家蚕微孢子虫葡萄糖-6-磷酸脱氢酶基因(G6PDH)的克隆及表达特征分析

Cloning and Expression Profile Analysis of G6PDH Gene of Nose-ma bombycis

葡萄糖-6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase,G6PDH)是戊糖磷酸途径的第一个关键酶,催化葡萄糖-6-磷酸形成6-磷酸葡萄糖酸-δ-内酯。家蚕微孢子虫有较为完整的戊糖磷酸途径,该途径除提供糖酵解的中间代谢物外还可形成还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)从而抵抗生物体内的氧化刺激。为了解G6PDH基因在微孢子虫生长发育过程中的作用,根据家蚕微孢子虫G6PDH基因序列设计特异引物克隆了该基因,并对其序列特征进行生物信息学分析,进一步采用荧光定量PCR分析G6PDH基因在家蚕感染不同时间点的中肠组织中的转录水平。结果表明,克隆的家蚕微孢子虫G6PDH基因编码区全长1 305 bp,编码434个氨基酸,预测的G6PDH蛋白无信号肽,分子质量为50.8 kD,等电点为5.22,没有跨膜结构域,存在磷酸化位点,不存在N-糖基化位点及O-糖基化位点;家蚕微孢子虫感染家蚕后,从2 h起G6PDH基因表达量开始升高,至12 h时达到最大值,在24~72 h虽然有逐渐降低的趋势,但趋势并不明显,96 h后开始显著降低,至144 h表达量达到最低,而后在168 h时表达量又有所回升。推测G6PDH基因参与家蚕微孢子虫发芽、增殖以及成熟等整个生命周期。

Glucose-6-phosphate dehydrogenase( G6 PDH) is the first key enzyme in the pentose phosphate pathway and catalyzes glucose-6-phosphate to form 6-phosphoglucono-δ-lactone. There is a relatively complete pentose phosphate pathway in Nosema bombycis. This pathway can also produce reduced nicotinamide adenine dinucleotide phosphate( NADPH) and provide glycolysis intermediates to resist oxidation stimulation in vivo. In order to understand the role of G6 PDH in the growth and development of Nosema bombycis,we cloned the G6 PDH gene of Nosema bombycis by using specific primers according to its sequence retrieved from GenBank. Then we performed sequence analysis by bioinformatics tools and expression profile analysis by real-time fluorescent quantitative PCR. The results showed that the cloned fulllength coding sequence of G6 PDH gene of Nosema bombycis is 1 304 bp and encodes 434 amino acids. The predicted G6 PDH protein has no signal peptide,the molecular weight is 50. 8 kD,and the isoelectric point is 5. 22. There is no transmembrane domain,no O-glycosylation site and N-glycosylation site but there are phosphorylation sites. The results of real-time fluorescent quantitative PCR showed that the G6 PDH gene starts to express from 2 h after silkworm infected with Nosema bombycis,and reaches the maximum at 12 h. Although there is a trend of decreasing gradually within 24-72 h,the trend is not obvious. After96 h,it starts to decrease significantly. The expression level reaches the minimum at 144 h,and then the expres-sion level of G6 PDH gene increases at 168 h. It is speculated that the G6 PDH gene may be involved in spore germination,proliferation and maturation of the whole life cycle.