优雅蝈螽水溶性和类膜结合型海藻糖酶编码cDNA的鉴定及其在体内的表达

海藻糖酶是一种二糖水解酶，催化海藻糖转换为葡萄糖，为昆虫包括发育、壳多糖合成及飞翔代谢在内的多种生理过程所必需。尽管某些昆虫的海藻糖酶基因已被鉴定，但优雅蝈螽的海藻糖酶编码序列尚未见报告。本研究采用RACE结合多重PCR技术，分离鉴定优雅蝈螽的水溶性海藻糖酶（GgTre1）和类膜结合型海藻糖酶（GgTre2-like）的全长编码序列（cDNA），包括携带不同长度3′-非翻译区（3′-UTR）的3个GgTre1 cDNA 亚型（GenBank：No.KY400001-KY400003）和3个GgTre2-like cDNA 亚型（GenBank：No.KY400004-KY400006）。 3个GgTre1 cDNA序列分别为2 107，2 021和1 914 bp，具有相同长度的5′-UTR（33 bp），但3′-UTR 长度不同，分别为322，248和129 bp。GgTre1-2 cDNA含1 740 bp，编码579 个氨基酸残基组成的多肽链，分子量为67.29 kD；与之不同，根据cDNA演绎的GgTre1-1和GgTre1-3序列较GgTre1-2多4个氨基酸残基，多肽链的分子量为67.88 kD。3个GgTre2-like cDNA（GgTre2-like-1，-2和-3）序列全长分别为2 491，2 460 和2 381 bp。5′-UTR 均为284 bp，3′-UTR 分别为398，367和285 bp。GgTre2-like cDNA开阅读框为1 809 bp，编码602 氨基酸残基组成的多肽链，分子量为67.88 kD。实时定量PCR, 分析GgTre1和GgTre2-like基因在雌、雄个体（各20个）的组织特异性表达。结果显示，GgTre1 在卵巢和附腺表达量最高；GgTre2-like 主要在卵巢表达，在雄性肌肉和马氏管的表达量高于其他组织。上述结果表明，本研究从优雅蝈螽分离到3′-UTR长度不同的3个水溶性和3个类膜结合型海藻糖酶cDNA序列。结果还提示，GgTre1 在各组织的表达差异较大，而GgTre2-like 在各组织的表达相对稳定。不同长度3′-UTR的GgTre1 和 GgTre2-like 亚型的存在，以及不同长度的3′-UTR在翻译过程中的特殊作用，尚待今后研究证实。

Characterization of cDNAs Encoding Soluble and Membrane-bound-like Trehalases from Gampsocleis Gratiosa and Expression in vivo

Trehalase is a disaccharide hydrolysing enzyme. It catalyzes the conversion of glucose to trehalose, which is necessary for multiple physiological processes in insects, including the development, synthesis of chitin and metabolism during flying. Although trehalase genes have been identified, the cDNA encoding trehalase in Gampsocleis gratiosa has not been reported. The soluble trehalase gene (GgTre1) and membrane-bound-like trehalase gene (GgTre2-like) were isolated and their full-length sequences were identified using RACE method combined multiple PCR technology. Progressive lengthening of the 3′-UTRs in three GgTre1 cDNA isoforms (GenBank accession No. KY400001-KY400003) and three GgTre2-like cDNA isoforms (No.KY400004-KY400006) was observed. The lengths of three GgTre1 cDNA isoforms were 2 107, 2 021, 1 914 bp, respectively. All of the three isoforms of GgTre1 share the same 5′-untranslated regions (5′-UTRs) with 33 bp, but differ in the length of the 3′-UTRs with 322，248, and 129 bp, respectively. The GgTre1-2 with an ORF of 1 740 bp, encodes a 579 amino acids protein with a calculated molecular weight of 67.29 kD. In contrast, the ORFs of GgTre1-1/3 encode four extra amino acids, a 583 amino acids protein with a calculated molecular weight of 67.88 kD. The lengths of three GgTre2-like cDNAs (GgTre2-like-1, -2 and -3) were 2 491, 2 460, 2 381 bp. All of them share the same 5′-UTRs with 284 bp, but differ in the length of the 3′-UTRs with 398, 367, 285 bp, respectively. The GgTre2-like cDNA with an ORF of 1 809 bp, encodes a 602 amino acids protein with a calculated molecular weight of 67.88 kD. Tissue-specific expression of GgTre1 and GgTre2-like in G. gratiosa was evaluated in approximately 20 individuals of both genders by real-time quantitative PCR. GgTre1 was highly expressed in the ovary and accessory gland. GgTre2-like was mainly expressed in the ovary. The expression level of GgTre2-like was significantly higher in the muscle and Malpighian tubules than in other tissues in male carcass. Three GgTre1 cDNA isoforms and three GgTre2-like cDNA isoforms with different length of 3′-UTRs were isolated from G. gratiosa. These results also suggest that the expression level of GgTre1 varied considerably among tissues compared to that of the GgTre2-like gene, which presented relatively stable expression levels. However, why GgTre1 and GgTre2 like varies with different length of 3′-UTR, and the special functions of variation in 3′-UTR during the translation process needs further research.