美国科学院院报：生物源性非晶钙「ACC」——碳酸钙最易溶形式( 二 ) _蛋白质

图 2 A展示了 GAP 65 的推导氨基酸序列，包括预测的 N 端信号序列（氨基酸 1-20）。 GAP 65 的预测 pI 为 5.01 ，不富含任何特定氨基酸。
大约 4.6% 的 GAP 65 总氨基酸代表可能的磷酸化位点，而仅检测到三个预测的N-糖基化位点和两个推定的O-糖基化位点。
生物信息学分析还表明存在三个已知结构域（图 2 B) ，即几丁质结合结构域 2 (ChtBD2 氨基酸 29-102)、低密度脂蛋白受体 A 类结构域 (LDLa 氨基酸 122-159) 和多糖脱乙酰酶结构域 (氨基酸 195-332) .
图2

Deduced amino acid sequence of GAP 65 and bioinformatics analysis of its sequence. (A) Deduced amino acid sequence of the GAP 65 ORF. Bold indicates predicted signal sequence the asterisk indicates putative phosphorylation sites (NetPhos 2.0 server software) O indicates predicted O-glycosylation sites and N indicates predicted N-glycosylation sites (YinOYang 1.2 software). (B) The complete GAP 65 sequence showing the predicted signal sequence (S.S.) and the ChtBD2 LDLa and polysaccharide deacetylase domains (SMART and Pfam software). Numbers indicate the amino acid range of each domain.

其中，只有 LDLa 结构域具有预测钙结合能力。 C 终端的最后 216 aa 不提供任何已知功能的指示。
通过RT-PCR测试了GAP 65在几个靶组织中的表达（图3A ），仅在胃石上皮盘和表皮下组织中检测到表达。
GAP 65 的原位表达定位于内分泌诱导的蜕皮前的胃石盘的柱状上皮细胞，但在未经处理的蜕皮间小龙虾中未检测到（图3B）。
图3

Specific expression of GAP 65 and its localization to the columnar epithelium of the gastrolith during induced premolt. (A) Detection of GAP 65 expression during premolt by RT-PCR. RNA was extracted from gastrolith epithelial disk (Gas) hepatopancreas (Hep) subepidermal tissue (Sub) sperm duct (Spe) and stomach wall (Sto) tissues. Genomic control (Gen) was performed and Elongation factor 2 (Eft2) was used to confirm RNA extraction. (B) Localization of GAP 65 expression by in situ hybridization in induced premolt and intact intermolt males. (Left) H&E staining. (Center) Tissue probed with the negative control sense—GAP 65 probe. (Right) Tissue probed with the GAP 65 antisense probe with the far right image corresponding to an enlargement of a specific area (box in the antisense image). (Scale bar: 200 μm except for in the induced premolt sense probe images where it represents 100 μm and in the enlarged box where it represents 50 μm.)

注射蜕皮激素（类固醇蜕皮激素）并被GAP 65 dsRNA 沉默的小龙虾的转录水平显着低于注射蜕皮激素和 dsRNA 载体的小龙虾（图 4）。
在注射蜕皮激素和CqVg dsRNA（一种肝胰腺特异性基因，主要在生殖雌性中发现，作为序列特异性沉默的对照）的小龙虾中， GAP 65转录水平与在蜕皮激素和 dsRNA 载体注射组中检测到的水平相似。
图4

Relative transcript levels of GAP 65 in the gastrolith disk after GAP 65 silencing. Real-time RT-PCR relative quantification of GAP 65 transcript levels in the gastrolith disk of crayfish injected with (left to right): ecdysone and GAP 65 dsRNA (n = 7) ecdysone and the dsRNA vehicle (n = 6) ecdysone and C. quadricarinatus vitellogenin (CqVg) dsRNA (n = 3) or the ecdysone and dsRNA vehicles (n = 4). Different letters above columns represent statistically significant differences (P < 0.05 ± SE).

在对照组中， GAP 65转录水平高于蜕皮激素和GAP 65中发现的水平注射 dsRNA 的小龙虾仍低于注射蜕皮激素和 dsRNA 载体或注射蜕皮激素和CqVg dsRNA 的小龙虾中检测到的值。
在注射GAP 65 dsRNA 和蜕皮激素的小龙虾中可以看到胃石的形态畸形（图 5A ），而在仅注射蜕皮激素和 dsRNA 载体的小龙虾中，胃石看起来正常，没有畸形。
在对照组中，胃石似乎未发育。在注射了GAP 65 dsRNA 和蜕皮激素的小龙虾中，可以看到含有密度较低的矿物质的区域（箭头），尽管保留了胃石盘状结构。
图5

Morphological deformities of the gastroliths after GAP 65 silencing. Representative gastroliths dissected from crayfish injected with ecdysone and GAP 65 dsRNA (Left) ecdysone and the dsRNA vehicle (Center) or the ecdysone and dsRNA vehicles (Right). (A) Lateral view of the dissected gastroliths. (B) X-ray radiographs of the above gastrolith before dissection (dorsal view). (C) Mineral density assessment by color rendering of the x-ray images shown in B. The range of densities from the highest to the lowest levels is presented in yellow green blue and purple. Arrows point to areas in the gastrolith where less mineral can be detected.

在注射蜕皮激素和 dsRNA 载体的小龙虾中，胃石看起来正常，对矿物质密度没有影响（图 5 B和C）。