猪雌激素受体基因（ESR）点突变的PCR－SSCP检测

雌激素受体基因(ESR)是控制猪高产仔数的主效基因之一,具有明显的基因效应:BB型比AA型母猪胎总产仔数和产活仔数分别高出1.40～3.37和0.63～3.58头,是目前商品猪育种和生产中重点检测的主要基因之一。常规采用PCR－RFLPs方法区分该基因由点突变造成的3种不同的基因型。本研究建立1种基于PCR的SSCP(single－stranded conformation polymorphism)方法对猪ESR该位置的点突变进行检测,具有操作简便、灵敏度高和不需要酶切等优点,可以在育种实践中广泛应用。 Abstract:ESR is a major gene controlling litter size in swine.The sows of BB genotype produce 1.40～3.37 more piglets of the total number born and 1.07～2.40 piglets of the number born alive respectively comparing with those of AA genotype.ESR has been one of the widely detected genes in pig breeding and production.Usually the point mutation of ESR gene was detected by PCR-RFLP approach.The present study established a novel method based on PCR-SSCP,with the advantage of easy maniputation,high sensitivity and no necessity for restriction enzyme digestion.This method may be applied for commercial detection of the point mutation of ESR gene in swine breeding.     

撒坝猪血清酯酶多态性与繁殖性能关系的研究

采用垂直板聚丙烯酰胺凝胶电泳法(PAGE), 对115头撒坝猪的血清酯酶(ES)多态性进行了检测,计算了该位点的基因型频率、基因频率和位点多态杂合度(h),并用二因素有互作的最小二乘模型对血清酯酶多态性与繁殖性能的关系进行了分析。结果表明,撒坝猪血清酯酶3种基因型AA、AB和BB的频率分别为0.2696、0.5826和0.1478。两个等位基因A和B的基因频率分别为0.5609和0.4391。该位点的杂合度为0.4926。在3种基因型中,不同基因型母猪的繁殖性能在产仔数、仔猪初生窝重、20日龄窝重、断奶仔猪数和断奶窝重等性状上存在着显著差异(P<0.05);公、母猪不同基因型交配组合在产仔数、断奶仔猪数、仔猪断奶体重和断奶窝重等性状上亦有显著差异(P<0.05)。显示出猪的血清酯酶多态性可望作为繁殖性能选种的遗传标记。 Abstract：　Serum esterase polymorphisms of 115 Saba pigs were investigated by using the method of vertical polyacrylamide gel electrophoresis. The genotype frequency, gene frequency and heterozygosity of this locus were calculated. The relationship between the serum esterase polymorphism and reproductive performance was analyzed by the least square analysis of two factors (ES genotype of boar and sow) with interaction. The results demonstrated that the genotype frequency of AA, ABandBBwas 0.2696, 0.5826 and 0.1478 respectively, the gene frequency of the alleleAandBwas 0.5609 and 0.4391 respectively. The heterozygosity of this locus was 0.4926. There are significant differences (P<0.05) on litter size, litter weight at birth, litter weight at 20 days, litter size at weaning and litter weight at weaning of different genotypic sows. The significant differences (P<0.05) were showed on litter size, litter size at weaning, weaning weight and litter weight at weaning of different genotypic mating combinations. It indicated that the serum esterase polymorphism was expected to be the genetic marker of pig reproductive performance.     

PCR-RFLP检测LDL受体基因TaqⅠ多态性位点的研究

应用聚合酶链反应(PCR)扩增人类LDL受体基因外显子4-内含子4-外显子5片段,PCR产物为1.55kb,DNA片段经序列鉴定后,进行TaqI酶切位点的RFLP分析。结果显示:中国汉族人群LDL受体基因中存在着TaqⅠ酶切位点多态性; 200个LDL受体等位基因中TaqⅠ酶切位点出现的频率为0.515,该点频率较为适中, 可作为中国汉族人群LDL受体基 因的遗传标志来进行家族性高胆固醇血症(FH)的基因诊断。所建立起的LDL受体基因TaqⅠ位点的PCR -RFLP方法具有快速、简便的特点,在FH的基因诊断上有应用价值。 Abstract:To develop rapid and sensitive technique for detectin the TaqI polymorphism at the human LDL receptor gene in Chinese,the exon4-intron4-exon5 of the human LDL receptor gene was amplified by polymerase chain reaction(PCR).The PCR products were directly analysed by restriction fragment length polymorphisms(RFLP).The results showed that the TaqI polymorphism is associated with the LDL receptor gene in Chinese of Han nationality;The frequency of T= allele (presence of TaqI cutting site)is 0.515 in 200 LDL receptor alleles.This technique may be used for rapid and sensitive screening of the LDL receptor gene for the TaqI polymorphism.     

