Molecular characterization of Dkkl1 gene and its response to exogenous hormone treatment in Pelodiscus sinensis
-
摘要:
Dkkl1基因在哺乳动物睾丸发育和精子发生过程中扮演着重要角色，然而其在龟鳖类动物中的研究十分有限。为探索龟鳖类Dkkl1基因的潜在功能和作用机制，克隆了中华鳖 (Pelodiscus sinensis) Dkkl1基因的cDNA片段，分析其序列特征、表达模式以及对外源性激素处理的响应。克隆获得的Dkkl1基因cDNA序列长度为823 bp，其中3'-非编码区(UTR)为67 bp，5' UTR为90 bp，开放阅读框为666 bp，共编码222个氨基酸。中华鳖Dkkl1蛋白是一种稳定性较差、亲水性较强的碱性蛋白，其二级结构和三级结构主要以α-螺旋和无规则卷曲为主。氨基酸序列同源性比对结果显示其与中华草龟 (Chinemys reevesii) Dkkl1蛋白的相似性较高 (81%)，与棱皮龟 (Dermochelys coriacea) Dkkl1蛋白的同源性较低 (70%)。通过RT-PCR和RT-qPCR分析发现，中华鳖Dkkl1 mRNA在 3 冬龄成体精巢中极显著性高表达 (P<0.001)，而在其余体组织中几乎不表达。并且随着年龄的增长，中华鳖精巢中Dkkl1基因的表达量逐渐上升，并在3冬龄时达到顶峰。此外，17β-雌二醇 (E2) 和17α-甲基睾酮 (17α-MT) 处理均可显著抑制成体中华鳖精巢中Dkkl1基因的表达 (P<0.05)。研究表明，Dkkl1基因可能在中华鳖睾丸发育和精子发生过程中起着重要作用。
Abstract:Dkkl1 gene plays an important role in mammalian testicular development and spermatogenesis, but the research of Dkkl1 gene in turtles is still limited. Therefore, in order to explore its potential function and mechanism of action in turtles, we cloned a cDNA fragment of Dkkl1 gene from Chinese soft-shelled turtle (Pelodiscus sinensis), and analyzed its sequence characteristics, expression pattern and response to exogenous hormone treatment. The cDNA of Dkkl1 gene was 823 bp in length, with 3' UTR of 67 bp, 5' UTR of 90 bp, open reading frame of 666 bp, and encoded 222 amino acids in total. Dkkl1 protein is a kind of alkaline protein with poor stability and high hydrophilicity. The secondary and tertiary structures of Dkkl1 protein are mainly dominated by α-helix and irregular coil. Amino acid sequence homology comparison showed high similarity with Chinemys reevesii Dkkl1 protein (81%), but low homology with Dermochelys coriacea Dkkl1 protein (70%). RT-PCR and RT-qPCR analyses reveal that Dkkl1 mRNA was significantly highly expressed in spermathecae of 3-winter-age P. sinensis adults (P<0.001), while it was hardly expressed in the rest of somatic tissues. Moreover, the expression of Dkkl1 gene in P. sinensis spermathecae gradually increased with age and peaked at 3-winter-age. In addition, both 17β-estradiol and 17α-methyltestosterone treatments significantly inhibited the expression of Dkkl1 gene in adult P. sinensis spermathecae (P<0.05). The results suggest that the Dkkl1 gene might play an important role in the process of testicular development and spermatogenesis in P. sinensis.
