过量表达RdreB1BI对草莓果实品质及相关基因的影响

doi:10.3864/j.issn.0578-1752.2018.07.013

摘要/Abstract

摘要： 【目的】研究外源基因RdreB1BI转入对‘红颊’草莓果实品质及相关基因表达的影响，揭示RdreB1BI在草莓果实品质调控中的作用及分子机理。【方法】以5个转RdreB1BI株系及非转基因‘红颊’草莓全红期果实为材料，测定果实纵横径、单果重、可溶性糖、可溶性蛋白、抗坏血酸等风味物质及花青苷、总黄酮、总酚等着色物质的含量。应用BLASTn、GENEFINDER和PlantCARE等生物信息学方法分析外源基因和7个次生代谢产物合成途径相关基因（RdreB1BI、Fractin、FvC4H、FvCCR2、FvGST、FvF3H、FvDFR和FvMYB306）的结构，并预测基因启动子作用元件。采用qRT-PCR定量法检测相关基因表达量，应用7300 system软件和2^-△△Ct法分析数据。对生理生化及分子数据进行方差及相关性分析，综合讨论RdreB1BI的转入对‘红颊’草莓果实品质及相关基因表达的影响。【结果】转基因‘红颊’草莓株系与野生型果实单果重的范围为11.75—15.42 g，纵径和横径的范围分别为35.12—40.42 mm及28.73—32.6 mm，仅株系8果实纵径显著大于株系7，其余指标间差异不显著。其中转基因株系1和7的花青苷含量显著高于野生型；转基因株系1、7及8总黄酮含量显著高于野生型；各转基因株系的总酚含量均显著高于野生型。转基因株系1、7和8果实的可溶性糖含量显著高于野生型（21.70 mg·g^-1 FW），分别为野生型的2.87、3.39和3.35倍。可溶性固形物含量与可溶性糖含量呈正相关（r=0.811^*），但样品果实的可溶性固形物含量间不存在显著性差异。转基因株系草莓成熟果实果肉中氨基酸含量为0.2580—0.3950 g/100 g FW，野生型果实的氨基酸含量为0.5151 g/100 g FW，显著高于各转基因株系草莓果实；转基因株系1和7的可滴定酸含量显著高于野生型；转基因株系1果实的抗坏血酸含量为168.35 mg/100 g FW，显著高于野生型（92.50 mg/100 g FW）。转基因株系9及10果实的可溶性蛋白含量分别为25.97和25.86 mg·g^-1 FW，显著高于野生型（22.93 mg·g^-1 FW）。RdreB1BI、FvCCR2cinnamoyl-CoA reductase 2-like）和FvMYB306（myb-related protein 306）在各转基因株系中表达量显著高于野生型。分析发现差异表达基因启动子区域包含多种高等植物顺势调控元件，主要有对真核生物起增强基因转录效率的CAAT-box和核心启动子元件TATA-box。同时还包含G-Box、G-box、MBS、ARE、5UTR Py-rich stretch等能影响各基因对光的响应、在苯丙烷代谢途径中起调节作用的顺式作用元件。【结论】RdreB1BI调控相关基因参与转化体果实发育和成熟，提高光的有效性，活化黄酮类生物合成通路关键基因，差异基因中均包含大量光诱导响应元件，FvCCR2及FvMYB306的表达促进了总黄酮、酚类物质及花青苷等次生代谢物的合成。转入RdreB1BI使草莓果实的多项营养成分和着色物质含量显著增加，改善了草莓的果实品质。

