Molecular Cloning and Expression Analysis of NPY Gene in Silurus lanzhouensis

doi:10.7668/hbnxb.20193911

Abstract

Abstract:

In view of the low conversion rate of artificial domestication and survival rate of seedling cultivation of Silurus lanzhouensis,in order to use gene editing technology to create and cultivate new germplasm of S.lanzhouensis that was easy to domesticate,the effects of neuropeptide Y gene(NPY) of S.lanzhouensis were studied.The results showed that the full cDNA sequence of the NPY gene was 775 bp in length,with an open reading frame(ORF)of 288 bp,which encoded 95 amino acids and contained a transmembrane helix and a signal peptide binding site for pancreatic hormone recognition.The NPY protein sequence of S.lanzhouensis had the highest consistency with that of Silurus meridionalis,which was 98.95%.The Real-time Fluorescent Quantitative PCR results showed that NPY mRNA expression was the highest in brain tissue,followed by the hypothalamus,and the lowest in muscle.The expression level of NPY mRNA in male tissues of different ages was higher than that in female tissues;and the expression of NPY mRNA decreased with age in both sexes.Localization of brain cells by in situ hybridization found that NPY was mainly expressed in the cytoplasm,and abundant expression was found in the preoptic area,tectum,pituitary,the lentiform nucleus of cerebellum and the caudate nucleus of the medulla oblongata in different parts of the brain.Further verification of starvation compensation showed that the expression of NPY in brain tissue increased significantly after starvation,and decreased significantly after feeding.The above results showed that NPY gene might play an important role in the growth and feeding process of S.lanzhouensis.It could be used as an important verification of the subsequent functional gene editing and the creation of new germplasm,and had important theoretical significance and technical support for accelerating the breeding of new varieties of S.lanzhouensis.

Key words: Silurus lanzhouensis, NPY gene, Bioinformatics, Gene cloning, In situ hybridization, Tissue expression

LAI Zhanglong, RUAN Chaoling, XIAO Wei, LIU Yanbin, YU Zhaoxi, LIU Kai, SAI Qingyun, TIAN Yonghua, ZHANG Feng, YANG Ruilan, LIU Ting, YANG Liqiang, WANG Yutao, LIAN Zongqiang. Molecular Cloning and Expression Analysis of NPY Gene in Silurus lanzhouensis[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 227-238. doi: 10.7668/hbnxb.20193911.

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http://www.hbnxb.net/EN/Y2023/V38/I5/227

Figures/Tables 10

Tab.1 Primers used in this study

引物名称 Primer name	序列(5'-3') Sequence (5'-3')	用途 Usage
NPY-5gsp1	TGTATCCTGATGCTTGTTACAGATG	5'RACE-PCR
NPY-5gsp2	GTAACCATTTCTGATAATGTCATTATGAT	5'RACE-PCR
NPY-3gsp1	TTTGTGATCTCATATGTAGTCCATCTGT	5'RACE-PCR
NPY-3gsp2	CCATCTGTAACAAGCATCAGGATAC	5'RACE-PCR
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM short primer	CTAATACGACTCACTATAGGGC	RACE通用引物
NPY-qF	TCCTGCTGGTTGTATGTTTGGGTG	荧光定量PCR
NPY-qR	TGCCTCGTGATGAGGTTGATGTAG	荧光定量PCR
β-actin F	AAGATCATTGCCCCACCTGA	荧光定量PCR
β-actin R	CCTGCTTGCTGATCCACATC	荧光定量PCR
NPY-F	AACTAACTATGCGTCCTCG	中间序列克隆
NPY-R	TTGGTGTAGCCTTGTGCC	中间序列克隆
	AGACGCACACGTTTGCAAGAAAAC
	AACCAGCAGGATGCACGCGAC
	GGATATCCTTCCACCACTGCACC
	CCCCTCTCCTGGGTTCTGCGG
NPY-Mix	CGCAGTGTAATACTTCGCTAGATCCTCA	原位杂交
	CCTCGTGATGAGGTTGATGTAGTGTCT
	ATCAGCGTTTGACCTTTTACCATACC
	CCAAACAGCAGGTCTGGGGATAA
	ATATCTGGACCGCAGTTGCATCTC

Fig.1 Electrophoresis result of amplified product of NPY in S.lanzhouensis M.DNA Marker DL3000。

Fig.2 Syntenic analysis for NPY genes in S.lanzhouensis and H.sapiens Has.Homo sapiens; Sla.Silurus lanzhouensis.

Fig.3 Syntenic analysis for NPY genes in S.lanzhouensis and other teleost species Sla.Silurus lanzhouensis;Ipu.Ictalurus punctatus;Dre.Danio rerio;Cau.Carassius auratus.

Fig.4 Amino acid sequence homology of NPY gene of S.lanzhouensis

Fig.5 NPY gene phylogenetic tree

Fig.6 Tissue distribution and expression of NPY gene Different letters mean significant differences at P<0.05;the same letters mean no significant differences at P>0.05.

Fig.7 Expression of NPY gene in the male and female S.lanzhouensis at different ages 1.1-year-old;2.2-year-old;3.3-year-old;different letters indicated that there were significant differences among all sexes in different age groups(P<0.05),while the same letters indicated that there was no significant difference(P>0.05).

Fig.8 The cellular localization of NPY gene in the brain of S.lanzhouensis A.Cellular localization of NPY mRNA in the coronal section of the brain;B.Cellular localization of NPY mRNA in the disappearing section of the brain;C.Partial magnification at the arrow of Fig.A;D.Partial magnification at the arrow of Fig.B.

Fig.9 Expression of NPY mRNA in S.lanzhouensis under the condition of starvation and refeeding CK.Control group;R.Re-feeding;different letters showed significant difference in different treatment time(P<0.05),while the same letter indicated no significant difference(P>0.05).

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