Coding Region Cloning of Yak (Bos grunniens) Lkb1 Gene and Its Expression Analysis in Skeletal Muscle
LEI Zhao-Xiong1, BAI Xue1, 2, LIN Ya-Qiu1, 2, LI Jian1, 2, ZI Xiang-Dong1, 2, XIONG Xian-Rong1, 2, XIONG Yan1, 2, *
1 College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China; 2 Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Southwest Minzu University, Chengdu 610041, China
Abstract Research shows that Liver kinase b1 (Lkb1) plays key roles in mouse (Mus musculus) skeletal muscle development and energetic metabolism processes. Yak (Bos grunniens) is regarded as the specific livestock in Qinghai Tibet Plateau and its adjacent regions, whose growth and development of skeletal muscle is one of most important factors effecting on meat performance. To explore the underlying functions of Yak Lkb1 regulation on skeletal muscle development, growth and energy metabolism, its CDS region was firstly cloned using cDNA as template from longissimus dorsi muscle (L-muscle) by PCR. Next, the physic-chemical properties of Lkb1 amino acid sequence and evolutionary conservation among species were predicted by bioinformatic methods. Lastly, the tissues expression pattern in Jinchuan Yak was detected by quantified PCR and subsequently the expression difference between Jinchuan and Maiwa Yak in skeletal muscle was analyzed. The result showed that the CDS of Yak Lkb1 gene was 1 317 bp (GenBank No. MH412713), encoding 438 amino acids. ProtScal and TMpred software predicted that Lkb1 was a hydrophilic protein with characters of nuclear/cytoplasm translocation, existed transmembrane domain in the region of 231~253 (from inside to outside) and 238~255 (from outside to inside) amino acids. Phosphorylation/dephosphorylation sites were analyzed by NetPhos2.0 Server showed that Lkb1 had 6 phosphorylation/dephosphorylation sites in the N-terminal of this protein, including Ser13, Ser18, Thr7, Tyr8, Tyr19 and Tyr23. Further, functional prediction by Protfun2.2 Server showed Lkb1 might regulating energy and fatty acid metabolism. In addition, the secondary structure of Lkb1 was mainly α-helices and random coil, α-helices with 33.79% and random coil with 44.75%, respectively. Moreover, the conservation analysis showed that Yak Lkb1 gene has highest homology comparison of sheep (Ovis aries) and goat (Capra hircus), both with 97.5% comparability of nucleotide sequence and 99.54% of amino acids sequence. The amino acid homology of Lkb1 comparing to chicken (Gallus gallus), pig (Sus scrofa), dog (Canis lupus familiaris), zebrafish (Danio rerio), panda (Giant panda), gorilla (Pan troglodytes), human (Homo sapiens) and chimpanzee (Rattus norvegicus) were 79.73%, 95.89%, 84.05%, 83.31%, 92.71%, 81.51%, 99.54% and 87.47%. Therefore, the Lkb1 protein had high homology among species. However, the comparability of Lkb1 between Yak and mouse just reached at 45.48% and 44.19% of nucleotide and protein sequence, respectively. Interestingly, mRNA level of Lkb1 gene was enriched in skeletal muscle, compared to that of in liver and fat tissues. Furthermore, the differential expression detection in L-muscle and semitendinosus muscle (S-muscle) from Jin chuan and Maiwa Yak revealed that Jinchuan Yak had significantly higher mRNA level of Lkb1 gene in S-muscle than that of Maiwa Yak, but its mRNA level was indistinguishable in L-muscle between these 2 strains (P<0.05). Thus, it's speculated that Lkb1 might be involved in skeletal muscle growth and energy metabolism. Collectively, these data will provide reference to elucidate the function and mechanism in Yak skeletal muscle biology in the further study.
LEI Zhao-Xiong,BAI Xue,LIN Ya-Qiu, et al. Coding Region Cloning of Yak (Bos grunniens) Lkb1 Gene and Its Expression Analysis in Skeletal Muscle[J]. 农业生物技术学报, 2019, 27(1): 71-79.
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