杧果MiOFP1基因的表达与功能分析

Expression and functional analysis of MiOFP1 gene in mango

【目的】卵形家族蛋白(ovate family proteins,OFPs)在植物生长发育及逆境响应过程中扮演重要角色。前期通过杧果成花基因酵母文库筛选,获得了一个MiOFP1基因,为明确其功能,对MiOFP1的表达模式和转基因功能开展了研究。【方法】在本研究中分析了杧果MiOFP1的启动子序列;通过实时荧光定量PCR技术分析MiOFP1在杧果不同组织器官和不同生长发育期叶片中的表达模式;转化构建好的超量表达载体并侵染拟南芥研究MiOFP1的功能。【结果】四季蜜杧MiOFP1启动子包含激素响应元件:ABA响应元件、GA响应元件、SA响应元件和乙烯响应元件,逆境响应元件:盐响应元件、脱水响应元件、MYC转录因子和MYB转录因子结合位点。组织特异性表达分析显示,MiOFP1在各组织器官中均有表达,且在童期实生树和成年期嫁接树的茎中表达量最高,在成熟果实中表达量最低;嫁接树不同成花发育时期表达分析结果显示,MiOFP1在营养生长期的叶中表达量最高,在成花诱导期和花发育期表达水平较低。转基因功能研究显示,超量表达MiOFP1的拟南芥出现晚花表型,抽薹期叶片中成花抑制基因FLOWERING LOUS C(FLC)的表达水平显著上调,而成花促进基因FLOWERING LOCUS T(FT)的表达水平显著下调。逆境胁迫处理显示,ABA处理显著抑制拟南芥种子的萌发与根的伸长,但通过转基因显著提高了拟南芥种子的萌发率,降低了拟南芥根长对ABA的敏感性。进一步分析显示,MiOFP1显著提高了拟南芥在ABA处理后的脯氨酸含量和过氧化物酶活性,上调了ABA代谢相关基因的表达水平。【结论】明确了杧果MiOFP1抑制成花,且降低了转基因植株对ABA的敏感性,为进一步探索杧果MiOFP1参与杧果成花和逆境胁迫应答的分子机制奠定基础。

【Objective】Ovate Family Proteins(OFPs)play roles in the growth and development,hormone response and stress response of plants.In our previous study,the MiOFP1 gene was obtained from a screen mate and plate library of mango flowering genes.However,the information and functional characteristics of the MiOFP1 gene in mango were limited.To clarify the function of the MiOFP1 gene,its expression pattern and transgenic function were studied.The exploration of the MiOFP1 gene would provide a reference for further exploring the molecular mechanism of the MiOFP1 gene in mango flowering and resistance to adversity.【Methods】To learn more about the expression and regulation characteristics of the MiOFP1 gene,the promoter sequences in the 2000 bp region upstream of the coding region were analysed,and cis-acting elements of the promoter were analysed by NEW PLACE and illustrated via TBtools.The expression of the MiOFP1 in different tissues and different growth stages was verified and analysed by quantitative real-time PCR(qRT-PCR).The transgenic function of the MiOFP1 was investigated by overexpression of it in Arabidopsis thaliana.The phenotypes of homozygous plants of the T3 generation were observed,and the expression of the FLOWERING LOCUS T(FT)and FLOWERING LOUS C(FLC)in Arabidopsis thaliana was determined.The seeds of the T3 generation and seedlings of the T4 generation were treated with ABA,the germination rate and root length were observed and photographed,the physiological indexes,such as proline(Pro)content and peroxidase(POD)activity,and the expression of ABA-related genes,such as ABA-INSENSITIVE1(ABI1),MOTHER OF FT AND TFL(MFT),DESICCATION-RESPONSIVE PROTEIN 29B(RD29B)and Ninecis-epoxycarotenoid dioxygenase 3(NCED3),were determined.All data were analysed with IBM SPSS Statistics Version 22(SPSS,Inc.,Chicago,IL,USA),and the statistical validity was analysed by oneway ANOVA.Duncan’s multiple range test was applied.【Results】The promoter sequences of the MiOFP1 contain many hormone response elements,such as abscisic acid(ABA),gibberellin(GA),salicylic acid(SA)and ethylene response elements,and stress response elements,such as Slat,Dehydration,MYB and MYC binding sites.The results of tissue-specific expression analysis showed that the MiOFP1 gene was expressed in all tissues and organs in different development stages of mango,with the highest expression in the juvenile and adult stems and the lowest expression in the ripe fruit.The temporal expression pattern analysis showed that the MiOFP1 was highest in the vegetative growth stage but lower in the flower induction stage and flower development stage and gradually increased in the leaves during the fruit developmental stage.The transgenic Arabidopsis overexpression of the MiOFP1 gene showed that the bolting time and flowering time of three overexpressing lines were 2-4 days later than those of WT.The detection of the expression level of endogenous flowering genes in Arabidopsis showed that the MiOFP1 significantly increased the expression level of the flowering inhibiting gene AtFLC in transgenic Arabidopsis but significantly decreased the expression level of the flowering promoting gene AtFT.The ectopic expression of the MiOFP1 in transgenic plants resulted in higher tolerance to ABA.More specifically,the transgenic plants showed high ABA level tolerance manifested as increased seed germination and seedling growth.In the medium without ABA,the transgenic seeds germinated slightly faster than WT seeds;on the medium supplemented with ABA,not only containing 2μmol·L^(-1) ABA but also containing 5μmol·L^(-1) ABA,the germination of all seeds was inhibited,and the germination of transgenic seeds was significantly faster than that of WT seeds.For root length,no significant difference was observed in growth phenotype or root length between the transgenic Arabidopsis overexpression of the MiOFP1 gene and the WT plants under normal growth;however,the transgenic plants showed significantly greater root elongation than WT plants under 5μmol·L^(-1) ABA and 10μmol·L^(-1) ABA conditions.These results suggested that heterologous overexpression of the MiOFP1 could promote root elongation in Arabidopsis.Furthermore,under 20μmol·L^(-1) ABA stress,the Pro content and POD activity were increased,and the transcript levels of the several stress-responsive genes(AtABI1,AtMFT,AtRD29B and NCED3)in the transgenic lines were significantly upregulated compared with those in wild-type plants.In addition,the ectopic expression of the MiOFP1 in Arabidopsis resulted in insensitivity to ABA.【Conclusion】Promoter sequence analysis showed that the MiOFP1 promoters contained multiple hormone and stress cis-regulatory elements.Tissue expression analysis showed that the MiOFP1 was mainly expressed in juvenile and adult stems.The temporal expression analysis showed that the MiOFP1 was highly expressed in the vegetative growth stage.Furthermore,the overexpression of the MiOFP1 had a significant impact on the bolting time and flowering time.On the other hand,compared with those in WT plants,the overexpression of the MiOFP1 would improve root length,survival rate,Pro content,POD activity,and the expression of the ABA-related genes under ABA conditions.We speculate that it is the key gene that affects mango flowering correlation and participates in the ABA response.