The association of RNF34 3′UTR-588 G>A and RNF128 I1-2380C>T with carcass and meat quality traits of Chinese Simmental-cross steers

Ying Hai Jin. E-mail: jinyh@ybu.edu.cn Simple Summay: An experiment was performed to investigate the role of single nucleotide polymorphisms of the gene RNF34 3′UTR-588 G>A and RNF128 I1-2380C>T with carcass and meat quality traits of Chinese Simmental-cross steers. Sequencing and restriction enzyme digestion was performed to detect genotypes of RNF34 3′UTR-588 G>A and RNF128 I1-2380C>T. The associations of novel single nucleotide polymorphisms in intron regions of the RNF128 gene and in the 3′UTR region of RNF34 and meat quality traits of Chinese Simmental-cross steers were analyzed. Statistical analyses revealed that SNP of RNF128 was significantly associated with dressed weight, forepaw weight, carcass depth, carcass brisket depth, hind legs length (P<0.05), etc. And RNF34 were significantly associated with testis weight, kidney weight, tare weight (P<0.05), etc. Our findings suggest that polymorphisms in RNF34 and RNF128 might be important genetic factors that influence carcass and meat quality in beef cattle. Thus, they might be useful markers for meat quality traits in Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 August 2020 doi:10.20944/preprints202008.0518.v1


INTRODUCTION:
With the rapid development of molecular biology techniques, identification of single nucleotide polymorphisms has been widely used to study the effects of genetic mutations on animal performance, which in turn could be used for a molecular marker-assisted approach to breeding and production.
RNF34 and RNF128 play an important role in many biological processes. Several papers showed that E3 ubiquitin-protein ligases are master regulators of energy metabolism and adaptive thermogenesis in brown fat cells. And RNF34 is a bonafide E3 ubiquitin-protein ligase for PGC-1α and negatively regulates brown fat cell metabolism. RNF34 binds to the C-terminal region of PGC-1α and targets it for degradation independently of the previously identified N-terminal phosphor degron motif [1,2] .In brown fat cells, knockdown of RNF34 has several effects including increased endogenous PGC-1α protein levels, increased uncoupling protein 1 (UCP1) expression and increased oxygen consumption [3,4] . However, the opposite effects are observed in brown fat cells that ectopically express wild-type RNF34 instead of its ligase activity-defective mutant form [5]. Interestingly, cold exposure and β3-adrenergic receptor signaling, conditions that induce PGC-1α expression, suppress RNF34 expression in brown fat cells, indicating a physiological relevance for this E3 ligase in the thermogenesis process [6] [7] The RNF128 gene plays an important role in a series of cellular pathways and processes such as DNA repair, cell cycle regulation, apoptosis, and inflammatory response [7]. T-cell activation is tightly regulated in order to avoid autoimmunity. GRAIL protein, encoded by RNF128 and related to energy metabolism in T-cells, is an E3 ubiquitin-protein ligase associated with T-cell tolerance. Interestingly, ubiquitination and degradation of CD40L by RNF128 is one cause of T-cell incompetence [8,9] . In recent years, expressions of RNA34 and RNA128 showed a significant difference between adult cattle Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 August 2020 doi:10.20944/preprints202008.0518.v1 and younger ones [2].
Therefore, it is possible that the RNA34 and RNA128 maybe candidate genes influencing carcass and meat quality of beef cattle. But, at present, only a little information is available on the genetic polymorphism of bovine RNA34 and RNA128 genes and the effect of the genetic variants of RNA34 and RNA128 genes remain inconclusive. Hence, in the present study, SNPs of RNF34 and RNF128 were examined with respect to their association with carcass and meat quality traits in Chinese Simmental-cross steers. The results of this study may provide useful evidence to MAS in the process of pure breeding, crossbreeding, and the preservation of important genetic resources.

Ethics statement
Animal experiments were conducted in strict accordance with the guidance for the care and use of laboratory animals by the Jilin University Animal Care and Use Committee (permit number: SYXK (Ji) 2008-0010/0011). All production traits were measured with standardized methods.

Materials
The animals of Simmental-cross steers for this study were taken from the Inner Mongolian Baolongshan cattle farm. Blood samples (10 mL each) were collected from the jugular vein using an anticoagulant (Acid citrate dextrose, ACD) followed by storage at -80℃. DNA was extracted from 1 mL of extracted whole blood using a DNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's protocol.

• Trait measurements
Carcasses were stored in refrigerated rooms at temperatures ranging from 0 to 4 C˚ for 24 h before the carcass and meat traits were measured. Trait measurements were made based on the GB/T17238-1998 cutting standards for fresh and chilled beef of China (China Standard Publishing House).
Final body weight, living QIB, and ribeye area were recorded before slaughter. All visceral indicators, including the weight of the spleen, large intestine, small intestine, heart, liver, kidney and fat belly, were weighed after slaughter. Other carcass properties were also recorded, including the carcass weight, slaughter rate, net weight of bone, head weight, tare weight, fat color score, hind legs circumference, hind legs width, and carcass brisket depth etc. The described measurements were determined strictly according to established measurement standards. Carcass traits were shown in table1.

