甜瓜α-甘露糖苷酶基因的克隆、表达特性及功能分析
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摘要
甜瓜是具有重要商业价值的园艺作物,对于消费者来说,它的品质是一个重要的质量属性。甜瓜果实中含有香味挥发物、可溶性糖、有机酸、色素、次生代谢物等,可以提供很多人类的日常饮食营养,因此受到人们的喜爱。甜瓜果实不耐贮运,货架期相对较短,在运输和销售过程中伴随着果实的成熟和软化,损失加剧。而且,采摘后果实过度软化引起皱缩、干燥和霉变,严重降低了甜瓜果实的质量和感观性状。
α-甘露糖苷酶(α-mannosidase)是一类关键性糖苷酶,由很多成员组成。它的功能是修剪糖蛋白糖链中的不同甘露糖基,它不仅在糖蛋白的形成过程中发挥重要作用,并且参与果实的成熟与软化。
为了研究甜瓜α-甘露糖苷酶基因(α-Man)在果实发育成熟中的功能,本论文以甜瓜品种河套蜜瓜为研究材料,克隆了甜瓜果实中的α-Man基因全长cDNA。测定了甜瓜根、茎、叶、子房等不同组织和不同发育时期的果实中α-甘露糖苷酶活性,结果表明在子房中的酶活性最低,根、茎、叶组织中的酶活性较高,约为子房中活性的3～5倍。授粉后10～30d的果实中,其活性显著下降,而授粉后40d果实果皮颜色变黄,其活性有所增加。RT-qPCR分析结果显示,α-Man基因在叶片中的表达量最高,子房中表达量最低,叶片中表达量约为子房中的表达量的6倍,授粉后10-30d α-Man基因表达量显著下降,而授粉后40d果皮颜色变黄,其表达量有所回升
利用农杆菌介导的瞬时表达技术,分别将含α-Man基因的超表达载体pPZP221-Man、RNAi载体pART-Manl和pART-Man2的农杆菌菌液注射至成熟期甜瓜果实,进行瞬时表达,并对其进行了表达特性分析。3-5天时,注射超表达载体菌液的果实在注射部位出现了提前变黄的表型。5-7天时,注射RNAi载体菌液的果实在注射部位仍保持绿色(推迟变黄)。α-甘露糖苷酶活性测定结果显示,与非注射部位相比,注射超表达载体的组织α-甘露糖苷酶活性较高,而注射RNAi载体的组织α-甘露糖苷酶活性较低。RT-qPCR分析表明,在瞬时超表达α-Man基因的果实组织中,α-Man基因和ACS6等成熟相关基因表达水平增高,而在瞬时抑制α-Man基因表达的果实组织中,这些基因表达水平有所下降,表明α-Man基因具有促进果实成熟的功能。
应用花粉管通道法转化技术,分别将pPZP221-Man (Oe-Man)和pART-Man1俾(Ri-Man)载体导入甜瓜,经PCR检测证明外源基因已经整合到受体植物的基因组中,分别获得了T1代和T2代转基因植株。与未转基因对照甜瓜相比,转Oe-Man T1代甜瓜果实提前10d成熟,α-Man基因及成熟相关基因表达量显著提高,α-甘露糖苷酶活性也显著升高；转Ri-Man1T2代甜瓜果实成熟期和采后贮藏期显著延长,α-Man基因和成熟相关基因表达量以及α-甘露糖苷酶活性下降。结果表明,甜瓜α-Man基因在甜瓜果实耐贮藏基因工程中具有重要应用价值。
Melon is a commercially important fruit crop, and its texture is an important quality attributes for consumers.
Melon fruit contains aroma volatiles, soluble sugar, organic acids, pigments, and other secondary metabolites, which provides the human daily diet with valuable nutrients. Therefore it is one of the most vital fruits for human.
Melon fruit is not suitable for storage and transportation. Excessive softening exacerbates the damage incurred during handling and sales process, and decreases the shelf life of fruits. The excessive softening causes drying, shrinkage, and pathological disorders, so that it severely reduces the quality and acceptability of melon fruits.
Alpha-mannosidase (a-Man) is a key glycosidase enzyme. The enzyme family consists of many members. The enzyme function is cutting different mannose in the glycoprotein carbohydrate chains, which play an important role not only in the formation process of glycoproteins, but also in the fruit ripening and softening.
