瘤背石磺磷脂酶C基因的克隆及在不同声波刺激下的表达

磷脂酶C(Phospholipase C,PLC)是一类与膜蛋白相关的酶,在信号转导途径中起作用。栖息于潮间带的瘤背石磺(Onchidium reevesii)能够感知当地的潮汐规律,为了探究瘤背石磺中磷脂酶C在潮汐感知中的作用和分子机制,本实验以瘤背石磺神经环为实验材料,利用RACE-PCR技术得到了其cDNA全长,并进行生物信息学分析和实时荧光定量PCR实验。结果显示,瘤背石磺PLC基因的cDNA全长为2 578 bp,开放阅读框1 881 bp, 5′非编码区337 bp, 3′非编码区340 bp,并且在末端存在AATAAA加尾信号,共编码627个氨基酸;氨基酸序列比对结果显示,瘤背石磺PLC氨基酸序列与福寿螺的磷脂酶C样蛋白2氨基酸序列匹配度最高,与传统形态学的分类相吻合。在实验室内模拟潮汐产生的音波来刺激瘤背石磺,使用实时荧光定量PCR检测OrPLC基因在瘤背石磺神经环中的表达,结果显示:OrPLC在刺激声波频率为40 Hz和70 Hz的时候表达量基本稳定;声波频率为160 Hz和310 Hz时表达量较低;在声波频率为220 Hz和280 Hz时,处于高表达的状态,说明该基因在瘤背石磺的低频感知中发挥重要作用。该实验为进一步了解瘤背石磺潮汐感知功能研究奠定了理论支撑。

Cloning and expression of the phospholipase C gene in Onchidium reevesii under different frequency sound wave stimulation

Phospholipase C (Phospholipase C, PLC) is a class of enzymes related to membrane proteins, which can cleave phospholipids before phosphate groups and play an important role in signal transduction pathways. Onchidium reevesii that inhabits the intertidal zone can perceive the local tidal laws, in order to explore the role and molecular mechanism of phospholipase C in O. reevesii in tide sensing. In this experiment, we used the ganglion of O. reevesii as the experimental material. The cDNA sequence of PLC gene was cloned by RACE-PCR technology, and bioinformatics analysis and qRT-PCR experiment were performed. The results showed that the full length of the cDNA of the PLC gene consists of 2,578 base pairs, the open reading frame(ORF) was 1,881 bp, the 5"non-c-oding (UTR) region was 337 bp, the 3"non-coding region (UTR) was 340 bp, and there was an AATAAA tailing signal at the end, which encoded a total 627 amino acids. The relative molecular weight of approximately is 71.473KDa, and the theoretical isoelectric point is 6.52. The amino acids with the highest proportion in the amino acid chain are serine (Ser, 9.4%) and leucine (Leu, 9.1%). The result of amino acid sequence comparison showed that the PLC gene of O. reevesii has the highest match with the phospholipase C-like protein 2 protein sequence of Pomacea canaliculata, with a similarity of 88%, followed by the phospholipaseC-likeprotein2 protein sequence of Crassostrea virginica, it is consistent with the classification of traditional morphology. By simulating the low frequency sound in the tide in the laboratory to stimulate the tumor, the expression of PLC gene at different frequencies was detected by fluorescence quantitative PCR. The results showed that the expression of PLC was basically stable when the stimulation sound wave frequency was 10 Hz to 70 Hz. When the frequency is increased to 100 Hz-160 Hz, the activity decreases. When the sound wave frequency reaches 220 Hz and 280 Hz, it is in a state of high expression, indicating that this gene plays an important role in the low-frequency perception of lumps. This experiment lays a theoretical support for further understanding of the tidal sensing function of the O. reevesii