ISSN 0026 8933, Molecular Biology, 2010, Vol. 44, No. 4, pp. 536540. Pleiades Publishing, Inc., 2010.

GENOMICS AND TRANSCRIPTOMICS

UDC 577.21

A Novel SNP of Lactalbumin Gene in Chinese Dairy Goats1

R. N. Maa, C. J. Denga, X. M. Zhanga, X. P. Yuea, X. Y. Lana, H. Chena,b, and C. Z. Leia
a

Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A & F Yangling University Yangling, Shaanxi 712100, China b Institute of Cellular and Molecular Biology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, China; e mail: leichuzhao1118@126.com
Received October 26, 2009; in final form, December 17, 2009

AbstractThe lactalbumin ( LA) plays a key role in lactose synthesis in mammary glands of domestic animals. Mutations in the LA gene are associated with the milk traits in dairy cattle. In our study, a novel SNP: NO_X06366: g.875 C > T was detected in 708 dairy goat individuals268 of the Xinong Saanen breed and 440 of Guanzhong breed, which revealed a synonymous mutation in the exon 1 of LA gene. The Poly merase Chain ReactionSingle Strand Conformation Polymorphism (PCR SSCP) and sequencing tech niques showed that there were three genotypes: CC, CT and TT. Moreover, the 2 test showed that the geno type frequencies of the two breeds were in good agreement with the HardyWeinberg equilibrium (P > 0.05). The relationship of the polymorphism of dairy goat LA gene with the milk trait and the body size trait was revealed. Individuals with the CC genotype were significantly smaller at chest circumference than those with CT (P < 0.05) in both breeds. But the milk trait and other body size traits of the two dairy goat breeds had no significant association with genotypes studied. DOI: 10.1134/S0026893310040059 Key words: dairy goat, lactalbumin gene, PCR SSCP, SNP, body size traits, milk trait

The Xinong Saanen breed (SN) and the Guan zhong breed (GZ) are well defined dairy goat breeds in China. The composition of goat milk is most similar to that of humans among the milk of animals. LA is one of the major serum proteins in ruminant milk and plays a key role in lactose synthesis in the mammary gland [1] and thus is a potential quantitative trait locus for dairy cattle [2]. It induces lactose synthesis in the mammary gland by interacting with the UDP galac tosyltransferase, thus forming the heterodimer of the lactose synthetase enzyme. In human milk it also pro vides a major source of amino acids [3]. Many envi ronmental factors including diet, exercise, stage of lactation and stress during labor have been shown to affect milk composition [4, 5]. However, genetic vari ations of the LA gene have also been reported in cat tle, goats and humans [3]. The primary amino acid sequence of the bovine LA was first determined by Gordon [6] and Brew [7]. The LA is a small (Mr 14 200) acidic (pI 45) Ca2+ binding milk protein that consists of 123 amino acids [8]. It is one of the two components required for lac tose synthesis. It catalyzes the final step in lactose bio synthesis in the lactating mammary gland [9]. It has been found that some forms of LA can induce apo ptosis in tumor cells [10], which suggests that this pro
1 The article is published in the original.

tein can fulfill many other important biological func tions. Goat LA contains 123 amino acids [11]. The bovine LA gene is located on chromosome 5 (Gen Bank acc. no: X06366) it consists of four exons span ning 3.0 Kb (Fig. 1). The human LA gene consists of four exons spanning 2.5 Kb [12] and is located on chromosome 12 [13], and in sheep the LA gene has been localized on chromosome 3 [14]. However, there were no reports about dairy goat LA gene. Polymorphisms of the LA gene in dairy goats have received far less attention than that of dairy cattle, pos sibly because of the lack of important economic values in the dairy goat industry. This study focuses on genetic variants of the LA gene in order to better understand its functional significance in dairy goats. We identified variant forms of the LA gene and ana lyzed the relationship between the SNPs and the milk and body size traits in two dairy goat breeds from China. EXPERIMENTAL Sample collection and DNA extraction. Two dairy goat breeds (SN and GZ) from China were used in this study. All of 708 blood samples were collected from three dairy goat breeding farms (Shaanxi province dairy goat breeding centre in Sanyuan county; North west A&F University dairy goat breeding farm; and

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A NOVEL SNP OF LACTALBUMIN GENE IN CHINESE DAIRY GOATS exon II

(100.0%)

537

exon I
(94.7%)

exon III

exon IV

intron I 5' UTR

intron II

intron III 3' UTR

lactalbumin gene of bovine

3090 bp

Fig. 1. The structure of the bovine LA gene.

