Expression of Hoxc13 Gene in Skin Tissue of Tan Sheep at Different Stages

Li’na MA Zhengwei ZHAO Jin WANG Qing MA

Abstract ［Objectives］The expression differences of Hoxc13 gene in the skin tissue of Tan sheep at different stages were analyzed.

［Methods］ qRT-PCR was used to analyze the mRNA expression level of Hoxc13 gene in the skin tissue of Tan sheep at different stages (35, 55 d), and the relative quantitative method was used to analyze the expression level.

［Results］ The content of Hoxc13 gene in the skin tissue of Tan sheep at 55 d was significantly higher than that in the skin tissue at 35 d. Hoxc13 gene was differentially expressed in the skin tissue of Tan sheep lambs at different stages (35, 55 d).

［Conclusions］ This study provides basic data for further exploration of the growth and development mechanism of Tan sheep.

Key words Hoxc13; Tan sheep; qRT-PCR; mRNA

Received: May 20, 2022  Accepted: July 27, 2022

Supported by Study on the Mechanism of DLX3/DLX4 Gene Regulation of Coat Bending in Tan Sheep (2021A0491); Breeding of Mutton and Fur Dual-purpose Tan Sheep Lines (2018NYYZ04).

Li’na MA (1985-), female, P. R. China, assistant research fellow, master, devoted to research about livestock genetic breeding.

*Corresponding author. E-mail: maqing1973@126.com.

Tan sheep is a unique fine breed of light-fur sheep in China, and the "Ermao period" has a long-standing reputation and is well-known at home and abroad. The "Ermao period" refers to the period when the fur stripped from lambs of Tan sheep which is slaughtered at about 35 d after birth, 8-9 cm long, and has very high economic value. However, after the Ermao period, with the increase of Tan sheep’s age, the hair strands gradually become thicker and longer, and such excellent characteristics of fur will gradually disappear, as the hair strands will become loose, and the crimp ears will not be beautiful［1］.  Hoxc13 (homeobox c13) belongs to the Abd-B class of the Hox (Homeobox) gene family and is closely related to hair follicle formation and hair growth. The expression of hair structural proteins KP (keratin) and KAP (keratin-associated protein) are both tightly regulated by Hoxc13, and the expression level of Hoxc13 directly affects the characteristics of hair and is also crucial for maintaining the normal shape of hair follicles［2］. Hoxc13 is well known as a transcriptional regulator related to hair follicle generation and development, and it can control hair follicle development and hair growth by regulating the expression of the keratin gene. To this end, the expression level of Hoxc13 gene in the skin tissue of Tan sheep was analyzed by qRT-PCR at different stages.

Materials and Methods

Sample collection

The skin tissue of Tan sheep was collected at the ages of 35 and 55 d. Each group was assigned with 10 sheep, and the sample size was about 1 cm2. The collection sites were all at the posterior edge of the left scapula. The collected samples were rinsed with sterile saline, immediately placed in a liquid nitrogen tank for storage, taken to the laboratory, and stored at -80 ℃ for later use. The experimental animals were all from the Tan sheep breeding farm in Yanchi, Ningxia.

Primer design and synthesis

Referring to the sheep Hoxc13 gene sequence and 18s rRNA gene sequence published by GenBank, one pair of primers were designed in each of their conserved regions using Primer 5.0, and was synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 1.

RNA extraction

Total RNA from tissue samples was extracted with Trizol. Specifically, ① a tissue sample was ground with a mortar, added in a centrifuge tube with 1 ml of Trizol, and stood for 5-10 min; ② 0.2 ml of chloroform was added, and the mixture obtained was mixed vigorously up and down for 15-30 s, and then stood for 3 min (ice bath or room temperature); ③ centrifugation was performed at 4 ℃ and 12 000 rpm or 15 min; ④ the water phase was pipetted into a new EP tube, and added with 0.5 ml of isopropyl alcohol, and the obtained system was mixed well and cooled in an ice bath for 10 min; ⑤ centrifugation was performed at 4 ℃ and 12 000 rpm or 15 min; ⑥the supernatant liquid was poured out and precipitated with 75% ice ethanol (1 ml), and mixed well; ⑦ centrifugation was performed again at 4 ℃ and 12 000 rpm or 15 min; and ⑧ the precipitate was dried. The obtained RNA was finally dissolved with DEPC water for concentration determination and stored at -80 ℃.

Reverse transcription

TIANScript RT KIT was used for reverse transcription. The experimental operation was carried out according to the product instructions. Specifically, the first part of the reaction system (50 μl) was added to a reaction tube according to the table below, and mixed well.

The reverse transcription system (Table 4) was heated at 70 ℃ for 5 min, and quickly cooled on ice for 2 min. The reactionliquid was collected after a short centrifugation, and added with the components in Table 5.

