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脱落酸(abscisic acid,ABA)能促进草莓(Fragaria×ananassa)果实的发育成熟,葡萄糖苷酶(β-glucosidase,BG)能通过水解ABA葡萄糖酯(ABA glucose ester,ABA-GE),把非活性的ABA转换成有活性的ABA,因此,BG参与果实发育进程。但是目前还没有遗传学证据来揭示BG对果实发育的作用机理。为了研究BG在果实发育中的作用,本实验以草莓品种甜查理为试材,通过PCR分子克隆的方法分离到3个编码BG的基因Fa BG1、Fa BG2和Fa BG3;首先对3个基因进行生物信息学、时空表达分析,以及测定果实内源ABA含量的变化;其次通过分子调控Fa BG3基因的表达量影响果实内源BG的活性,分析草莓果实的生理指标以及与ABA信号路径、色素代谢和细胞壁代谢相关基因的表达水平。实验结果表明,BG蛋白含有一个Bgl B区域,是能水解其他的复合物的特异区域。随着草莓果实的发育成熟,ABA的含量逐渐升高,Fa BG1基因的表达量一直很低,Fa BG2的表达量呈总体的下降趋势,只有Fa BG3的表达量与ABA含量变化相一致。利用RNA干扰技术调低草莓果实中Fa BG3基因的表达量也下调了Fa BG1和Fa BG2的表达量,同时降低了果实中BG的活性,导致果实中ABA含量、果实色素和可溶性固形物含量降低,提高了果实的硬度等生理指标,与生理指标相关的如色素代谢基因查尔酮合酶(chalcone synthase,CHS)、查尔酮异构酶(chalcone isomerase,CHI)、类黄酮-3-羟化酶(flavonoid-3-hydroxy-lase,F3H)、类黄酮-3-O-葡萄糖基转移酶(UDP Glc-flavonoid 3-O-glucosyl transferase,UFGT)和二氢黄酮醇-4-还原酶(dihydroflavo-nol-4-reductase,DFR),果实软化基因果胶甲酯酶(pectin methylesterase,PE)、伸展蛋白(expansin,EXP)、多聚半乳糖醛酸酶(polygalacturonase,PG)和果胶酸裂解酶(pectate lyases,PL),果实香味代谢基因醌氧化还原酶(quinone oxidoreductase,QR)、酰基转移酶(alcohol acyltransferase,AT)和ABA信号路径中的基因抗副球菌素蛋白(pyrabatin resistance,PYR)、ABA不敏感体1(ABA insensitive 1,ABI1)、ABI3、ABI4和ABI5的表达量等分子指标也出现了不同程度的变化,最后导致了果实延迟成熟。与此相反,Fa BG3基因超表达的草莓果实出现了提前着色成熟的现象。果实生理及分子指标的变化可揭示BG调控草莓果实发育进程的机理。
Abscisic acid (ABA) can promote the maturation of Fragaria × ananassa fruit. The activity of β-glucosidase (BG) can be increased by hydrolyzing ABA glucose ester (ABA-GE) Of ABA into active ABA, therefore, BG is involved in the process of fruit development. However, there is no genetic evidence to reveal the mechanism of action of BG on fruit development. In order to study the role of BG in fruit development, we used the strawberry variety Sweet Charlie as test material and isolated three genes encoding BG, Fa BG1, Fa BG2 and Fa BG3 by PCR molecular cloning. Bioinformatics, spatiotemporal expression analysis, and the determination of endogenous ABA content in fruit. Secondly, the expression of Fa BG3 gene was influenced by molecular regulation of endogenous BG activity in fruit. The physiological indexes of strawberry fruit and the correlation with ABA signal pathway, And cell wall metabolism related gene expression levels. The experimental results show that the BG protein contains a Bgl B region, which is a specific region that can hydrolyze other complexes. With the maturation of strawberry fruit, the content of ABA gradually increased, the expression level of Fa BG1 gene was always low, the expression level of Fa BG2 showed an overall downward trend, and only the expression level of Fa BG3 was consistent with the change of ABA content. Down-regulation of Fa BG3 gene expression in strawberry fruits by RNAi also decreased the expression of Fa BG1 and Fa BG2 and decreased the activity of BG in the fruits, resulting in a decrease of ABA content, fruit pigment and soluble solids contents , Increased the hardness of the fruit and other physiological indicators, and physiological indicators such as the pigment metabolism genes chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxy Flavonoid-3-hydroxy-lase (F3H), UDP Glc-flavonoid 3-O-glucosyl transferase (UFGT) and dihydroflavonol- 4-reductase dihydroflavo-nol-4-reductase (DFR), fruit softening gene pectin methylesterase (PE), expansin (EXP), polygalacturonase (PG) and pectic acid The expression of pectate lyases (PL), quinone oxidoreductase (QR), alcohol acyltransferase (AT), and pyrabatin resistance (PYR) ), ABA insensitive 1 (ABI 1), the expression of ABI3, ABI4 and ABI5 and other molecular indicators also showed varying degrees of change, and finally led to delayed maturation of fruit. In contrast, strawberry fruit over-expression of Fa BG3 gene appeared premature coloring maturation. Physiological and molecular indicators of fruit changes reveal the mechanism of BG regulation of strawberry fruit development.