豌豆蚜與桃蚜 C002 與 forkhead 基因於發育時期之分析

Abstract

蚜蟲以刺吸植物韌皮部汁液維生，利用唾腺分泌產生膠狀唾液與水狀唾液，以協助取食。在吸食汁液的同時會獲得植物體的病原菌，唾腺能夠協助傳播植物病原菌。解析蚜蟲唾腺之形成以及參與形成的基因，能夠作為未來抑制唾腺形成，以及減少蚜蟲取食危害應用之基礎。蚜蟲胚胎唾腺之形成僅在古老文獻有所著墨，並未有分子標記之依據。C002 為蚜蟲特有之唾腺基因，過去研究顯示 C002 會協助蚜蟲將口針刺入篩板，在蚜蟲唾腺中大量被表現，抑制表現除了會使蚜蟲壽命減短，甚至會影響子代數目。影響子代的原因是否與 C002 基因在胚胎發育中有關並未有深入研究。因此，我於本研究中即是利用整體原位雜合技術，以及擬南芥轉殖株於基因默化之應用，探討 C002 mRNA 在豌豆蚜與桃蚜胚胎發育過程中如何被表現，以及此表現與唾腺發育之關係。與此同時，我也選殖保守表現於動物外胚層的基因 forkhead (fkh)， fkh 基因已被證實會在果蠅唾腺中被表現，利用 fkh mRNA 整體原位雜合結果，以研究同樣發育自外胚層的唾腺，希望能夠建立蚜蟲胚胎發育。整體原位雜合結果顯示 C002 mRNA 被表現於晚期發育之胚胎主唾腺上，此時蚜蟲唾腺已完成發育，顯示 C002 與唾腺發育並無相關。而 fkh 於胚胎之前口凹與後口凹以及頭部外胚層被表現，此結果顯示蚜蟲 fkh 基因與其他動物相同，表現與調控外胚層之發育，同時參與原始唾腺之形成。利用 fkh mRNA標記胚胎之前口凹與後口凹，發現蚜蟲前口凹形成早於後口凹，與果蠅相反。在 RNA 干擾技術的應用方面，由於餵食擬南芥轉殖株在基因抑制上效果不彰，因此未能在功能取向上，研究 C002 基因於蚜蟲胚胎發育過程所扮演的角色。

Aphids are phloem-feeding insects and produce two types of saliva from the salivary glands. When taking fluid from the plants, aphids can meanwhile transmit pathogens into the plants. Salivary glands of aphids play a critical role both in pathogen transmission and also stylet penetration. It is worthy to reveal how salivary glands form in aphids. The formation of salivary glands can provide the foundation for reducing the damage caused by aphids. C002, an aphid-specific salivary gland gene, was identified to regulate stylet’s contact with the sieve elements. The knockdown of C002 will shorten the life span of aphids and even reduce fecundity of aphids. I thus aim to study how C002 mRNA is expressed during embryogenesis and how it relates to the formation of the salivary glands. forkhead (fkh), a conserved gene in animals, is another target for monitoring the development of the salivary glands in aphids. Results of whole mount in situ hybridization showed that C002 mRNA was expressed in the principal glands of late-stage embryos but expression of fkh had a broader range in the embryonic anterior and alimentary tract. fkh is expressed by ectodermal stomodeum and proctodeum, which is the same as the expression pattern of Drosophila fkh. However the formation of ectodermal stomodeum in aphids is prior to ectodermal proctodeum that is just the opposite in Drosophila. After feeding the dsC002 transgenic Arabidopsis, the knockdown effect of C002 is not significant.