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuⅡ/la, Taq Ⅰ/2b-3, Taq Ⅰ/5b-7, and Xba Ⅰ/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuⅡ/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq Ⅰ/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq Ⅰ/2b-3 and Xba Ⅰ/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq Ⅰ/8 and A2 (10.Tkb) in EcoR Ⅴ/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq Ⅰ/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu Ⅱ/la, there was no significant difference between Chinese and Japanese.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

绵羊产羔性状主效基因检测研究

以绵羊BMP15基因和BMPR-IB基因为候选基因,以湖羊、中国美利奴单胎品系、中国美利奴肉用和毛用多胎品系为研究对象,采用PCR-RFLP方法对候选基因进行单核苷酸多态性（SNP）位点检测和基因型分析,同时研究基因对绵羊产羔数的影响。对BMP15基因进行SNP检测,结果未发现多态性位点;对BMPR-IB基因进行多态性检测,结果发现了一个A746 G SNP位点。依据A746 G SNP位点进行基因型分析,结果在各品种（系）羊中发现了3种基因型,即BB、B+和++。等位基因型频率在各品种（系）间差异极显著（P<0.001）,在湖羊中以BB基因型为主,在中国美利奴单胎品系中以++基因型为主, 而在中国美利奴肉用和毛用多胎品系中以B+基因型为主。BMPR-IB A746G位点的变异明显影响绵羊的产羔数,与++基因型母羊相比, BB和B+基因型母羊产羔数明显较多。研究结果同时表明,利用BMPR-IB基因型可以很好的预测母羊的产羔数。研究获得的这些结果强烈表明BMPR-IB为影响绵羊的产羔数的主效基因,可以用于对绵羊产羔数的选择。Abstract: The current study was designed to detect SNPs within BMP15 and BMPR-IB gene and investigate the effect of the genes on sheep litter size. Four sheep lines, HU-Yang, Chinese M erino monotocous, Chinese Merino multiparous for wool production and Chinese Merino multiparous for mutton production, were used in this study. Litter sizes were recorded for each ewe in the four lines. Primers for BMP15 and BMPR-IB gene were designed from database sheep sequence and polymorphisms were detected by PCR-RFLP method. The results showed that there was no polymorphism with BMP15 gene among the four lines, and there was an A / G SNP with BMPR-IB gene at base 746 among the four lines. Three types of genotype (BB, B+ and ++), based on A / G locus, were found within each line. The frequencies of genotypes were significantly different among the lines (P<0.001), with BB genotype primarily existing in HU-Yang, ++ genotype in Chinese Merino monotocous line, and B+ genotype in Chinnese Merino multiparous lines. The A / G mutation influence significantly the sheep litter sizes, and the BB and B+ ewes had significant higher litter sizes than ++ ewes. The results of present study showed simultaneously that the genotype of BMPR-IB was a perfect predictor of the sheep litter sizes. These results intensively indicated that BMPR-IB is a major gene to affect litter size in sheep, and could be used as the molecular genetic marker to select litter size in sheep.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

FSHβ subunit gene is associated with major gene controlling litter size in commercial pig breeds

An insertion fragment in porcine FSHβ subunit gene was cloned by PCR. Sequencing data show that the insertion is a retroposon of 292 bp siting in intronⅠ at the site between +809 and +810 base. Based on these results, a PCR programme was created to genotype animal individuals in different pig breeds at FSHβ locus and polymorphism of FSHβ gene was analyzed. With the combination of genotype and litter size of sows, it was demonstrated that FSHβ locus is closely associated with major gene controlling litter size in commercial pig breeds, such as Yorkshire, Landrace, Durco. Averagely the AA sows give more 1.5 piglets than BB sows do per litter.     

不同猪种肠毒素大肠杆菌（ETEC）F4受体微卫星标记遗传差异性研究The

采用13号染色体上与K88ab和K88ac受体基因连锁的2对引物（S0223和S0068）,研究沙子岭猪和大约克猪的遗传差异性。结果表明,2个猪种在2个基因座均存在多态性,其基因杂合度和Shannon信息指数存在很大差异,而中外猪种的K88ab和K88ac受体基因也存在遗传差异,这2对引物可望作为K88ab和K88ac受体基因的遗传标记。Abstract: The genetic variation of ETEC F4 receptor in Shaziling and Yorkshire breeds were studied using two microsatellite markers(S0223 and S0068). The results showed that there were polymorphisms in the two markers, and there were great variations of the gene heterozygosity and Shannon information index in the two breeds. It was also reported that there were differences in K88ab and K88ac receptors in Chinese native breeds and foreign breeds, so the two markers might be the genetic markers of F4 receptor gene.     