-
20世纪80年代开始，澳大利亚、美国、英国、丹麦等国对食品微生物生长、失活、残存和风险评估模型进行了广泛研究[1-3]，目的是使用微生物预测模型描述不同物理和化学条件下微生物变化情况，客观评价食品在加工、流通、销售、贮藏过程中各种影响因子对食品安全和品质的影响，运用数学模型对微生物的动态变化进行快速有效预测和评价。近年来，美国、英国、丹麦等国致力于构建微生物特征数据库，开发了Food Micromodel, Pathogen Modeling Program, Seafood Safety and Spoilage Predictor等专家系统，用于食品品质预测和安全评估，实现关键控制点分析，协作管理者进行管理。
食品微生物生长模型通常有Monod, Gompertz, Baranyi, Logistic等模型，其中修正的Logistic和Gompertz模型被广泛采用。Baranyi和Robert[4]综合Logistic模型和Michaeli-Menton模型开发了Baranyi模型，该模型相对来说比较复杂。本研究中养殖大黄鱼(Pseudosciaena crocea)取自福建闽东三都湾养殖区(26°35′~26°55′N)，2005年3月海水温度变化幅度为10.5~11.1℃。依据以前对0、5℃恒温冷藏过程中采用感官、挥发性盐基氮(TVBN)、菌落总数(TVC)进行品质变化研究的基础上，本文采用修正的Logistic和Gompertz模型拟合细菌生长曲线，经非线性回归分析，求出预测模型的动力学参数，建立了细菌生长预测模型，并对2种模型的适用性进行了分析。
1. 材料与方法
1.1 试验材料
大黄鱼在福建省闽东三都湾养殖场捕获(2005年3月)，立即放入冰水中冷休克。选用大小基本一致的个体(300~400 g·尾-1)。
1.2 贮藏实验
到达实验室后，将鱼腹部朝上装入下有篦子能沥水的塑料盆中，盖上有漏气孔的盖，分别放入高精度低温培养箱(Sanyo MIR 153, 日本)中，控制贮藏温度在0、5±0.1℃。每隔适当时间取出试样鱼进行感官鲜度评价、TVBN、TVC测定，综合确定产品货架期，本次研究未给出和分析感官、TVBN实验数据。
1.3 样品处理
随机抽取2尾试样鱼，先进行生鱼感官评价，然后去鳞去内脏去腮洗净，用干净吸纸擦干。沿脊骨剖切，取半条鱼肉(带鱼皮)，用组织捣碎机打碎，用于TVBN和TVC测定；其余半条鱼蒸熟后用于感官评价。
1.4 感官鲜度评价
由6名经过训练的评价员组成感官评价小组，评价生鱼的气味和蒸熟后鱼的气味和味道。采用3分法进行评分，0为最好品质，1为鲜鱼的鲜香味消失，0~1为高品质期，2为明显出现臭味和异味即可接受界限[5]。当半数或以上评价员评价2或以上时，即为货架期终点(感官拒绝点)。
蒸熟时将带头的半条鱼分别用铝箔包好，待锅中水沸腾后，放入锅内的金属篦子上，盖上锅盖蒸20 min，打开锅盖后立即进行感官评价。
1.5 菌落总数计数
称取鱼肉浆10.0 g，加入90 mL 0.1%蛋白胨无菌生理盐水，高速振荡后，以10倍稀释将鱼肉浆稀释，取3个浓度合适的稀释液0.1 mL，涂布于标准琼胶培养基(中国科学院上海昆虫科技开发公司康乐培养基有限公司)平板表面。每个稀释液涂布2个平皿，25℃培养48 h。
1.6 细菌生长曲线拟合
0、5℃大黄鱼贮藏实验得到的细菌增殖动态数据，采用修正的Gompertz方程和Logistic方程[6]描述其生长动态。修正Gompertz方程如下：
$$ \lg N(t)=A+C \times \exp \{-\exp [-B \times(t-M)]\} $$ (1) 式中t为时间(h)，N(t)为t时的菌数(lgCFU·g-1)，A为初始菌数N0(lgCFU·g-1)，C为最大菌数Nmax与N0之差(lgCFU·g-1)，M为1/2Nmax时的时间(h)，B为M时比生长速率(h-1)，最大比生长速率μmax＝BC/e。
修正Logistic方程如下：
$$ \lg N(t)=A+C / \exp \{1+[-B \times(t-M)]\} $$ (2) 式中t为时间(h)，N(t)为t时的菌数(lgCFU·g-1)，A为最小菌数N0(lgCFU·g-1)，C为最大菌数Nmax与N0之差(lgCFU·g-1)，B为最大比生长速率μmax (h-1)，M为1/2Nmax时的时间(h)。
实验数据用Statistica (Release 5.5)统计软件采用最小平方差法进行非线性回归。
1.7 细菌生长模型的可靠性评价
细菌生长动力学模型求得的预测值，与大黄鱼贮藏实验所得的细菌生长的实测值比较，依据均方根[7]评价建立的生长动力学预测模型的可靠性。均方根用下式表示：
$$ \text { RMS }=\left[\frac{\sum_i\left(\lg N_{i, \text { predicted }}-\lg N_{i, \text { obsered }}\right)^2}{n}\right]^{0.5} $$ (3) 式中lgNi, predicted为冷藏实验中菌落总数预测值(lgCFU·g-1)，lgNi, observed为菌落总数实测值(lgCFU·g-1)，n为测试次数。
2. 结果
2.1 0℃冷藏大黄鱼细菌生长模型