Qianyang Saanen dairy goat breeding farm in Shaanxi province). Genomic DNA was extracted from the blood by the standard phenol chloroform method. The first fetal milk yield and body size traits were also collected and measured at same three breeding farms. PCR conditions. Basing on the sequence of the bovine LA gene (GenBank acc. no. X06366) and its construc tion (Fig. 1), a pair of primers including the complete exon 1 and partially the intron 1 was designed with primer 5.0. The primer pair was as follows: Forward, 5' GGGGTAAC CAAAATGATGT 3', Reverse, 5' ATATGGCAGACCA CAGAGTATC 3'. This pair of primers was used to amplify the PCR product of about 364 bp for the LA gene. 25 l of the reaction mixture contained 50 ng genomic DNA, 0.5 M of each primer, 1 buffer (including 1.5 mM MgCl2), 200 M dNTPs and 0.625 units of Taq DNA polymerase (MBI). The cycling protocol was 4 min at 95C, 34 cycles of denaturation at 94C for 30 s, annealing at 53.0C for 40 s, extension at 72C for 40 s, with a final extension at 72C for 10 min. SSCP polymorphism and sequencing. 5 l aliquots of the PCR products were mixed with 5 l of the denatur ing solution (95% formamide, 25 mM EDTA, 0.025% xylenecyanole and 0.025% bromophenol blue), heated for 10 min at 98C and then chilled on ice. Denatured DNA was subjected to polyacrylamide gel electrophoresis (PAGE) (80 73 0.75 mm) in 1 Tris borate EDTA (TBE) buffer at a constant volt age (190 V) for 2.53.0 h. The gel was stained with 0.1% silver nitrate [15]. After the polymorphism was detected, the PCR products from different electro phoresis patterns were sequenced in both directions in an ABI PRIZM 377 DNA sequencer. The sequences were analyzed by the DNASTAR 5.0 package. Statistical analysis. Based on the genotypic numbers of the LA gene locus in the analyzed breeds, geno typic frequencies and allelic frequencies according to the HardyWeinberg equilibrium were calculated. Differences for genotypic frequencies in the LA gene locus were analyzed by the 2 test and the rela
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tionship of the genotypes with the milk and body size traits (Body length, Height at withers and Chest cir cumference) in the two dairy goat breeds (n = 708) were calculated using the SPSS software (version 16.0). An adjusted linear model was created and considered the effects of the sire, the dam for the same sire, age, and genotype, as well as the correlation between the sire and the genotype. The adjusted linear model: Yijklm = + Si + Dij + Ak + Gl + (SG)il + (AG)kl + Eijklm, where Yijklm is the trait measured for each of the ijklmth animal, is the overall population mean, Si is the fixed effect associated with the sire, Dij is the fixed effect associated with jth dam for the sire i, Ak is the fixed effect due to the kth age, Gl is the fixed effect associated with the lth genotype, (SG)il is the interac tion between the ith sire and the 1th genotype, (AG)kl is the interaction between the kth age and the 1th genotype, and Ejiklm is the random error. Effects associated with the farm and season of birth (spring vs. fall) are not considered in the linear model, since the preliminary statistical analysis indicated that these effects do not have a significant influence on the variability of traits in the analyzed breeds. Population genetic indexes, including homozygosis (Ho), heterozygosis (He), effective allele numbers (Ne) and PIC (Polymorphism information content) were calculated using the Bot stein method [16]. RESULTS AND DISCUSSIONS The LA gene of the two breeds demonstrated poly morphic patterns by PCR SSCP and sequencing method. One mutation was detected in the exon 1 of the LA gene and no polymorphisms were detected in the partial intron 1 .The novel SNP revealed one tran sition: No_06366: g.875 C > T. In detail, the C > T mutation in the 875 nucleotide position of GenBank No_X06366 revealed a synonymous mutation in LA. Three genotype patterns were found and named CC, CT and TT (Fig. 2) in this study. The allele frequency of the LA C in the two analyzed breeds

538 (a)

MA et al.

(b)

(c)

Fig. 2. The sequencing maps of the novel SNP in exon 1 of LA gene. Sample chromatograms of heterozygous (b) and homozygous (a and c) genotypes are shown. The denotes the location of the polymorphism.

