Study On MLPH, The Candidate Gene Of Dilute Coat Color In Goat

On the basis of cattle(NC₀07301)and dog(NC₀06607)melanophilin sequences from GenBank,Intron 1 to exon 16 of goat melanophilin gene fragments was amplified(except for two gap which were about 12.5KB and 7KB,in intron 2 and intron 9 respectively).The total length of determined sequence was 19289bp,which had been handed out to GenBank, the Accession Number was EU316218.76 point mutations were found in the determinged goat melanophilin gene sequence,of which 18 was in exon,and 58 was in intron.These mutations in exon were named as follows:2486A＞C,5620A＞G,8731C＞G,8743G＞A,8753G＞A,8765C＞T,8782C＞T,11512G＞A,11584G＞A,13998G＞A,14012A＞G,17308T＞C,18904A＞G,18927G＞T,18951A＞G,18929T＞C,18955A＞C,and 18977A＞G.The missense mutation in that were Ala8731Gly,Arg8743His,Pro8782Leu,Asp11512Asn,Gly11584SerandLys14012Arg. The 58 mutation in intron were 1468G＞T,2260G＞A,2517G＞A,5644C＞T,7935T＞C,7958C＞T,7972G＞A,8067G＞C,8068C＞T,8151G＞A,8242G＞A,8580T＞A,8587C＞A,8610G＞A,8645C＞T,9109G＞A,9385T＞A,8892T＞A,8923C＞G,9009G＞A,9125G＞A,9179G＞C,9613T＞C,9656A＞C,10028A＞G,9918C＞T,10745G＞T,10831C＞T,10515C＞T,10813C＞T,10925T＞G,11362G＞A,11395A＞G,11421T＞C,11832C＞A,13542T＞C,13774A＞C,13782C＞G,13797A＞G,13898A＞C,14992G＞A,15144G＞A,15146A＞G,15592T＞C,15897T＞C,17041C＞T,17078T＞C,16710T＞A,16711C＞A,16761G＞A,16878G＞A,17041C＞T,17047T＞C,17078T＞C,17200C＞A,17885C＞T,18634T＞A,and 18740C＞T.Nine SNPs at position 8580T＞A,8587C＞A,8610G＞A,8645C＞T,8731C＞G,8743G＞A,8753G＞A,8765C＞T and 8782C＞T exhibited completed linkage.In our sequenced 108 goat individuals,92 were wildtype homozygote and 16 were heterozygote. So we made a hypothesis that the mutation allele might be or might be linked with a recessive lethal gene,which took highest 38.46%gene frequency in Leizhou black goat.we determined a missense mutation A→G at position 11584.Meanwhile the PCR-RFLP of this mutation was performed in 304 individuals from 9 goat breeds/strains,results showed that allele A was the superior allele(83.88%).Allele G was found mostly in Chengdu Ma goat(39.58%)and Nanjing Brown goat(in eluding three strains)and homozygote genotype GG was only found in these breeds/strains.We mader a conclution that allele G might be a candidate site for the particular dilute coat color(tan)found in Nanjiang Brown goat and Chengdu Ma goat. In the 5'UTR region,infrequently used poly(A)signal was found.We compaired poly(A) signal mong species.Result showed that the most highly conserved AATAAA was only found in Homo sapiens,Pan troglodytes,Mus musculus,and Rattus norvegicus,while another kind of variation(AATGAA)was found in Bos taurus and Monodelphis domestica. However,we failed to specify any possible hexanucleotide to be polyA signal or its mutation in other species shown in Table 2,partly because relatively high variance exist in MLPH gone among species and partly because there might be some special molecular mechanism for splicing other than that in other genes.As it came to the stop codon usage,most species used TAA as stop codon:Capra hircus, Ovis_aries,Bos taurus,Equus caballus,Sus scrofa,Homo sapiens,Pan troglodytes,and Mus musculus.Three species use TGA as stop codon:Felis catus(including normalvariant and truncated melanophilin),Canis familiaris,and Rattus norvegicus.TAG as stop codon was only found in Monodelphis domestica and Gallus gallus.Great variance of peptide leght among species was aslo observed.It spans from 487aa (Ornithorhynchus anatinus)to 617aa(Gallus gallus)with mean value 577.7aa and standard deviation of 34.6aa.For artiodactyla,it ranges from 577aa(Sus scrofa)to 610aa (Ovis aries).By aligning the four sequences of artiodactyla,we found that a 4aa insert (QPQP)in Capra hircus from position 193 to 196,and a 26aa insert (SLDWKDPHSKELPVPSETCRPFPCAQ)in Ovis aries from position 237 to 262.Two deletions were detected in Sus scrofa:one was 6aa(QEAGIL)after position 249,and the other was laa(E)after position 384 with the peptides 100%conserved in the three non-deletion species.Generally speaking,high variance of the MLPH gene exits among species.Comparing with cattle,the protein identities are as follows:goat 90.82%,sheep 88.52%,horse 66.39%, cat 64.10%,dog 64.02%,pig 69.69%,human 59.61%,chimpanzee 55.03%,house mouse 53.97%,Norway rat 53.50%,opossum 28.20%,Omithorhynchus 34.67%and chicken 39.37%.For the mRNA sequences,the overall mean distance(p-distance module)& Std.Err are 0.447 and 0.014 among goat,sheep and 11 manunals.When the values above were recalculated using the corresponding protein data,we got larger variation:the overall mean distance was 0.523 with Std.Err 0.025 among mammals.The disparity index test matrix was computed using the UPGMA method based on mRNA sequences among species using Mega3.1 software.When the disparity index test matrix was reconstructed according to the translated protein data,only 38 out of 91 pairs multiple comparison of hypothesis rejection at 5% level were observed.Both the mean analysis and the disparity index test matrix analysis showed that more differentiation occurred at protein sequence rather than in mRNA level.By the way,ESTs as the random sequencing results of cDNA,have a significant meaning.ESTs from goat Skin with anagen hair follicle and mammary gland were analyzed synthetically and.The results showed that 48 out of 392 ESTs from Cashmere goat skin with anagen hair follicle are genes coding for keratin or keratin associated protein. However there are not any ESTs of this kind from goat mammary gland,but some genes coding for immunoglobulin,choline transporter-like protein,MHC and so on,among which immunity and enzyme related genes take the most.The most remarkable analogical genes according to the ESTs from two different tissues code for ribosome proteins.The proportion from skin with anagen hair follicle is 15.1%and mammary gland 21.6%.It should be pointed out that 17 out of 26 contigs whose ESTs are from both skin and mammary gland code for ribosome proteins,as high up to 65.4%.