Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (7): 1482-1490.doi: 10.3864/j.issn.0578-1752.2020.07.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Sequence Structure and Expression Characteristics Analysis of AGTR2 in Bovine Follicle

ZhiWei ZHU1,ShuNing HOU1,QingLing HAO1,JiongJie JING2,LiHua LÜ2,PengFei LI1   

  1. 1.College of Life Science, Shanxi Agricultural University, Taigu 030801, Shanxi
    2.College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2019-01-28 Accepted:2020-01-13 Online:2020-04-01 Published:2020-04-14

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Abstract: 【Objective】 The aim of study was to identify molecular characteristics and three-dimensional structure of AGTR2 in the bovine follicular development, and the function was analyzed by combining expression characteristics of AGTR2 in different physiological follicles.【Method】Ovaries in the bovine follicular were observed by B-type ultrasonography (twice a day), removed from cows when DF and SF appeared, and then separated DF and SF; GCs were isolated, total RNA was extracted and detected by RT-PCR, specific primers were amplified and sequenced, and CDS region sequence structure was obtained; the bioinformatics method was used to analyze its sequence structure, relationship and three-dimensional structure; the qRT-PCR primers of AGTR2 and reference gene RPLP0 were designed to analyze differential expression level of AGTR2 in DF and SF; DF and SF of another cow were fixed with 4% paraformaldehyde, positive, negative control groups and experimental groups were set, expression level and localization of AGTR2 were analyzed by immunohistochemistry. 【Result】The results showed that total length of AGTR2 CDS region was 1 089 bp, encoding 362 amino acids; BLAST analysis of amino acid sequence of AGTR2 and the corresponding amino acid sequence of other 24 animals obtained by NCBI database indicated that the sequence had the highest similarity with buffalo (99.4%) and 92.0%-98.9% with other animals; The three-dimensional structure and functional domain analysis showed that AGTR2 possessed 7 parallel alpha helical structures across the cell membrane, which was a typical G protein-coupled receptor; the results of qRT-PCR analysis showed that expression level of AGTR2 mRNA in DF was significantly higher than SF (P<0.01), and the differential expression multiple was up to 7.47 times. The immunohistochemical analysis showed that AGTR2 was expressed in GCs and membrane cells layer of DF and SF, and the specific color intensity showed that expression of AGTR2 in SF membrane cells was higher than DF. 【Conclusion】AGTR2 belonged to G protein-coupled receptor and conforms to basic characteristics of CART receptor, and the study laid a foundation for further study on mechanism of AGTR2 regulating signal pathway and hormone secretion during bovine follicular development, meanwhile, it was of great significance for identification of CART receptor and in-depth explanation of mechanism of CART regulating bovine follicular development.

Key words: bovine, follicular development, CART, G protein-coupled receptor, AGTR2

Table 1

Primer sequences used in this study"

引物名称
Primer name		引物序列
Sequence (5′ to 3′)		退火温度
Tm (℃)		大小
Size (bp)
RT-PCR
AGTR2-FATGAAGACCAACTTCTCCCTTGCCAC58.61089
AGTR2-RTTAAGACACAAAGGTCTCCATTTCTCTAAGGG62.9
qRT-PCR
AGTR2-R-FTTCCCTTCCATGTTCTGACC59.996
AGTR2-R-RAAGGAAGTGCCAGGTCAATG60.1
RPLP0-FCAACCCTGAAGTGCTTGACAT59.3115
RPLP0-RAGGCAGATGGATCAGCCA57.1

Fig. 1

Electrophoresis pattern of RT-PCR product"

Fig. 2

AGTR2 sequence analysis"

Fig. 3

Multi-species amino acid sequence alignment"

Fig. 4

Phylogenetic tree based on amino acid sequences of AGTR2"

Fig. 5

Predicted 3D structure and functional domains of AGTR2"

Fig. 6

qRT-PCR analysis of AGTR2 mRNA in bovine DF vs. SF Superscript ** indicate significantly different at the level of 0.01"

Fig. 7

Immunohistochemical analysis of AGTR2 in bovine DF vs. SF (400×) A, B, C: Control group, experiment group and negative control group of DF, respectively; D, E, F: Control group, experiment group and negative control group of SF, respectively; GC: Granulosa cells; TC: Theca cells; Scale 20 μm"

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