大熊猫苦味受体T2R2基因的克隆与序列分析

Based on bitter taste receptor T2R2 gene sequence of domesticated dog(AB249685), one pair of primers were designed and used to amplify an approximately 1.1 kb DNA fragment from genomic DNA sample of giant panda by using PCR. The PCR products were ligated into the pMD-18T vector, and then transformed into competent cells of E.coli DH5α. The identified positive clone was sequenced. The result showed that the T2R2 gene of giant panda was 1 008 bp in length, and contained complete exon, and 915 bp, encoding 304...     

Association with litter size of new polymorphisms on ESR1 and ESR2 genes in a Chinese-European pig line

The objective of this study was to search for polymorphisms in the coding region of the estrogen receptors 1 and 2 (ESR1 and ESR2 )and to analyze the effects of these variants and the well known intronic ESR1 Pvu II polymorphism on litter size in a Chinese-European pig line. We identified five silent single nucleotide polymorphisms (SNP) in the ESR1 cDNA: c.669T > C (exon 3), c.1227C > T (exon 5), c.1452C > T (exon 7), c.1665T > C and c.1755A > G (exon 8). One pair of these SNP (c.1665T > C and c.1755A > G) co-segregated in the analyzed line, and the SNP c.669T > C showed the same segregation pattern as the Pvu II polymorphism. These polymorphisms were tested in this study, although the c.1452C > T SNP within exon 7 was not analyzed due to its low informativeness. In the ESR2 cDNA, one missense SNP was found within exon 5, which caused an amino acid substitution in the coded protein: "c.949G > A (p.Val317Met)" and was tested on sow litter size. Information on 1622 litter records from 408 genotyped sows was analyzed to determine whether these SNP influenced the total number of piglets born (TNB) or the number of born alive (NBA). The polymorphisms ESR1: [Pvu II; c.669T > C], ESR1: [c.1665T > C; c.1755A > G] and ESR2: c.949G > A showed no statistically significant association with litter size. However, the ESR1: c.1227T allele was significantly associated with TNB. The additive substitution effect was estimated to be 0.40 piglets born per litter (P < 0.03), and no dominance effects were observed. This SNP could be useful in assisted selection for litter size in some pig lines, as a new genetic marker in linkage disequilibrium with the causative mutation.     

中国人dystrophin基因RFLPs的初步研究

We have studied the RFLPs distribution and frequency of dystrophin gene in Chinese by using 14 subclones of complete 14 kb cDNA for the dystrophin gene as hybridization probes. Allelic fragments are detected in hybridization patterns of Pvu II/1a, Taq I/2b-3, Taq I/5b-7, Xba I/10. Among them, the allelic fragments (26 kb and 3.8 kb) in Pvu II/2b-3 patterns and the allelic fragments (10 kb and 8.4 kb) in Taq I/5b-7 patterns are the new RFLPs which have never been reported. From the comparison of our data with those of Caucasian and Japanese respectively and their statistical analysis, we can obtain the results as follows: There is remarkable difference (p less than 0.01) of the allelic fragment frequency in Taq I/2 b-3 (A1 = 3.4 kb, fre. 0.04; A2 = 3.2 kb, fre. 0.96) and Xba I/10 (A1 = 7.4 kb, fre. 0.41; A2 = 6.7 kb, fre. 0.59) between Chinese and Caucasian. The frequency of the allelic fragments A2 in Taq I/8 (A1 = 6.5 kb, A2 = 5.6 kb) and EcoR V/9 (A1 = 11.8 kb, A2 = 10.7 kb) are high in Caucasian, but have not been detected in Chinese. These differences are also highly significant. But the B1B2 allelic frequencies in Taq I/5 b-7 (B1 = 3.2 kb, B2 = 1.6 kb) are the same. There is no significant difference in the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1 a between Chinese and Japanese. Preliminary results suggest that there probably are high frequencies for spontaneous neutral mutations in the evolution process of the huge dystrophin gene (about 2,300 kb). In the meantime, the neutral mutation frequencies of various sectional sequences have remarkable differences, and that of some sectional sequences of the gene between Chinese and Caucasian may also have remarkable differences.     

Is there a relationship between prion protein genotype and ovulation rate and litter size in sheep?