0℃冷藏期间菌落总数实测值和预测值见表 1。开始贮藏4~5 d，细菌生长缓慢，菌落总数小于6.0 lgCFU·g-1，这是由于暖带海域水温高于温带海域，中温菌数量多, 冷藏过程不耐低温，生长受到抑制，甚至死亡。同时一些嗜冷菌逐渐适应低温环境，随着贮藏期的延长，细菌生长加快，进入指数生长期，好冷菌增殖速度逐渐达到高峰，细菌数呈几何级数增加，腐败终点依据感官评分、TVBN、TVC(7.31 lgCFU·g-1)确定产品货架期为409 h。
表 1 0℃冷藏大黄鱼菌落总数Table 1 Total viable counts of P.crocea stored aerobically at 0℃lgCFU·g-1 时间/h
time实测值
observed valuesGompertz预测模型predictive model Logistic预测模型predictive model 预测值
predicted values残存值
residual values预测值
predicted values残存值
residual values0 5.20 5.20 0.00 5.21 -0.01 49 5.10 5.20 -0.10 5.24 -0.14 76 5.25 5.21 0.04 5.29 -0.04 123.5 5.34 5.42 -0.08 5.48 -0.14 167 6.05 5.95 0.10 5.89 0.16 214 6.50 6.55 -0.05 6.52 -0.02 262 6.89 6.96 -0.07 6.99 -0.10 286 7.10 7.08 0.02 7.12 -0.02 334 7.34 7.22 0.12 7.23 0.11 383 7.22 7.29 -0.07 7.27 -0.05 409 7.31 7.31 0.00 7.28 0.03 图 1、方程4和方程5是采用修正Gompertz方程和Logistic方程回归，得到的0℃冷藏大黄鱼中菌落总数变化曲线和方程，生长动力学参数见表 2。
表 2 0℃冷藏大黄鱼细菌生长动力学参数Table 2 Kinetic parameters of bacteria growth on P.crocea stored aerobically at 0℃参数
parametersN0
(lgCFU·g-1)Nmax
(lgCFU·g-1)μmax(h-1) M(h) Ns
(lgCFU·g-1)货架期/h
shelf lifeGompertz模型model 5.20 7.33 0.013 170.88 7.31 409 Logistic模型model 5.20 7.26 0.024 195.56 注：Ns为感官终点细菌数
Note: Ns denotes population at the time of organoleptic rejection.$$ \begin{aligned} & \quad\quad \lg N(t)=5.20+2.13 \times \exp \{-\exp [-0.015 \times \\ & (t-170.85)]\} \end{aligned} $$ (4) $$ \begin{aligned} & \quad\quad \lg N(t)=5.20+2.06 /\{1+\exp [-0.024 \times(t- \\ & 195.56)]\} \end{aligned} $$ (5) 2.2 5℃冷藏大黄鱼细菌生长模型
5℃冷藏期间菌落总数实测值和预测值见表 3。贮藏初期，细菌生长缓慢，进入指数生长期，细菌增殖速度快于0℃冷藏大黄鱼中细菌增殖速度(μmax见表 2，表 4)，货架期终点菌落总数为7.34 lgCFU·g-1，产品货架期为291 h。
表 3 5℃冷藏大黄鱼菌落总数Table 3 Total viable counts of P.crocea stored aerobically at 5℃lgCFU·g-1 时间/h
time实测值
observed valuesGompertz预测模型predictive model Logistic预测模型predictive model 预测值
predicted values残存值
residual values预测值
predicted values残存值
residual values0 5.20 5.20 -0.00 5.22 -0.02 49 5.41 5.21 0.20 5.29 0.12 76 5.24 5.29 -0.05 5.39 -0.15 123.5 5.79 5.82 -0.04 5.81 -0.02 167 6.60 6.50 0.10 6.48 0.18 216 6.90 7.03 -0.14 7.08 -0.18 264 7.40 7.30 0.10 7.30 0.10 291 7.34 7.37 -0.03 7.34 -0.00 表 4 5℃冷藏大黄鱼细菌生长动力学参数Table 4 Kinetics parameters of bacteria growth on P.crocea stored aerobically at 5℃参数
parametersN0
(lgCFU·g-1)Nmax
(lgCFU·g-1)μmax(h-1) M(h) Ns
