 rescues feedback inhibition of fatty acid synthesis in hydroxylase-expressing seeds.Plant Physiology,2016,171(1):179-191.
[26]Qu J,Ye J,Geng Y F,et al.Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing.Plant Physiology,2012,160:738-748.