柞蚕己糖激酶基因的组织表达特征及在解除滞育和蛹发育期的表达与酶活性变化

Tissue-specific Expression and Enzyme Activity Variations of Antheraea pernyi Hexokinase Genes During Diapause Termination and Pupa Development

昆虫能够将消化吸收的葡萄糖在脂肪体中转化为海藻糖或作为糖原储存,当机体需要能量时,海藻糖再转化为葡萄糖进入糖酵解途径,己糖激酶(hexokinase,HK)是在此转化过程合成中间代谢物葡萄糖-6-磷酸的限速酶。为研究柞蚕蛹解除滞育及发育过程中的糖类代谢规律,克隆了柞蚕2个己糖激酶同工酶基因,分别命名为Ap HK1和Ap HK2(Gen Bank登录号:KY624592,KY624593)。2个基因的ORF全长分别为1 455 bp、1 362 bp,编码484、453个氨基酸。系统进化分析柞蚕HK1和HK2蛋白分布于2个不同的进化分支,不同种类昆虫的同一类型HK具有更高的同源性。柞蚕5龄幼虫各组织中,2个HK基因在脂肪体中的表达水平都较高,Ap HK2在各组织中均有表达,分布比Ap HK1更广,且在中肠、肌肉内也有较高表达。以一化性柞蚕滞育蛹的脂肪体组织为材料,采用qRT-PCR分析长光照(17 h/d)处理解除柞蚕蛹滞育及蛹发育过程中HK基因的表达规律,结果表明:2个HK基因的表达量在处理后21 d明显升高,其中Ap HK2的表达量升高了3.1倍;化蛹后42 d,Ap HK1的表达量升高了2.8倍,而Ap HK2的表达量无显著变化。同时测定蛹脂肪体中己糖激酶的活性在长光照处理下高于对照组,前期呈现上升趋势,处理后28 d达到最高值,此后逐渐下降并于处理后35 d趋于稳定;测定蛹血淋巴中葡萄糖含量呈先下降后上升的趋势,这与脂肪体中己糖激酶的活性具有相关性。研究结果显示柞蚕滞育蛹解除滞育及发育过程中己糖激酶基因表达上调,在蛹发育中期己糖激酶酶活力及葡萄糖含量之间呈现出互补关系,即己糖激酶的活性增强,血淋巴中葡萄糖的含量减少。

Insects can turn glucose from digestion into trehalose or reserve it as glycogen in fat body. Trehalose can be converted into glucose used for glycolysis when energy is needed. Hexokinase （HK） is a rate-limiting en- zyme that catalyzes synthesis of glucose-6-phosphate which is an intermediate metabolite of this conversion process. In order to study glycometabolism during pupa diapause termination and pupa development in Antheraea pernyi, two hexokinase isozyme genes were cloned, and named as ApHK1 （GenBank accession No. KY624592）and ApHK2 （GenBank accession No. KY624593）. Their full-length open reading frames are 1 455 bp and 1 362 bp, en- coding 484 and 453 amino acids respectively. Phylogenetic analysis indicated that ApHK1 and ApHK2 are located in two different evolutionary clades, and the same HK isoform from different insects has higher identity. In different tissues of the 5th instar A. pernyi larvae, the two HK genes have high expression level in fat body. ApHK2.was expressed in all tested tissues, showing wider distribution than ApHK1. Its expression in midgut and muscle was higher than in other tissues. Ex- pression levels of ApHK genes were detected in the fat body of univoltine diapause pupa by using real-time quantitative （qRT-PCR） under long photoperiod （17 h/d） condition. The results showed that the relative expression level of the two genes was increased obviously at 21 d after pupation, among which the expression of ApHK2 was increased by 3. 1 times. At 42 d after treatment, ApHK1 expression was increased by 2.8 times while relative expression of ApHK2 had no significant change. Hexokinase activity in fat body of pupae under long photoperiod treatment was higher than that of the control group. It increased in early stage, reached peak level at 28 d after treatment, and stabilized after decline at 35 d. Glucose content in pupa haemolymph exhibited a first-decline then-rise trend, which was correlated with hexokinase ac- tivity in fat body. These data show that the expression levels of HK genes are up-regulated during pupa diapause termi- nation and pupa development, and hexokinase activity has complementary relationship with glucose content during mid- dle stage of A. pemyi pupa development, i. e. glucose content in haemolymph decreases with increase of hexokinase activity.