(lgCFU·g-1)货架期/h shelf life Gompertz模型model 5.20 7.29 0.016 137.17 7.34 291 Logistic模型model 5.20 7.38 0.030 154.99 图 2、方程6和方程7是采用修正Gompertz方程和Logistic方程回归，得到的5℃冷藏大黄鱼中菌落总数变化曲线和方程，生长动力学参数见表 4。
$$ \begin{aligned} & \quad\quad \lg N(t)=5.20+2.29 \times \exp \{-\exp [-0.019 \times \\ &(t-137.15)]\} \end{aligned} $$ (6) $$ \begin{aligned} & \quad\quad \lg N(t)=5.20+2.06 /\{1+\exp [-0.024 \times(t- \\ & 195.56)]\} \end{aligned} $$ (7) 2.3 细菌生长模型评价
修正Gompertz﹑Logistic模型含有4个参数，参数值和RMS值见表 2﹑表 4﹑表 5。从表 5看出，2种模型的相关系数均大于0.99, 表示2种模型均能很好拟合实验数据。0℃贮藏Gompertz﹑Logistic模型的RMS分别为0.077和0.138, 5℃贮藏Gompertz﹑Logistic模型的RMS分别为0.100和0.114, 2种预测模型相比，Gompertz的RMS均较小，其预测结果更为理想。
表 5 非线性回归方程分析Table 5 Analysis of nonlinear estimation equation参数
parameters均方根(RMS)
root mean square相关系数(R)
correlation coefficient0℃ 5℃ 0℃ 5℃ Gompertz模型model 0.077 0.100 0.994 0.993 Logistic模型model 0.138 0.114 0.992 0.995 3. 讨论
(1) Whiting和Buchanan[7]依据预测模型的发展阶段分为菌数增殖变化模型、环境要素模型和专家系统模型。细菌生长曲线通常呈不对称S形，使用Logistic模型定量描述细菌生长时，不能有效表示细菌生长的延滞期，Gibson等[8, 9]修正了Logistic模型和Gompertz模型，能更好的描述细菌生长情况，然而受食品成分构成、外界环境因子影响，数学模型的适用性显现出较大差异。许多研究者[9, 10]对不同数学模型的性能和适用性进行分析与比较，结果显示相同的数学模型针对不同的研究对象预测结果存在差异。即使如此，大家普遍认为Logistic模型和Gompertz模型的预测效果较好，并得以广泛应用，例如修正的Gompertz模型被应用于预测微生物软件程序Food Micromodel和Pathogen Modeling Program等[9]，Dalgaard等[11, 12]把修正的Logistic模型用于新鲜和气调包装鱼的微生物品质分析, 建立了3参数和4参数预测模型。
(2) 建立在恒温条件下的微生物生长模型，很难有效预测在实际生产、流通、贮藏、消费过程食品微生物的生长变化情况。在实际过程中温度是随机波动的，无法直接用数学式来描述时间-温度的变化规律，现在多根据实际过程把时间-温度的变化设定为多个短的假设为恒温的时间间隔，然后使用分段的数学模型来描述微生物的生长[13]。本研究建立的数学模型可以快速有效预测大黄鱼0、5℃恒温冷藏过程细菌生长变化情况，有待进一步研究建立符合实际流通过程的波动温度预测模型。
-
![]()
图 1 中华鳖 Dkkl1 基因核苷酸序列及其推导的氨基酸序列
Figure 1. Nucleotide of P. sinensis Dkkl1 gene and its encoded protein sequence
![]()
图 2 中华鳖与其他物种的 Dkkl1 氨基酸序列同源相似性比对
注：所有的氨基酸序列来自于NCBI数据库：巴西红耳龟，XM_034791724.1；绿海龟，XM_037888484.1；三趾箱龟，XM_026659251.1；中华草龟，XM_039510142.1；西部锦龟，XM_024113109.1；棱皮龟，XM_038381465.2。黑色阴影为氨基酸序列比对相同的位点，灰色阴影为相近位点。
Figure 2. Homologous comparison of Dkkl1 amino acid sequence of P. sinensis with other species
Note: All amino acid sequences come from NCBI database: T. scripta elegans, XM_034791724.1; C. mydas, XM_037888484.1; T. carolinatriunguis, XM_026659251.1; C. reevesiis, XM_039510142.1; C. picta bellii, XM_024113109.1; D. coriacea, XM_038381465.2. The black and gray shadows represent the same and similar sites, respectively.