The system was gently pipetted and released to mix well. Next, it was incubated at 25 ℃ for 10 min and at 42 ℃ for 50 min, and then heated at 95 ℃ for 5 min. The reaction system was diluted to 50 μl with RNase-Freedd H2O and stored at -20 ℃.

Statistical analysis methods

An ABI7500 fluorescence quantitative PCR instrument (model 7500) produced by American ABI Compan was used for determination, and the relative quantitative analysis of the data was carried out using the 2-△△CT method:

F=2-［The average Ct value of the target gene in the experimental group-The average Ct value of housekeeping genes in the experimental group-［The average Ct value of the target gene in the control group-The average Ct value of housekeeping genes in the control group

Detection process of real-time PCR

Reaction system of real-time PCR

The amplification program was: 95 ℃ for 15min, and (95 ℃, 15 s, 58 ℃, 30 s, 72℃, 30 s)×45 cycles. The real-time reaction system contained 2*Super Real PreMixPlus 10 μl, upstream primer (10 μM) 0.6 μl, downstream primer (10) 0.6 μl, cDNA 100 ng, 50*ROX Reference Dy 0.4 μl, and sterile purified water to 20 μl.

Real-Time PCR detection

The cDNA of each sample was diluted by 10 times, and 2 μl was used as a template, which was amplified with primers of the target gene and the internal reference gene, respectively. Meanwhile, melting curve analysis was carried out at 60-95 ℃, and the cDNA of each sample was detected 3 times.

Experimental Results

RNA electrophoretogram

On a 1% agarose gel, 5 μl of RNA was electrophoresed. The results are shown in Fig. 1 as 28S, 18S and 5S, with the 5S band being lighter, indicating no significant RNA degradation. Detected by a UV spectrophotometer, the ratios of OD260/OD280 were between 1.8 and 2.0, indicating that the RNA purity was high.

Expression of Hoxc13 gene in skin tissue at different stages

In the quantitative detection test, the internal reference gene was 18S rRNA, and real-time fluorescent quantitative PCR with SYBR Green II dye was adopted for verification. In specific, the mRNA expression of Hoxc13 gene in the skin tissue of Tan sheep was analyzed at 35 and 55 d by the relative quantitative method with the 18s rRNA gene as the internal reference gene.

According to the original detection results of real-time PCR, the relative quantitative results of the target gene of various samples were calculated with the relative quantitative calculation formula of 2-△△ct, that is, the differences of the target gene of various samples relative to the control sample in the mRNA transcription level (with 1 as the control sample). The results showed that the relative quantitative result of Hoxc13 gene in the skin tissue at 35 d was (0.58±0.21), and the relative quantitative result of the skin tissue at 55 d was (1.20±0.20). It could be seen that the expression of Hoxc13 gene in the skin tissue of Tan sheep at 55 d was extremely significantly higher than that in the skin tissue at 35 d (P<0.01).

Conclusions and Discussion

The study of genes related to hair follicle growth and development has become a research hotspot in recent years. In particular, whether some genes are expressed and their expression intensity during hair follicle development can reveal the potential role of these genes in hair follicle growth and development, and lay a foundation for finding candidate genes related to hair quality. Hoxc13 plays an important role in controlling hair formation［2］. WU et al.［3］ found that Hoxc13 gene expression and hair follicle morphogenesis in the skin have the same trend of change during embryonic development. Zhao et al.［4］ compared the relative expression of Hoxc13 gene among individuals of white cashmere goats with different cashmere yields in Northern Shaanxi by fluorescence quantitative PCR method during anagen and telogen. It was found that KAP9.2 gene had an inhibitory effect on villus growth and Hoxc13 had a regulatory effect on KAP9.2 gene expression during the periodical growth of villi. Du et al.［5］ found that Hoxc13 gene was expressed in various tissues and organs of Subo Merino sheep and there were differences in expression. The expression of Hoxc13 gene in skin was higher than that in muscle, kidney, heart, spleen, lung and liver tissue, and Hoxc13 gene may be closely related to the process of hair follicle development. WU et al.［6］ found that Hoxc13/β-catenin gene was related to follicle activity. The expression of Hoxc13 gene increased sharply from July to November, and the overexpression of Hoxc13 gene decreased the expression of HFDRGs, which have negative effects on hair follicles. Therefore, Hoxc13 gene has a positive effect on hair follicle development. As a key transcription factor in the process of hair follicle growth and development, Hoxc13 can regulate the growth and development, differentiation, migration, proliferation and apoptosis of hair follicles and hair follicle-related cells by controlling keratin and keratin-associated proteins, thereby controlling mammalian hair texture. Meanwhile, Hoxc13 gene is also affected by upstream genes, related proteins, chemical preparations, etc., and they jointly control the formation and growth of mammalian hair［7］. The Ermao characteristic of Tan sheep is the characteristic of Tan sheep’s growth and development in a special period (birth to about 30 d), which has been established in the embryonic stage of Tan sheep. Its formation may be related to the expression and regulation of genes related to the development of hair follicles and hair fibers in the embryonic stage［8］. Luo［9］ reported that Hoxc13 transcription factor Hoxc13 positively regulated KRT38 and KRT84 genes, and negatively regulated KRT1 gene. The expression of Hoxc13 gene in cashmere goats’ skin is consistent with the change trend of skin thickness during fetal period; and in adult cashmere goats’ skin, the expression of Hoxc13 gene is significantly correlated with the secondary hair follicle activity cycle. Hoxc13 gene has undergone adaptive evolution during the origin of mammalian coat. In-vitro and in-vivo studies have shown that Hoxc13 plays an important role in the morphogenesis and periodic growth of hair follicles in cashmere goats’ skin［10］. Ma［11］ amplified the Hoxc13 gene fragment of cashmere goat and Mongolian sheep. The sequence alignment showed that the Hoxc13 gene of mammalian was very conserved and had a very high homology. The results of real-time PCR showed that the expression level in the lamb skin of cashmere goats two weeks after birth was higher than that in the embryo skin of day 70, and no polymorphisms were detected in the three loci of Hoxc13 gene including two exons, which is in line with the relatively conservative genetic characteristics of the gene. Studies have obtained the CDS sequence of Hoxc13 gene of Tibetan cashmere goats. There is one missense mutation and two synonymous mutations in exon 1 of Hoxc13 gene, and there is no difference in the expression of Hoxc13 at different altitudes［12］.