关键词: 草莓, RdreB1BI, 过量表达, 果实品质, 启动子

Abstract: 【Objective】 The effect of exogenous RdreB1BI transformation to the fruit quality and related gene expression in ‘Benihoppe’ strawberry were analyzed to reveal the function and molecular mechanism of quality regulation in transgenic strawberry fruit. 【Method】 The edible organs on five RdreB1BI transgenic strawberry lines and non-transgenic ‘Benihoppe’ strawberries (fruits in full red stage) were used as material to test and determine the vertical diameter, transverse diameter, weight, contents of flavor substance (soluble sugar, soluble protein, ascorbic acid) and coloring materials (anthocyanin, flavonoids and total phenol). The structure of the exogenous gene and seven secondary metabolites synthesis pathway-related genes (RdreB1BI, Fractin, FvC4H, FvCCR2, FvGST, FvF3H, FvDFR and FvMYB306) and the elements of gene promoter were analyzed and were predicted by bioinformatics methods such as BLASTn, GENEFINDER, and PlantCARE. The expression of related genes was detected by qRT-PCR. The data were analyzed by 7300 system software and 2^-△△Ct method. The variance and correlation analysis of physiological, biochemical and molecular data were analyzed. The effects of RdreB1BI on the fruit quality and related gene expression of ‘Benihoppe’ strawberry were discussed comprehensively. 【Result】 The fresh weight of transgenic and non-transgenic strawberries was ranging from 11.75 to 15.42 g. The vertical and transverse diameters were ranging from 35.12 to 40.42 mm and 28.73 to 32.6 mm, respectively. Only the volume diameter of transgenic line 8 was significantly higher than that of line 7, and there were no significant differences between the other samples. The contents of anthocyanin in line 1 and line 7 were significantly higher than those of the control. The contents of total phenol in transgenic line1 and line 7 were significantly higher than that in control. The contents of total phenol in each line were significantly higher than that in the control. The soluble sugar contents in the transgenic line 1, line 7 and line 8 were significantly higher than that of the control (21.70 mg?g^-1 FW), which were 2.87, 3.39 and 3.35 times of the control. There was a positive correlation between soluble solids contents and soluble sugar contents (r=0.811*), but there was no significant difference in the soluble solids contents of the samples. The contents of amino acids in the fruiting fruits of the transgenic lines were ranging from 0.2580 to 0.3950 g/100 g FW. The content of amino acids in the control was 0.5151 g/100 g FW, which was significantly higher than that of the transgenic lines. The contents of titratable acid in the transgenic line 1 and line 7 were significantly higher than that in the control. The content of ascorbic acid in line 1 was 168.35 mg/100 g FW, which was significantly higher than that of the wild-type of 92.50 mg/100 g FW. The contents of soluble protein in transgenic line 9 and line 10 were 25.97 and 25.86 mg?g^-1 FW, respectively, which was significantly higher than that of control (22.93 mg?g^-1 FW). The expression levels of RdreB1BI, FvCCR2 (cinnamoyl-CoA reductase 2-like) and FvMYB306 (myb-related protein 306) in the transgenic lines were significantly higher than that of the wild-type. It was found that the promoter region of differentially expressed genes contained a variety of higher plant cis-acting elements, mainly were CAAT-box and TATA-box, which had enhanced transcription efficiency of eukaryotes. It also included some the cis-acting elements such as G-Box, G-box, MBS, ARE, 5UTR Py-rich stretch, which can affect the response of each gene to light and play a regulatory role in the phenylpropane metabolic pathway. 【Conclusion】 RdreB1BI regulated the related genes involved in the development and maturation of transformants, improved the effectiveness of light, activated the key genes of flavonoid biosynthetic pathway. Differentially expressed genes contained a large quantity of light-induced response elements. The expression of FvCCR2 and FvMYB306 promoted the synthesis of secondary metabolites such as flavonoids, phenols, anthocyanins and total phenols. The transformation of RdreB1BI gene resulted in a significant increase in the nutrient and coloring material contents and improved the fruit quality in strawberry.

Key words: strawberry (Fragaria×ananassa Duch.), RdreB1BI, overexpression, fruit quality, promotor

阎依超，万春雁，古咸彬，郭成宝，陈月红，高志红. 过量表达RdreB1BI对草莓果实品质及相关基因的影响[J]. 中国农业科学, 2018, 51(7): 1353-1367.

YAN YiChao, WAN ChunYan, GU XianBin, GUO ChengBao, CHEN YueHong, GAO ZhiHong. Effect of RdreB1BI Gene Overexpression on Fruit Quality and Related Gene Expression in Strawberry[J]. Scientia Agricultura Sinica, 2018, 51(7): 1353-1367.

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