![]()
图 3 中华鳖 Dkkl1 基因在 3 冬龄成体组织中的表达情况
注：***. P<0.001；M. DL2000 Marker。
Figure 3. Expression of Dkkl1 gene in tissues of 3-winter-age adult P. sinensis
Note: ***. P<0.001; M. DL2000 Marker.
![]()
图 4 中华鳖 Dkkl1 基因在不同发育时期精巢中的表达情况注：**. P<0.01表示差异极显著。
Figure 4. Expression of P. sinensis Dkkl1 gene in different developmental periods of testis
Note: **. P<0.01 represents very significant difference.
![]()
图 5 中华鳖精巢中 Dkkl1 基因对 17β-雌二醇和17α- 甲基睾酮处理后的表达响应
注：不同小写字母表示组间存在显著性差异(P<0.05)。
Figure 5. Expression response of Dkkl1 gene in testis of P. sinensis to E2 and 17α-MT treatment
Note: Different lowercase letters represent significant differences between groups (P<0.05).
表 1 中华鳖 Dkkl1 cDNA 克隆和表达分析的引物
Table 1 Primers used for cDNA cloning and expression analysis of P. sinensis Dkkl1
名称
Name序列 (5'—3')
Sequences (5'–3')用途
PurposeDkkl1-F GGGACAGGGAAGGGAAAC cDNA克隆 Dkkl1-R GCAAGAAAGACCAAGGAGTAGG Dkkl1-qF ATGGCTAGCAGCCTGTGTCT 实时荧光定量和半定量分析 Dkkl1-qR GACCTGGCAAAGAGATGGAG EF1αF ACTCGTCCAACTGACAAGCCTC 内参基因 EF1αR CACGGCGAACATCTTTCACAG 
下载: 导出CSV
-
[1] GLINKA A, WU W, DELIUS H, et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction[J]. Nature, 1998, 391(6665): 357-362. doi: 10.1038/34848
[2] HU Y, LIU M Y, XU S W, et al. The clinical significance of Dickkopf Wnt signaling pathway inhibitor gene family in head and neck squamous cell carcinoma[J]. Med Sci Monit, 2020, 26: e927368.
[3] DOUCET D, BRUBAKER C, TURNER D, et al. Factors affecting the role of canonical Wnt inhibitor Dickkopf-1 in cancer progression[J]. Front Oncol, 2023, 131114822.
[4] KANEKO K J, DEPAMPHILIS M L. Soggy, a spermatocyte-specific gene, lies 3.8 kb upstream of and antipodal to TEAD-2, a transcription factor expressed at the beginning of mouse development[J]. Nucleic Acids Res, 2000, 28(20): 3982-3990. doi: 10.1093/nar/28.20.3982
[5] YAN Q X, WU X P, CHEN C R, et al. Developmental expression and function of DKKL1/Dkkl1 in humans and mice[J]. Reprod Biol Endocrinol, 2012, 10: 51. doi: 10.1186/1477-7827-10-51
[6] KOHN M J, SZTEIN J, YAGI R, et al. The acrosomal protein Dickkopf-like 1 (DKKL1) facilitates sperm penetration of the zona pellucida[J]. Fertil Steril, 2010, 93(5): 1533-1537. doi: 10.1016/j.fertnstert.2009.06.010
[7] KANEKO K J, KOHN M J, LIU C, et al. The acrosomal protein Dickkopf-like 1 (DKKL1) is not essential for fertility[J]. Fertil Steril, 2010, 93(5): 1526-1532. doi: 10.1016/j.fertnstert.2009.06.011
[8] 颜秋霞, 唐爱发, 来永庆, 等. 精子顶体相关基因DKKL1在正常人和男性不育患者睾丸组织中的表达差异[J]. 实用医学杂志, 2014, 30(1): 36-39. doi: 10.3969/j.issn.1006-5725.2014.01.014 [9] 颜秋霞, 马义, 陈润强, 等. 弱精子症患者精子中DKKL1的表达[J]. 南方医科大学学报, 2018, 38(3): 324-328. doi: 10.3969/j.issn.1673-4254.2018.03.13 [10] CHEN H, HUANG Y F, LIU T F, et al. Characteristics of seasonal spermatogenesis in the soft-shelled turtle[J]. Anim Reprod Sci, 2020, 214106307.