The function of a-mannosidase gene was analyzed during fruit ripening in melon. The full-length cDNA of a-Man was cloned.
Activity of the a-mannosidase was analyzed in different organization of melon (roots, stems, leaves, ovary) and in fruit of different development stages. The results showed that a-mannosidase activity was about3-5times higher in the root, stem, leaf tissue than that in the ovary. The enzyme activity was significantly decreased10-30days after pollination (DAP) in melon fruit. When the fruit peel changed into yellow color on the40DAP, the enzyme activity increased.
The real-time quality PCR (RT-qPCR) analysis showed that a-Man was highly expressed in the leaves. The level of the expression was the highest (6times highest) in the leaves. However it was the lowest in the ovary. The level of expression in the fruits was significantly decreased at10-30DAP in melon fruit. When the fruit peel was changed into yellow color at40DAP, the expression increased.
The function of a-mannosidase gene was analyzed during melon fruit ripening by transient expression system. The full-length target gene was constructed into overexpression vector pPZP221and two target gene fragments were constructed into RNAi vector pART27, then transformed into mature green melon fruits by Agrobacterium respectively. The injection site of the fruit transformed by overexpression vector emerged yellow phenotype from3to5days after injection. The fruits injected bacteria RNAi vectors remain green at the injection site (delayed yellowing) from5to7days after injection. Analysis of a-mannosidase activity showed that the enzyme activity in the tissue transformed by overexpression vector was higher than the non-injected tissue, and it was lower in the tissue transformed by RNAi vectors.
RT-qPCR analysis showed that the expression of the a-Man and ACS6etc. genes related to fruit ripening was increased in the injection site of fruit tissues transformed by overexpression vector. However, these genes expression level decreased in the injection site of fruits transformed by RNAi vectors. These results indicated that a-Man gene can promote melon fruit ripening.
pPZP221-Man (Oe-Man) and pART-Manl (Ri-Man) vectors were transformed into melon by pollen tube pathway transformation. It was initially confirmed that the foreign gene was integrated into the genome of the recipient plant by PCR. T1-and T2-generation transgenic plants were obtained. Fruit of Oe-Man T1-generation fully matured earlier10days than non-transgenic fruit. RT-qPCR analysis indicated that the expression of a-Man gene and ripening-related gene was significantly higher than non-transgenic controls. The fruit ripening and postharvest storage period of Ri-Man T2melon fruit have significantly longer, while the a-Man gene expression and a-Man activity decreased correspondingly.
These results showed that a-Man gene could play an important role in genetic engineering of melon fruit storability
α-甘露糖苷酶(α-mannosidase)是一类关键性糖苷酶,由很多成员组成。它的功能是修剪糖蛋白糖链中的不同甘露糖基,它不仅在糖蛋白的形成过程中发挥重要作用,并且参与果实的成熟与软化。
为了研究甜瓜α-甘露糖苷酶基因(α-Man)在果实发育成熟中的功能,本论文以甜瓜品种河套蜜瓜为研究材料,克隆了甜瓜果实中的α-Man基因全长cDNA。测定了甜瓜根、茎、叶、子房等不同组织和不同发育时期的果实中α-甘露糖苷酶活性,结果表明在子房中的酶活性最低,根、茎、叶组织中的酶活性较高,约为子房中活性的3～5倍。授粉后10～30d的果实中,其活性显著下降,而授粉后40d果实果皮颜色变黄,其活性有所增加。RT-qPCR分析结果显示,α-Man基因在叶片中的表达量最高,子房中表达量最低,叶片中表达量约为子房中的表达量的6倍,授粉后10-30d α-Man基因表达量显著下降,而授粉后40d果皮颜色变黄,其表达量有所回升
利用农杆菌介导的瞬时表达技术,分别将含α-Man基因的超表达载体pPZP221-Man、RNAi载体pART-Manl和pART-Man2的农杆菌菌液注射至成熟期甜瓜果实,进行瞬时表达,并对其进行了表达特性分析。3-5天时,注射超表达载体菌液的果实在注射部位出现了提前变黄的表型。5-7天时,注射RNAi载体菌液的果实在注射部位仍保持绿色(推迟变黄)。α-甘露糖苷酶活性测定结果显示,与非注射部位相比,注射超表达载体的组织α-甘露糖苷酶活性较高,而注射RNAi载体的组织α-甘露糖苷酶活性较低。RT-qPCR分析表明,在瞬时超表达α-Man基因的果实组织中,α-Man基因和ACS6等成熟相关基因表达水平增高,而在瞬时抑制α-Man基因表达的果实组织中,这些基因表达水平有所下降,表明α-Man基因具有促进果实成熟的功能。
应用花粉管通道法转化技术,分别将pPZP221-Man (Oe-Man)和pART-Man1俾(Ri-Man)载体导入甜瓜,经PCR检测证明外源基因已经整合到受体植物的基因组中,分别获得了T1代和T2代转基因植株。与未转基因对照甜瓜相比,转Oe-Man T1代甜瓜果实提前10d成熟,α-Man基因及成熟相关基因表达量显著提高,α-甘露糖苷酶活性也显著升高；转Ri-Man1T2代甜瓜果实成熟期和采后贮藏期显著延长,α-Man基因和成熟相关基因表达量以及α-甘露糖苷酶活性下降。结果表明,甜瓜α-Man基因在甜瓜果实耐贮藏基因工程中具有重要应用价值。
Melon is a commercially important fruit crop, and its texture is an important quality attributes for consumers.