was 0.423 and 0.447, respectively (Table 1). The 2 test (Table 1) showed that the genotype frequencies of the two breeds were found to agree with the HardyWein berg equilibrium (P > 0.05). The population genetic parameters were calculated for the present breeds (Table 2). The differences between the expected and observed heterozygosity were all less than 0.5 within the two breeds. The PIC values of the two breeds of SN and GZ were 0.3691 and 0.3721, respectively. Accord ing to the classification of PIC (PIC < 0.25, low poly morphism; 0.25 < PIC < 0.5, intermediate polymor

phism; PIC > 0.5, high polymorphism), the two ana lyzed breeds possessed intermediate polymorphisms. The GZ has higher PIC than SN, which reflected that in the analyzed samples the GZ has a higher polymor phism than the SN. Association of the novel mutation in the LA gene with the milk trait (the first fetal milk yield) and body size traits in SN were analyzed (Table 3). We found that SN individuals with the CC genotype were signif icantly smaller in the chest circumference than those with the CT (P < 0.05) genotype. The relationship between the novel mutation and body size traits in GZ was analyzed too (Table 4). Individuals from the GZ breed with the CC genotype were also smaller in the chest circumference than those with the CT (P < 0.05) genotype. No other statistically significant differences on milk traits and body size traits (P > 0.05) were observed between the CC, TC, and TT genotypes in the two breeds. Therefore, the presence of the transition (No_X06366: g.875 C > T) might influence the chest circumference at dairy goats. A lot of research regarding polymorphisms, milk trait and body size traits has been conducted on cattle. The associations between genetic variations of milk com position and its production are of great significance to the dairy industry. Variants resulting in the changes of the amino acid sequence have been detected earlier in many genes. In addition, variants containing changes in the noncoding regions have been detected. The 5' flanking region of the LA gene is a control region, thus variations in this region of the gene could poten tially modify the LA gene expression by altering binding of the RNA polymerase and various transcrip tion factors that control gene expression through bind ing to this region of the gene [17]. In 1993, Bleck found that a SNP in the 5' flanking region of the Hol stein LA gene is associated with the increased milk yield [18]. SNPs can also affect the amino acid sequence or post translational modifications of the milk proteins and their bioactivity in humans [19]. Although this novel mutation is unlikely to affect the biological function of the LA gene, it highlights the need for further studies of the polymorphisms among dairy goat milk proteins.

Table 1. Genotype and gene frequencies at the dairy goat LA gene exon 1 locus Genotype frequency Breeds Sample TT SN GZ 268 440 0.261 (70) 0.252 (111) CT 0.631 (169) 0.602 (265) CC 0.108 (29) 0.145 (64) T 0.577 0.553 C 0.423 0.447
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A NOVEL SNP OF LACTALBUMIN GENE IN CHINESE DAIRY GOATS Table 2. Genetic diversity of the LA gene exon 1 locus in the two breeds Breed SN GZ Sample 268 440 Ho 0.5117 0.5057 He 0.4883 0.4943 Ne 1.9543 1.9774 PIC 0.3691 0.3721

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Table 3. Association of the LA gene genotypes with milk and body size traits in the SN breed Genotype CC CT TT The first fetal milk yield (Mean S.E.) 626.133 25.431 617.817 10.480 612.305 16.266 Body height (Mean S.E.) 74.556 0.923 74.925 0.380 75.864 0.590 Height at withers (Mean S.E.) 82.778 1.042 82.201 0.429 83.046 0.666 Chest circumference (Mean S.E.) 87.556 1.522a 91.917 0.627b 90.955 0.973a, b

Note: Values with different superscripts within the same line differ significantly at P < 0.05 (a, b). S.E. standard error of means.

Table 4. Association of the LA gene genotypes with body size traits in the GZ breed Genotype CC CT TT Body height (Mean S.E.) 73.543 0.752 74.021 0.524 73.865 0.556 Height at withers (Mean S.E.) Chest circumference (Mean S.E.) 81.876 0.856 82.112 0.523 81.958 0.678 86.598 1.325a 90.625 0.568b 89.954 0.841a, b

Note: Values with different superscripts within the same line differ significantly at P < 0.05 (a, b). S.E. standard error of means.

Our data indicates that there is a new variant (No_ X06366: g. 875 C > T) of the LA gene, which not only extends the spectrum of genetic variation of the LA gene, but possibly also contributes to the dairy goat breed ing. The polymorphism characterized in this study does not affect the biological function of the LA gene. This novel mutation is tightly associated with the chest circumference. The impact of SNPs on the goat milk protein variability represents a vast area for fur ther research. ACKNOWLEDGMENTS This work was supported by the National 863 Pro gram of the P. R. China (no. 2008AA10Z138), the 13115 Sci Tech Innovation Program of Shaanxi Province (2008ZDKG 11), Research Fund for the Doctor Program of Higher Education of China
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(no. 20080712001), The Young Topnotch Researcher Support Project of Northwest A&F University (no. QNGG 2009 007) and Natural Science Foun dation of Jiangsu Province (no. BK2008120). REFERENCES
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