[11] 何中央, 张海琪, 周凡, 等. 中华鳖 “浙新花鳖”[J]. 中国水产, 2017(3): 80-88. [12] YANG Y N, LI C, SONG W, et al. Purification, optimization and physicochemical properties of collagen from soft-shelled turtle calipash[J]. Int J Biol Macromol, 2016, 89: 344-352. doi: 10.1016/j.ijbiomac.2016.04.048
[13] 朱道玉, 吴红松. 中华鳖精巢发育的组织学观察[J]. 安徽农业科学, 2009, 37(22): 10522-10524, 10677. doi: 10.3969/j.issn.0517-6611.2009.22.079 [14] 朱道玉. 中华鳖性腺的发生与发育研究[J]. 水生生物学报, 2009, 33(5): 924-930. [15] LI W, ZHU J X, LEI L, et al. The seasonal and stage-specific expression patterns of HMGB2 suggest its key role in spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis)[J]. Biochem Genet, 2022, 60(6): 2489-2502. doi: 10.1007/s10528-022-10229-0
[16] LEI L, ZHU J X, CHEN C, et al. Expression and characterization of the Spats1 gene and its response to E2/MT treatment in the Chinese soft-shelled turtle (Pelodiscus sinensis)[J]. Animals, 2022, 12(14): 1858. doi: 10.3390/ani12141858
[17] ZHOU Y J, SUN W, CAI H, et al. The role of anti-Müllerian hormone in testis differentiation reveals the significance of the TGF-β pathway in reptilian sex determination[J]. Genetics, 2019, 213(4): 1317-1327. doi: 10.1534/genetics.119.302527
[18] THIRUMALAI A, PAGE S T. Androgens in male contraception[J]. Best Pract Res Clin Endocrinol Metab, 2022, 36(5): 101627. doi: 10.1016/j.beem.2022.101627
[19] HESS R A, SHARPE R M, HINTON B T. Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens[J]. Differentiation, 2021, 118: 41-71. doi: 10.1016/j.diff.2020.11.004
[20] DEAN R, MANK J E. The role of sex chromosomes in sexual dimorphism: discordance between molecular and phenotypic data[J]. J Evol Biol, 2014, 27(7): 1443-1453. doi: 10.1111/jeb.12345
[21] ROSATI L, AGNESE M, DI FIORE M M, et al. P450 aromatase: a key enzyme in the spermatogenesis of the Italian wall lizard, Podarcis sicula[J]. J Exp Biol, 2016, 219(15): 2402-2408. doi: 10.1242/jeb.135996
[22] VERDERAME M, LIMATOLA E, SCUDIERO R. Ectopic synthesis of vitellogenin in testis and epididymis of estrogen-treated lizard Podarcis sicula[J]. Gen Comp Endocrinol, 2016, 235: 57-63.
[23] PASSINI G, STERZELECKI F C, DE CARVALHO C V A, et al. 17α-Methyltestosterone implants accelerate spermatogenesis in common snook, Centropomus undecimalis, during first sexual maturation[J]. Theriogenology, 2018, 106: 134-140. doi: 10.1016/j.theriogenology.2017.10.015
[24] ROUGE M, DROUAULT M, HANOUX V, et al. Ex vivo effects of 17β-estradiol on the prepubertal rat testis[J]. Reprod Toxicol, 2023, 118: 108363. doi: 10.1016/j.reprotox.2023.108363
[25] ARTIMO P, JONNALAGEDDA M, ARNOLD K, et al. ExPASy: SIB bioinformatics resource portal[J]. Nucl Acids Res, 2012, 40(W1): W597-W603. doi: 10.1093/nar/gks400
[26] ZHANG Y, ZHANG Y, XU L, et al. Molecular cloning, tissue expression and polymorphism analysis of the Caveolin-3 gene in ducks[J]. Brit Poultry Sci, 2021, 62(1): 17-24. doi: 10.1080/00071668.2020.1817324
[27] KOHLI D K, BACHHAWAT A K. CLOURE: Clustal Output Reformatter, a program for reformatting ClustalX/ClustalW outputs for SNP analysis and molecular systematics[J]. Nucleic Acids Res, 2003, 31(13): 3501-3502. doi: 10.1093/nar/gkg502
[28] CHEN J S, ZHU N Y, KONG L, et al. First case of soft shell disease in Chinese soft-shelled turtle (Trionyx sinens) associated with Aeromonas sobria: A. veronii complex[J]. Aquaculture, 2013, 406/407: 62-67. doi: 10.1016/j.aquaculture.2013.05.006
[29] HUANG X, LIANG Y, ZHANG R, et al. Genome-wide identification of the PP2C gene family and analyses with their expression profiling in response to cold stress in wild sugarcane[J]. Plants (Basel), 2023, 12(13): 2418.