Melon fruit contains aroma volatiles, soluble sugar, organic acids, pigments, and other secondary metabolites, which provides the human daily diet with valuable nutrients. Therefore it is one of the most vital fruits for human.
Melon fruit is not suitable for storage and transportation. Excessive softening exacerbates the damage incurred during handling and sales process, and decreases the shelf life of fruits. The excessive softening causes drying, shrinkage, and pathological disorders, so that it severely reduces the quality and acceptability of melon fruits.
Alpha-mannosidase (a-Man) is a key glycosidase enzyme. The enzyme family consists of many members. The enzyme function is cutting different mannose in the glycoprotein carbohydrate chains, which play an important role not only in the formation process of glycoproteins, but also in the fruit ripening and softening.
The function of a-mannosidase gene was analyzed during fruit ripening in melon. The full-length cDNA of a-Man was cloned.
Activity of the a-mannosidase was analyzed in different organization of melon (roots, stems, leaves, ovary) and in fruit of different development stages. The results showed that a-mannosidase activity was about3-5times higher in the root, stem, leaf tissue than that in the ovary. The enzyme activity was significantly decreased10-30days after pollination (DAP) in melon fruit. When the fruit peel changed into yellow color on the40DAP, the enzyme activity increased.
The real-time quality PCR (RT-qPCR) analysis showed that a-Man was highly expressed in the leaves. The level of the expression was the highest (6times highest) in the leaves. However it was the lowest in the ovary. The level of expression in the fruits was significantly decreased at10-30DAP in melon fruit. When the fruit peel was changed into yellow color at40DAP, the expression increased.
The function of a-mannosidase gene was analyzed during melon fruit ripening by transient expression system. The full-length target gene was constructed into overexpression vector pPZP221and two target gene fragments were constructed into RNAi vector pART27, then transformed into mature green melon fruits by Agrobacterium respectively. The injection site of the fruit transformed by overexpression vector emerged yellow phenotype from3to5days after injection. The fruits injected bacteria RNAi vectors remain green at the injection site (delayed yellowing) from5to7days after injection. Analysis of a-mannosidase activity showed that the enzyme activity in the tissue transformed by overexpression vector was higher than the non-injected tissue, and it was lower in the tissue transformed by RNAi vectors.
RT-qPCR analysis showed that the expression of the a-Man and ACS6etc. genes related to fruit ripening was increased in the injection site of fruit tissues transformed by overexpression vector. However, these genes expression level decreased in the injection site of fruits transformed by RNAi vectors. These results indicated that a-Man gene can promote melon fruit ripening.
pPZP221-Man (Oe-Man) and pART-Manl (Ri-Man) vectors were transformed into melon by pollen tube pathway transformation. It was initially confirmed that the foreign gene was integrated into the genome of the recipient plant by PCR. T1-and T2-generation transgenic plants were obtained. Fruit of Oe-Man T1-generation fully matured earlier10days than non-transgenic fruit. RT-qPCR analysis indicated that the expression of a-Man gene and ripening-related gene was significantly higher than non-transgenic controls. The fruit ripening and postharvest storage period of Ri-Man T2melon fruit have significantly longer, while the a-Man gene expression and a-Man activity decreased correspondingly.
These results showed that a-Man gene could play an important role in genetic engineering of melon fruit storability
引文
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