The identification of an association between polymorphisms of the prion protein (PrP) gene and susceptibility to scrapie has enabled the development of breeding programmes to increase natural resistance to scrapie. It is, however, imperative to identify if such selection would affect important reproduction and production traits. The objective of this study was to determine if there is a relationship between polymorphisms at codons 136, 154 and 171 of the PrP gene and ovulation rate or litter size in sheep. Data were collected from a mixed-aged flock of Belclare ewes, over a 9-year period. Ovulation rate was determined annually using laparoscopy by counting the number of corpora lutea at each of two consecutive oestrous cycles, one immediately before and one after mating (2418 records from 366 ewes). Litter size was recorded at parturition (875 records from 353 ewes). The five common PrP alleles were present in the population and 14 PrP genotypes were represented among the animals studied. There was no significant overall effect of PrP genotype on ovulation rate or litter size and pairwise comparisons among genotypes did not reveal any significant differences for either trait. These data suggest that breeding programmes based on selection for specific polymorphisms of the PrP gene will not influence ovulation rate or litter size, at least in the breed studied.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

GDF9 as a candidate gene for prolificacy of Small Tail Han sheep

Growth differentiation factor 9 (GDF9) which controls the fecundity of Belclare, Cambridge, Santa Ines, Moghani, Ghezel and Thoka ewes was studied as a candidate gene for the prolificacy of Small Tail Han sheep. According to the sequence of ovine GDF9 gene, six pairs of primers were designed to detect single nucleotide polymorphisms of two exons of GDF9 gene in both high fecundity breed (Small Tail Han sheep) and low fecundity breed (Dorset sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Only the products amplified by primers 2-1 and 2-2 displayed polymorphisms. For primer 2-1, three genotypes (AA, AB and BB) were detected in both sheep breeds. Sequencing revealed one silent mutation (G477A) in exon 2 of GDF9 gene in the BB genotype in comparison with the AA, which was known as G3 mutation of GDF9 gene in Belclare and Cambridge ewes. The relationship of least squares means for litter size was AA?>?AB?>?BB in Small Tail Han sheep (P?>?0.05). For primer 2-2, two genotypes (CC and CD) were detected in both sheep breeds. Sequencing revealed one novel single nucleotide mutation (G729T) in exon 2 of GDF9 gene in the CD genotype in comparison with the CC, which resulted in an amino acid change (Gln243His). The ewes with mutation heterozygous genotype CD had 0.77 (P?相似文献   

圈卷产色链霉菌尼可霉素生物合成基因-sanK的克隆及功能研究

利用染色体步移策略,以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌中克隆到了一个大约１０ｋｂ的ＤＮＡ片段。对其中１．８ｋｂ的ＰｖｕⅡ－ＳａｃⅡ片段进行了序列分析,结果表明：此片段中含有一个具有１１７０个核苷酸的完整开放阅读框,起始密码子为４４７位的ＡＴＧ,终止密码子为１６１４位的ＴＧＡ,推测其编码一个３８９个氨基酸的蛋白质产物。利用ＢＬＡＳＴＸ程序进行了分析揭示,此基因编码一个肌氨酸单体     

雌激素受体基因和长白猪繁殖性能相关研究

通过检测612头长白母猪共2 239窝ESR基因的PvuⅡ酶切多态性,分析了不同ESR基因型和长白母猪繁殖性状的相关,以确定ESR基因在猪育种应用的可能性.头胎母猪、第2胎母猪和3胎以上母猪的资料分开统计.群体中B等位基因的频率很低,但似然比检验结果显示群体的基因频率处于哈代-温伯格连锁平衡状态.第1胎BB基因型母猪的总产仔数显著高于AA型母猪(12.05±0.82 vs 10.19±0.24)(P＜0.05),但BB基因型母猪的初生仔猪重显著低于AA型母猪(1.23±0.07vs 1.42±0.02)(P＜0.05).第3胎以上资料合并,BB基因型母猪的总产仔数显著多于AA和AB基因型母猪(11.98±0.63 vs10.90±0.48/10.92±0.51)(P＜0.05),产活仔数显著高于AA型母猪(10.31±0.58 vs 9.43±0.45)(P＜0.05);AB基因型母猪初生仔猪重显著低于AA型母猪(1.44±0.04 vs 1.48±0.04)(P＜0.05).所有资料合并,BB基因型母猪的总产仔数极显著高于AB型母猪(11.63±0.52 VS 10.63±0.42)(P＜0.01),显著高于AA型母猪(11.63±0.52 vs 10.70±0.40)(P＜0.05);BB基因型母猪的产活仔数显著高于AB和AA基因型母猪(10.15±0.50 vs 9.33±0.39/9.41±0.41)(P＜0.05).其余情况下各基因型母猪间繁殖性状间差异不显著(P＞0.05).总之,BB基因型母猪的总产仔数和产活仔数优于其他基因型母猪,但仔猪初生重较低.ESR基因可以作为遗传标记,用于本群长白猪产仔数的选择.