[30] GEOURJON C, DELEAGE G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Bioinformatics, 1995, 11(6): 681-684. doi: 10.1093/bioinformatics/11.6.681
[31] ANDREW W, MARTINO B, STEFAN B, et al. SWISS-MODEL: homology modelling of protein structures and complexes[J]. Nucl Acids Res, 2018, 46(W1): W296-W303. doi: 10.1093/nar/gky427
[32] 祝骏贤, 陈辰, 刘晓莉, 等. 中华鳖组蛋白H2A变体克隆及其在卵母细胞中的表达分析[J]. 水生生物学报, 2021, 45(6): 1207-1213. [33] YANG Y F, WU J H, LIN R L, et al. Seasonal spermatogenesis, epididymal storage, and creatine kinase expression in Pelodiscus sinensis[J]. Anim Reprod Sci, 2023, 249: 107198. doi: 10.1016/j.anireprosci.2023.107198
[34] di FIORE M, SANTILLO A, FALVO S, et al. Molecular mechanisms elicited by d-aspartate in leydig cells and spermatogonia[J]. Int J Mol Sci, 2016, 17(7): 1127. doi: 10.3390/ijms17071127
[35] JIANG Y J, LUO H R, HOU M X, et al. Aromatase inhibitor induces sex reversal in the protogynous hermaphroditic rice field eel (Monopterus albus)[J]. Aquaculture, 2022, 551: 737960. doi: 10.1016/j.aquaculture.2022.737960
[36] ZHU Q, HAN C, LIU S, et al. Development and gene expression analysis of gonad during 17α-methyltestosterone-induced sex reversal in mandarin fish (Siniperca chuatsi)[J]. Aquac Rep, 2022, 23: 101049. doi: 10.1016/j.aqrep.2022.101049
[37] ASAD F, NAZ S, ALI T, et al. Effect of natural and synthetic androgen hormone on sex reversal of Nile tilapia (Oreochromis niloticus)[J]. Braz J Biol, 2023, 84: e272413.
[38] XU G F, HUANG T Q, GU W, et al. Effects of letrozole and 17α-methyltestosterone on gonadal development in all-female triploid rainbow trout (Oncorhynchus mykiss)[J]. Aquac Res, 2021, 52(6): 2460-2469. doi: 10.1111/are.15095
[39] STEWART M K, MATTISKE D M, PASK A J. Exogenous oestrogen impacts cell fate decision in the developing gonads: a potential cause of declining human reproductive health[J]. Int J Mol Sci, 2020, 21(21): 8377. doi: 10.3390/ijms21218377
[40] CHAVES-POZO E, LIARTE S, VARGAS-CHACOFF L, et al. 17Beta-estradiol triggers postspawning in spermatogenically active gilthead seabream (Sparus aurata L.) males[J]. Biol Reprod, 2007, 76(1): 142-148. doi: 10.1095/biolreprod.106.056036
[41] WALKER W H. Androgen actions in the testis and the regulation of spermatogenesis[J]. Adv Exp Med Biol, 2021, 1288: 175-203.
[42] DAKHOVA O, O'DAY D, KINET N, et al. Dickkopf-like1 regulates postpubertal spermatocyte apoptosis and testosterone production[J]. Endocrinology, 2009, 150(1): 404-412. doi: 10.1210/en.2008-0673
[43] JUNIOR M Z, NAUFAL M R, SETIAWATI M, et al. The sex ratio and testosterone levels in tilapia immersed in different doses of 17α-methyltestosterone[J]. J Akuakultur Indonesia, 2017, 16(1): 51-59. doi: 10.19027/jai.16.1.51-59
计量
- 文章访问数: 176
- HTML全文浏览量: 54
- PDF下载量: 47
粤公网安备 44010502001741号
