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Journal of Zhejiang University (Agriculture and Life Sciences)  2020, Vol. 46 Issue (5): 604-610    DOI: 10.3785/j.issn.1008-9209.2019.12.271
Animal sciences & veterinary medicine     
Expression profile of β-defensin genes and the effect of stocking density on them in Chinese alligator blood
Yao LU(),Shengguo FANG()
MOE Key Laboratory of Biosystems Homeostasis and Protection/State Conservation Center for Gene Resources of Endangered Wildlife, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
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Abstract  

In order to investigate the expression profile of β-defensin genes in the blood of Chinese alligator and the effect of stocking density on them, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of β-defensin genes in blood samples from 41 seven-year-old Chinese alligators and study the defensin gene expression level differences between sexes and populations with different stocking densities. The results showed that only the AsBD5 and AsBD8 genes expressed in the blood of Chinese alligator, and there was a significantly positive correlation between their expression levels. There was no significant sex-biased difference of the defensin gene expression levels. However, their expression levels were significantly different between the two populations with different stocking densities. The defensin gene expression levels were significantly higher in the Chinese alligator population with higher stocking density, which indicating that the stocking density has some effect on the immune status of the captive Chinese alligator.



Key wordsChinese alligator      β-defensin gene      stocking density      reverse transcription quantitative polymerase chain reaction (RT-qPCR)     
Received: 27 December 2019      Published: 19 November 2020
CLC:  S 86  
Corresponding Authors: Shengguo FANG     E-mail: luyao2017@zju.edu.cn;sgfanglab@zju.edu.cn
Cite this article:

Yao LU,Shengguo FANG. Expression profile of β-defensin genes and the effect of stocking density on them in Chinese alligator blood. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(5): 604-610.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2019.12.271     OR     http://www.zjujournals.com/agr/Y2020/V46/I5/604


扬子鳄β防御素基因的血液表达谱及饲养密度对防御素基因表达的影响

为研究扬子鳄血液中β防御素基因家族的表达水平和饲养密度对群体防御素基因表达的影响,利用反转录荧光定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction, RT-qPCR)技术检测41头扬子鳄七龄鳄血液中β防御素基因的表达情况,并分析不同性别和不同饲养密度群体间防御素基因相对表达量的差异。结果表明:在扬子鳄群体血液中,表达的防御素基因有AsBD5AsBD8,并且这2个基因的表达量之间存在显著的正相关性。AsBD5AsBD8在雌性和雄性扬子鳄间的表达水平没有显著性差异;但是在不同饲养密度下,2个扬子鳄群体间的表达水平存在显著差异,其中饲养密度较高的扬子鳄群体,整体的防御素表达水平更高,表明饲养密度对扬子鳄圈养种群的免疫状态有一定的影响。


关键词: 扬子鳄,  β防御素基因,  饲养密度,  反转录荧光定量聚合酶链式反应 

处理

Treatment

饲养池面积

Breeding

area/m2

群体大小

Population

size

群体密度

Population

density/

(capita/m2

样本大小

Sample size

饲养池A69.7210.3016
饲养池B140.0770.5525
Table 1 Information of seven-year-old Chinese alligator populations and samples
Fig. 1 Electrophoretogram results of total RNA in blood (A) and tissues (B) of Chinese alligatorM: DL2000 DNA marker. In Fig. A, numbers of 1-8 standard for total RNA from blood samples of eight Chinese alligators, respectively; and in Fig. B, numbers of 1-8 standard for total RNA from heart, liver, brain, lung, spleen, stomach, intestine and muscle samples of the Chinese alligator, respectively.

基因名称

Gene name

引物序列(5′→3′)

Primer sequence (5′→3′)

产物大小

Product size/bp

退火温度

Annealing temperature/℃

扩增效率

Amplification efficiency/%

AsBD5F: TACGGCGAACGAGAGAC12358.393.27
R: TGCAGCCAAAGGAAATG
AsBD8F: ACCTTCTCTTCGCTGTTTCC13253.9101.23
R: CAATGTACTTGGTGTTCGGG
AsBD10F: GTGGCATGCAGAAGTCAACA10353.795.41
R: TTGAACAGCAAAGCAAGGCT
AsBD13F: GTAATTGCAACGGTGGGC12452.094.41
R: TTGCTGTGGTGTGGTCTCTC
AsBD101F: GTGAGGAGCAGATTGGGACT8652.096.24
R: CAGCCTGCTTGCCTGTATC
AsBD105γF: AGATGTCGCTGGTATGGGAT15853.092.94
R: TGGTGCTGGCTTAGAGGATT
AsBD106γF: CTTCTCTTTGCCCTGGTCC13558.596.62
R: CACATCGCCTTTTGTTTCC
AsBD107αF: TGGCAGCCCCAGAAAACAG10950.999.06
R: GCACAGGATCCCAACTTGACTT
AsBD108F: AAGCCCAGGGATCATGAAGA9858.5108.48
R: CGTTCAGCAGGTTACATGGC
GAPDHF: GGAAGATGTGGCGGGATG60134.097.28
R: TGTTGGGACACGGAATGC
Table 2 Primer information for qPCR
Fig. 2 Relative expression levels of β-defensin genes AsBD5 and AsBD8 in blood of seven-year-old Chinese alligators
Fig. 3 Relative expression levels of β-defensin genes for populations of Chinese alligators under different sexes (A) and stocking densities (B)Triple asterisks (***) indicate extremely high significant differences at the 0.001 probability level.
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