Hoxc13 gene was expressed in the skin tissue of Tan sheep aged 35 and 55 d, and there were differences in expression. The expression of Hoxc13 gene in 55-day skin was higher than that in 35-day skin tissue. Hoxc13 gene may be closely related to the process of hair follicle development.

Agricultural Biotechnology2022

References

［1］ LI XL, TAO JZ, DING W, et al. Analysis of wool traits and its relationship in tan sheep during the Ermao period［J］. China Animal Husbandry & Veterinary Medicine, 2019, 46(2): 442-448. (in Chinese).

［2］ WU JH, YAN ZW, HU SY, et al. Hoxc13 and the development of hair follicle［J］. Hereditas, 2010, 32(7): 656-662. (in Chinese).

［3］ WU JH, ZHANG WG, LI JQ, et al. Hoxc13 expression pattern in cashmere goat skin during hair follicle［J］ .Development Agricultural Sciences in China, 2009, 8(4): 491-496.

［4］ ZHAO ZD, XU HR, QU L, et al. Expression analysis of KAP9.2 and Hoxc13 gene in skin tissues during anagen and telogen of Shaanbei white cashmere goat［J］. Journal of Northwest A & F University: Natural Science Edition, 2012, 40(9): 24-28. (in Chinese).

［5］ DU JW, HE JM, CHEN CY, et al. Expression analysis of Hoxc13 gene in different tissues of Subo Merino sheep during growth and development［J］. Xinjiang Agricultural Sciences, 2019, 56(5): 936-944. (in Chinese).

［6］ WU JH, ZHANG YJ, ZHANG JX, et al. Hoxc13/β-catenin correlation with hair follicle activity in cashmere goat［J］. Journal of Integrative Agriculture, 2012, 11(7): 1159-1166.

［7］ YAO F, BAI SC, JIN RS, et al. Research progress on the Hoxc13 regulation in the development of mammalian hair follicle［J］. Chinese Journal of Animal Science, 2022, 58(1): 17-20. (in Chinese).

［8］ ZHAO ZW, MA Q, LI YK, et al. Research progress on curving characters of Tan sheep’s lamb hair［J］. The Chinese Livestock and Poultry Breeding, 2019(7): 96-97. (in Chinese).

［9］ lUO ZX. Study on the regulatory mechanism of Hoxc13 on goat keratin family-related genes［D］. Yangling: Northwest A&F University, 2018. (in Chinese).

［10］ WU JH. Expression pattern and function analysis of Hoxc13 gene in skin of cashmere goat［D］. Hohhot: Inner MongoliaAgricultural University, 2011. (in Chinese).

［11］ MA C. Study on Hoxc13 gene and KAP16 gene of Inner Mongolian cashmere goats［D］. Hohhot: Inner Mongolia Agricultural University, 2008. (in Chinese).

［12］ ZHANG D. A dissertation submitted to Sichuan Agricultural University in partial fulfillment of the requirements for the degree of master of science［D］. Ya’an: Sichuan Agricultural University, 2018. (in Chinese).