红肉脐橙（Citrus sinenesis Osbeck）果实生长发育过程中类胡萝卜素生物合成基因表达的原位检测
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摘要
柑橘是富含类胡萝卜素的水果,大多情况下果皮含量多于果肉。近年来以柑橘红肉突变体为对象的研究引起了越来越广泛的关注,这些突变体一方面赋予本身优良的外观,另一方面为柑橘果实芽变发生的机理研究以及柑橘果肉中的类胡萝卜素生物合成的研究提供了理想的材料。本研究以红肉脐橙(C.sinensis Osbeckcv 'Cara Cara')和清家脐橙(C.sinensis Osbeck cv 'Seika')为实验材料,利用RNA原位杂交技术分析了果实不同组织中类胡萝卜素生物合成相关基因的表达。主要研究结果如下:
1.通过对汁胞进行徒手切片和压片的显微观察,在红肉脐橙汁胞中检测到了红色线状有色体的存在。在番茄和红色葡萄柚中发现了类似的情形,随着果实发育的进行这种形状的有色体在红肉脐橙汁胞中有积累的趋势。
2.将不同发育时期的红肉脐橙和清家脐橙果实样品制成石蜡切片,利用特异性的cRNA探针进行原位杂交,结果表明:psy、pds、lcyb在红肉脐橙和清家脐橙黄皮层中均有表达;mRNA多积累在接近皮层的表层细胞内,在油胞及其周边组织细胞中未能检测到mRNA的积累;不同果实发育期mRNA的积累存在差异;白皮层和汁胞中相应的mRNA分布没有黄皮层组织中明显,但是可看出白皮层和汁胞中psy、pds、lcyb的表达量低于黄皮层组织中的表达量。
Citrus fruit is reported to be carotenoid abundant,normally more carotenoids content was found in the peel than that in the pulp.Some red-fleshed mutant such as 'Cara Cara' navel orange with abnormal lycopene accumulation and attracting appearance are reported to be good research material not only for the elucidation of bud sport mechanism in citrus fruits but also for the reveal of carotenoid biosynthetic pathway in citrus.In the present research,the red-fleshed 'Cara Cara' navel orange(C. sinenesis Osbeck) and the normal orange-fleshed "Seika" navel orange(C.Sinenesis Osbeck) fruit were used to investigate the in situ mRNA accumulating of carotenoid biosynthesis related genes on the cellular level at different ripening stage.Peel and pulp color development in 'Cara Cara' and "Seika" navel orange was detected with chromameter.The main results are as follows:
1.A special red thread-like structure in 'Cara Cara' juice sac was observed with hand-section preparation and fixation treatment under microscope observation.The structure is the same as the plastid structure in lycopene-abundant crop tomato(Lycopersicon esculentum Miller) and also visible in the red pummelo(Citrus grandis L.Osbeck.) pulp.The increasing density of the structure in developing 'Cara Cara' juice sac is correlated with the lycopene accumulation. 2.Paraffin-embed tissue section of flavedo,albedo,segment and juice sac dissected from 'Cara Cara' and 'Seika' navel orange fruits at different stages were prepared for the RNA in-situ hybridization,using the cRNA listed above,the accumulation of psy,pds,lcyb mRNA was visible at the epidermal cell under cortex in the flavedo part of 'Cara Cara' and 'Seika' navel orange,the expression level of such genes were different depending on the fruit maturing process.Seldom mRNA accumulation could be detected at the ocellus or the periphery cells in the flavedo. The expression profile in the albedo and juice sac cells was distance from the corresponding ceils in the flavedo,to our surprise,the mRNA accumulation in the vascular bundle was negligible.And generally,the mRNA expression in the albedo, segment and juice sac was lower than that in the flavedo.
1.通过对汁胞进行徒手切片和压片的显微观察,在红肉脐橙汁胞中检测到了红色线状有色体的存在。在番茄和红色葡萄柚中发现了类似的情形,随着果实发育的进行这种形状的有色体在红肉脐橙汁胞中有积累的趋势。
2.将不同发育时期的红肉脐橙和清家脐橙果实样品制成石蜡切片,利用特异性的cRNA探针进行原位杂交,结果表明:psy、pds、lcyb在红肉脐橙和清家脐橙黄皮层中均有表达;mRNA多积累在接近皮层的表层细胞内,在油胞及其周边组织细胞中未能检测到mRNA的积累;不同果实发育期mRNA的积累存在差异;白皮层和汁胞中相应的mRNA分布没有黄皮层组织中明显,但是可看出白皮层和汁胞中psy、pds、lcyb的表达量低于黄皮层组织中的表达量。
Citrus fruit is reported to be carotenoid abundant,normally more carotenoids content was found in the peel than that in the pulp.Some red-fleshed mutant such as 'Cara Cara' navel orange with abnormal lycopene accumulation and attracting appearance are reported to be good research material not only for the elucidation of bud sport mechanism in citrus fruits but also for the reveal of carotenoid biosynthetic pathway in citrus.In the present research,the red-fleshed 'Cara Cara' navel orange(C. sinenesis Osbeck) and the normal orange-fleshed "Seika" navel orange(C.Sinenesis Osbeck) fruit were used to investigate the in situ mRNA accumulating of carotenoid biosynthesis related genes on the cellular level at different ripening stage.Peel and pulp color development in 'Cara Cara' and "Seika" navel orange was detected with chromameter.The main results are as follows:
1.A special red thread-like structure in 'Cara Cara' juice sac was observed with hand-section preparation and fixation treatment under microscope observation.The structure is the same as the plastid structure in lycopene-abundant crop tomato(Lycopersicon esculentum Miller) and also visible in the red pummelo(Citrus grandis L.Osbeck.) pulp.The increasing density of the structure in developing 'Cara Cara' juice sac is correlated with the lycopene accumulation. 2.Paraffin-embed tissue section of flavedo,albedo,segment and juice sac dissected from 'Cara Cara' and 'Seika' navel orange fruits at different stages were prepared for the RNA in-situ hybridization,using the cRNA listed above,the accumulation of psy,pds,lcyb mRNA was visible at the epidermal cell under cortex in the flavedo part of 'Cara Cara' and 'Seika' navel orange,the expression level of such genes were different depending on the fruit maturing process.Seldom mRNA accumulation could be detected at the ocellus or the periphery cells in the flavedo. The expression profile in the albedo and juice sac cells was distance from the corresponding ceils in the flavedo,to our surprise,the mRNA accumulation in the vascular bundle was negligible.And generally,the mRNA expression in the albedo, segment and juice sac was lower than that in the flavedo.
引文
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