Cloning and tissue expression of alternative spliceosome in chicken G-protein alpha subunit gene

doi:10.3785/j.issn.1008-9209.2018.11.151

Journal of Zhejiang University (Agriculture and Life Sciences)

2019, Vol. 45

Issue (5): 603-610 DOI: 10.3785/j.issn.1008-9209.2018.11.151

Animal sciences & veterinary medicine

Cloning and tissue expression of alternative spliceosome in chicken G-protein alpha subunit gene

Xuedong ZHANG(

),Huanhuan WANG,Ying GE,Dandan SONG,Lei ZHANG,Qinghai LI,Lifeng LOU

Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China

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Abstract

In order to research deeply the transcription and splicing situations of chicken G-protein alpha subunit gene (GNAS), we used 5′ and 3′ rapid-amplification cDNA end (RACE) technology to clone and sequence the chicken GNAS gene, and used quantitative polymerase chain reaction (PCR) to detect the expression level of GNAS spliceosome in seven tissues of chicken， such as skin, pectoral muscle, heart, brain, liver, lung, and abdominal fat. The results showed that the chicken GNAS gene had two transcriptional spliceosomes of 1 554 bp and 1 796 bp, respectively. Both of spliceosomes included 12 exons, and only the length and position of their first exon were different, and the second to twelfth exons were the same. Clone 1 (1 554 bp) coded 417 amino acids, while clone 2 (1 796 bp) coded 379 amino acids. Protein alignment in the NCBI database showed that the similarity between the 379 amino acid sequence of the clone 2 and Gαs subunits of the known human and mouse GNAS genes was 93%. The 417 amino acid sequence of the clone 1 was more similar with the XLαs subunit, and their similarity was 87%. The gene expression detection showed that these two transcriptional spliceosomes had different degrees of expression in the seven tissues: the highest expression in the brain (P＜0.01), next in the skin (P＜0.01 or P＜0.05), and the lower expressions in the lung, pectoral muscle, heart and abdominal fat.

Key words： chicken G-protein alpha subunit gene spliceosome cloning expression

Received: 15 November 2018 Published: 05 December 2019

CLC:

S 831.2

Corresponding Authors: Xuedong ZHANG E-mail: bigzhengliang@hotmail.com

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	Xuedong ZHANG
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	Ying GE
	Dandan SONG
	Lei ZHANG
	Qinghai LI
	Lifeng LOU

Cite this article:

Xuedong ZHANG,Huanhuan WANG,Ying GE,Dandan SONG,Lei ZHANG,Qinghai LI,Lifeng LOU. Cloning and tissue expression of alternative spliceosome in chicken G-protein alpha subunit gene. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 603-610.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2018.11.151 OR http://www.zjujournals.com/agr/Y2019/V45/I5/603

鸡G蛋白α亚基基因可变剪接体的克隆和组织表达

为深入研究鸡G蛋白α亚基（G-protein alpha subunit, GNAS）基因的转录剪接情况，采用5′和3′cDNA末端快速扩增（rapid-amplification cDNA ends, RACE ）技术，对鸡GNAS基因进行克隆测序；并采用定量聚合酶链式反应（polymerase chain reaction, PCR）技术，检测鸡皮肤、胸肌、心、脑、肝、肺和腹脂等7种组织中GNAS基因剪接体的表达量。结果表明：鸡GNAS基因存在1 554和1 796 bp 2种转录剪接体，均含12个外显子，二者仅第1外显子的长度和位置不同，其余第2－12外显子相同。克隆子1（1 554 bp）编码417个氨基酸，克隆子2（1 796 bp）编码379个氨基酸。在NCBI数据库中进行蛋白比对发现，克隆子2的氨基酸序列与已知的人和小鼠的GNAS基因Gαs亚基同源相似度达93%，而克隆子1的氨基酸序列与XLαs亚基的同源性较高（相似度87%）。表达量检测表明，2种转录剪接体在7种组织中均有不同程度的表达，其中：在脑组织中的表达量最高，与其他组织间差异极显著（P＜0.01），在皮肤组织中的表达量（P＜0.01或P＜0.05）次之；而在肺、胸肌、肝、心、腹脂等组织中的表达量较低。

关键词： 鸡, G蛋白α亚基基因, 剪接体, 克隆, 表达

Table 1 RACE primer information of GNAS gene

Table 2 Primer information of gene expression detection

Fig. 1 PCR products of 5´ and 3´ RACE of chicken GNAS geneM: DL2000 marker; 1 and 2: PCR products of the first 5´ RACE circle; 3 and 4: PCR products of the second 5´ RACE circle; 5: PCR product of the first 3´ RACE circle; 6: PCR product of the second 3´ RACE circle.

Table 3 Information of the clones obtained from RACE of chicken GNAS gene

Fig. 2 Full-length sequences of two transcriptional spliceosomes of chicken GNAS geneA. 5´UTR and first exon of clone 1; B: 5´UTR and first exon of clone 2; C: The second to twelfth exons and 3´UTR of two spliceosomes. The upper part of each row is the nucleotide sequence of spliceosomes, and the lower part is the coding amino acid sequence of spliceosomes. The number and arrow upon nucleotides represent the start position of twelve exons. The underlines and wavy lines under nucleotides represent the position of primers used in expression detection of clone 1 and clone 2, respectively.

Fig. 3 Protein homology alignment results of two transcriptional spliceosomes of chicken GNAS geneA. Homologous cladogram of 417 aa sequence blasted with human and mouse; B. Homologous cladogram of 379 aa sequence blasted with human and mouse; C. Homologous cladogram of 417 aa and 379 aa sequences blasted with chicken.

Table 4 Protein identification (ID) and related information involved in homologous alignment

Fig. 4 Tissue expression level of two transcriptional spliceosomes of chicken GNAS geneDifferent lowercase letters above bars indicate significant differences at the 0.05 probability level, and different uppercase letters above bars indicate highly significant differences at the 0.01 probability level.


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