• Primers and PCR amplification
Primers were designed based on bovine RNF34 and RNF128 sequences (ENSTA) using Primer 5 software. Primer sequences are as follows. The PCRs were performed using the following cycling conditions: 95 °C for 5 min followed by 30 min, as shown in Fig.1.

• SNP detection and genotyping
Restriction endonuclease Bpu1102 was used to distinguish the genotypes of the PCR products of RNF34 gene. PCR products were digested with restriction endonuclease Spe I for genotyping of RNF128 gene. Restriction digestion reactions were conducted at 37 °C for 6 h. PCR digestion products were resolved on 3% agarose gels to distinguish the bands representing three different genotypes.
Genotypes and gene frequencies are shown in table 2.
• Statistical analysis SPSS 13.0 was used to calculate the relationship between genotype and production traits.
Genotype frequencies were calculated and were analyzed by significance testing. The genotypic effects of the RNF34 gene and RNF128 gene were determined using the general linear model (GLM) of SPSS.
The fixed model was as follows: Yijkl = u+ fysj + mk + eijkl, where Yijkl is the observed value of lth individual from the breed I, of genotype k, in the jth farm-year-season; u is the least square means of the observed values; fysj is the effective value of the jth farm-year-season; mk is the effective value of the genotype k; and eijkl is the random residual effect corresponding to the observed value.

• PCR amplification
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 August 2020 doi:10.20944/preprints202008.0518.v1 The PCR amplification products observed (intron 1) were consistent with the expected target fragments with good specificity. The PCR products were directly analyzed by restriction enzyme digestion as well as sequencing reactions.

• Restriction endonuclease analysis and sequencing of different genotypes
For SNP detection and genotyping, PCR amplification was first conducted on a mixture of randomly selected DNA samples followed by DNA sequencing that was performed by Sango (Shanghai, China). It was discovered that there was a single nucleotide polymorphism site at nucleotide 588 in the RNF34 gene 3'UTR region ( Figure 1). To allow for genotype identification, PCR products from 255 samples were digested with the Bpu1102 restriction enzyme and resolved on a 3% agarose gel. As shown in Associations of RNF34 polymorphisms with carcass traits were analyzed by one-way ANOVA.
Statistical analyses revealed that RNF34 3′UTR (c.+588 G>A) had a significant association with the carcass and meat quality traits, including tare weight, kidney weight, testis weight, fat color score (P<0.05), as shown in table 3.
Associations of RNF128 gene polymorphisms with carcass traits were analyzed by one-way ANOVA and LSD to allow for multiple comparisons to be conducted with respect to production traits.
As shown in table 4, there were significant differences between the different genotypes, involving dressed weight, forepaw weight, carcass depth, carcass brisket depth, the thickness of waist flesh, slaughter PH(P<0.05), and lung, trachea, hind legs length(P<0.01).

DISCUSSION
Meat quality is commercially important for the animal husbandry industry and is affected by the genetic background of the animals as well as management, nutrition and meat processing. Although, previous studies mainly focused on RNF34 and RNF128 were associated with cell differentiation and apoptosis [10]. RNF34 gene transcripts are highly enriched in BCB oocytes, suggesting that RNF34 may be involved in oocyte apoptosis [11,12]. And RNF34 plays an important role in the regulation of NOD1, RNF34 , NF-κB pathways, which supports the idea that RNF34 is a negative regulator of the NOD1 pathway through direct interaction and ubiquitination of NOD1 [13,14] [14] . negative regulator of T-cell receptor responsiveness and cytokine production [15,16]. But new important roles of RNF34 and RNF128 were founded in the present research.
In this study, SNPs of RNF34 and RNF128 were founded to play important roles in meat quality traits and growth traits. SNP of RNF34 3′UTR-588 G>A suggested that the AA genotype was significantly associated with testis weight and fat color score. The AG genotype was significantly associated with kidney weight. Furthermore, the average production data for cattle of genotype AA were lower than for those for cattle with genotypes AG or GG in pH after acid exhausted and fat color score.
The RNF128 gene I1-2380C>T was founded different genotypes exited significantly influence with the carcass traits. The TT genotype was significantly associated with carcass weight, the net weight of bone, bullwhip, mesenteric fat weight, and thickness of waist flesh. The TC genotype was significantly associated with slaughter PH. The CC genotype was significantly associated with hind leg circumference. The LSM of the carcass weight, the net weight of bone, bullwhip weight, mesenteric fat weight, the thickness of waist flesh, and hind leg circumference for the TC genotype was higher than that of the TT or CC genotypes. The LSM of slaughter PH for the TT genotype was higher than that for the TC or CC genotypes. So we can select the excellent meat traits by genotypes of RNF34 and RNF128. Thus, this study supports the development of a novel theory about the cultivation of excellent beef using molecular biology techniques.

Conclusion:
Our findings suggest that polymorphisms in RNF34 and RNF128 might be important genetic factors that influence carcass and meat quality in beef cattle. Thus, they might be useful markers for meat quality traits in future marker-assisted selection programs in beef cattle breeding and production.