摘要
【目的】脂氧合酶LOX是植物香气代谢脂氧合酶途径的起始酶,探究槟子(Malus pumila×M.asiatica)LOX2a基因在果实香气合成中的作用,为LOX2a基因在槟子果实中的功能研究奠定基础。【方法】以槟子转录组和挥发性物质检测数据为基础,筛选并克隆槟子LOX2a基因,利用生物信息学对其功能域、理化性质等进行预测,通过亚细胞定位分析LOX2a在细胞水平的表达位置,利用RT-qPCR检测LOX2a的时空表达水平,结合圆片温育和外源激素处理探究激素对LOX2a基因表达的调控作用。【结果】槟子LOX2a开放阅读框为2721 bp,编码906个氨基酸,含有一个保守的Lipoxygenase功能域,编码蛋白定位于细胞质;系统发育进化分析表明槟子LOX2a与苹果LOX2a同源性最高;槟子LOX2a在根部表达水平最高,种子中最低;在果实发育过程中花后30 d时表达水平最高,随后降低并维持在一定水平,到成熟(花后150 d)时表达水平上升;槟子LOX2a启动子上含有响应脱落酸、茉莉酸甲酯、赤霉素和水杨酸的顺式作用元件,且其表达受到这4种激素及乙烯的诱导。【结论】槟子LOX2a基因可能通过多种激素信号通路参与槟子果实的香气代谢过程。
【Objective】Binzi(Malus pumila×M.asiatica),an old local fruit species in Beijing,belongs to the Rosaceae family.Binzi is one of the varieties,and its fruit can give forth full aroma.Lipoxygenase(LOX),catalyzing the oxygenation of unsaturated fatty acids,contributes to the formation of fruit aroma.However,the LOX has not been reported in Binzi fruit,and the molecular mechanism of aroma formation remains unknown.In the present study,we firstly cloned the LOX2a gene from the Binzi,and identified its expression pattern in different tissues and at different fruit developmental stages.Moreover,the effect of phytohormone on LOX2a gene expression level was analyzed.【Methods】The Binzi fruits were collected on 17 May,16 June,17 July,15 August and 14 September in 2020.We screened one LOX gene based on the results of total volatiles and transcriptome data of Binzi fruit after harvest.The LOX2a protein sequences of different plants with higher homology were downloaded from the NCBI database,and phylogenetic tree was constructed by MEGA 5.1 software through NJ method(Neighbor-Joining).The protein conserved domain,protein secondary and tertiary structure,molecular weight,molecular formula,isoelectric point,and subcellular localization of Binzi LOX2a protein were analyzed using online software.In addition,cis-acting elements in LOX2a promoter region were predicted with PlantCARE software.Binzi LOX2a was cloned into the pCAMBIA-Super1300-GFP vector to construct the 35S::LOX2a-GFP recombinant vector.The recombinant vector and one control GFP vector were respectively transformed into Agrobacterium tumefaciens GV3101 competent cells,and then were transformed into tobacco(Nicotiana×tabacum)leaf cells.After 3 days,the subcellular localization of LOX2a was observed under a Confocal laser scanning microscope.The expression characteristics of LOX2a in different plant tissues(root,stem,leaf,flower and seed)and fruit at different(30,60,90,120and 150 d after flowering period)development stages were detected through the Real-Time fluorescence quantitative PCR.Finally,the fruit at 90 d after flowering period was chosen for further hormone treatment research.Fruit discs(10 mm in diameter and 1 mm in thickness)were prepared with a cork borer.The discs were immediately immersed in equilibration buffer(200 mmol·L-1Mannitol,50 mmol·L-1MES pH 5.5,10 mmol·L-1MgCl2,10 mmol·L-1EDTA,5 mmol·L-1CaCl2and 5 mmol·L-1Vc).The discs were respectively incubated in equilibration buffer with 100 mmol·L-1ABA,100 mmol·L-1MeJA,100 mmol·L-1SA and 200 mmol·L-1GA3.Meanwhile,the fruits were treated with 200 mmol·L-1ethylene and 1 m L·L-11-MCP.Then the expression levels of LOX2a gene after phytohormone treatment were determined by RT-qPCR.【Results】We screened out one LOX gene(named LOX2a)for further research based on the results of Binzi total volatiles and transcriptome.Binzi LOX2a contained 2721 bp of open reading frame and encoded 906 amino acids,which contained a conserved PLN02264(Lipoxygenase)domain.Binzi LOX2a sequence has been uploaded to the NCBI database,and its GenBank No.is ON952464.The physicochemical property results of Binzi LOX2a showed that its protein molecular formula was C4612H7222N1254O1341S27and isoelectric point was 6.97.The protein secondary and tertiary structure prediction results showed that the LOX2a protein mainly contained a-helix,extended strand,and random coil with the proportion of 33.33%,15.56%and 51.10%,respectively.The phylogenetic analysis showed that the Binzi LOX2a had the highest similarity with M.domestica LOX2a.The subcellular localization result showed that the green fluorescence in Nicotiana tabacum leaves transformed with 35S::LOX2a-GFP recombinant vector was only accumulated in the cytoplasm,which was consistent with the result from the Cell-PLoc 2.0 prediction.The results of RT-qPCR showed that LOX2a gene was expressed in roots,stems,leaves,flowers,fruits and seeds.And the expression of LOX2a was higher in roots,followed by the stems,leaves,and the lowest in seeds.The above results indicated the LOX2a gene expression had tissue expression specificity.During Binzi fruit development period,the highest expression level of LOX2a was detected from the 30 d after flowering period,and then increased at 150 d,which accompanied with the Binzi fruit aroma accumulation.The cis-acting elements of the LOX2a promoter consisted of the photo responsive element,hormone response element and stress related component.And the hormone response element included ABA,MeJA,GA and SA responsive element.Combining with fruit disc tissue incubation and hormone treatment,we determined the expression level of LOX2a using the RT-qPCR.It was found that the expression level of LOX2a was significantly higher after treatment with hormone than that in controls(p<0.05),indicating that the LOX2a gene expression was induced by ABA,MeJA,GA,SA and ethylene.【Conclusion】LOX2a gene may be involved in aroma metabolism of Xiangbizi fruit through multiple hormone signaling pathways.
作者
王庆华
高佳慧
王磊
吴文江
郭家选
吴国良
沈元月
WANG Qinghua;GAO Jiahui;WANG Lei;WU Wenjiang;GUO Jiaxuan;WU Guoliang;SHEN Yuanyue(College of Forestry,Henan Agricultural University,Zhengzhou 450002,Henan,China;Beijing Key Laboratory for Agricultural Appli-cation and New Technique,Beijing University of Agriculture,Beijing 102206,China;College of Horticulture,Henan Agricultural Uni-versity,Zhengzhou 450002,Henan,China;College of Agronomy,Henan Agricultural University,Zhengzhou 450002,Henan,China)
出处
《果树学报》
CAS
CSCD
北大核心
2022年第11期1977-1988,共12页
Journal of Fruit Science
基金
国家自然科学基金重点项目(32030100)
国家自然科学基金(32072516)。
