| 姜晓凡,刘阳嘉,刘 闯,谢莉萍,张荣庆.合浦珠母贝PU3基因的克隆及功能鉴定[J].现代生物医学进展英文版,2019,19(16):3001-3005. |
| 合浦珠母贝PU3基因的克隆及功能鉴定 |
| Cloning and Characterization of PU3 from the Oyster Pinctada fucata |
| Received:April 09, 2019 Revised:May 05, 2019 |
| DOI：10.13241/j.cnki.pmb.2019.16.001 |
| 中文关键词: 合浦珠母贝 生物矿化 FN3结构域 |
| 英文关键词: Pinctada fucata Biomineralization FN3 domain |
| 基金项目:国家自然科学基金项目（31872543，31372502，31502139，31572594） |
| Author Name | Affiliation | E-mail | | JIANG Xiao-fan | School of Life Sciences, Tsinghua University, Beijing, 100084, China | gintlefan@163.com | | LIU Yang-jia | School of Life Sciences, Tsinghua University, Beijing, 100084, China | | | LIU Chuang | School of Life Sciences, Tsinghua University, Beijing, 100084, China | | | XIE Li-ping | School of Life Sciences, Tsinghua University, Beijing, 100084, China | | | ZHANG Rong-qing | 1 School of Life Sciences, Tsinghua University, Beijing, 100084, China
2 Biotechnology and Pharmaceutical Research, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, 314006, China | |
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| 中文摘要: |
| 摘要 目的：克隆获得合浦珠母贝PU3基因的序列，并研究其在生物矿化中的功能。方法：使用RACE获得PU3基因的全长；利用实时荧光定量PCR的方法检测PU3基因在不同组织中的表达分布；利用实时荧光定量PCR的方法检测贝壳损伤修复过程中PU3基因的表达量的变化；通过RNAi实验，抑制PU3基因的表达，之后用扫描电子显微镜观察合浦珠母贝贝壳表面的变化。结果：合浦珠母贝PU3基因的cDNA全长为2361bp，编码618个氨基酸。氨基酸序列的功能结构域分析表明其含有4个FN3结构域。该基因在外套膜中高表达，且在外套膜边缘区的表达量高于外套膜中心区。在贝壳损伤修复的过程中，该基因的表达水平呈现上升的趋势。利用RNAi技术抑制PU3基因的表达后，贝壳的棱柱层结构发生了变化，缝隙变宽，且出现空洞。结论：PU3基因所表达的蛋白作为正调控因子参与生物矿化的过程，并主要作用于贝壳的棱柱层，抑制其表达会影响棱柱层的框架结构。 |
| 英文摘要: |
| ABSTRACT Objective: To obtain the sequence of PU3 gene from the oyster Pinctada fucata and study the function in biomineralization. Methods: The full length of PU3 gene was obtained by RACE. The expression levels of PU3 gene in different tissues were detected by real-time fluorescent quantitative PCR. The expression of PU3 gene through the shell notching experiment was detected by real-time fluorescent quantitative PCR. RNAi experiments was used to inhibit the expression of PU3 gene, and then the change in the surface of the shell of was observed by the scanning electron microscope. Results: The full-length cDNA of PU3 gene from Pinctada fucata is 2361 bp, encoding 618 amino acids. Functional domain analysis indicated that it contained four FN3 domains. PU3 is highly expressed in the mantle membrane, especially in the edge of mantle membrane. In the process of shell repairing, the expression level of PU3 gene showed an increasing trend. After using RNAi technology to inhibit the expression of PU3 gene, the prismatic layer of the shell has changed, the gaps became wider, and voids appeared. Conclusion: It is speculated that PU3 gene acts as a positive regulator in the process of biomineralization, and mainly influences the prismatic layer of the shell. Inhibiting its expression will affect the framework structure of the prism layer. |
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