山羊GH基因部分序列多态及生物信息学分析

1、山羊GH基因部分序列多态及生物信息学分析    生长激素(growth hormone，GH)是一种种属特异性激素，由垂体前叶分泌，为机体正常生长和泌乳所必需，在促进蛋白合成、调节糖和脂肪代谢过程中发挥着重要的作用。本试验采用PCR-SSCP技术，将GH基因作为目的基因，对黄淮山羊、长江三角洲白山羊以及波尔山羊168只个体进行多态性检测，分析山羊GH基因5侧翼区两个位点以及第2和第3外显子的遗传多样性，并分析了各多态位点与山羊体重性状之间的关系，探讨调控山羊生长发育的基因位点，为黄淮山羊、长江三角洲白山羊的选育和保种提供理论基础。在此基础上，进一步利用生

2、物信息学软件对GH基因的氨基酸组成、蛋白理化性质、5侧翼序列的转录因子结合位点以及序列的特征进行了分析，并进行了结构和功能的预测。研究结果如下： 1山羊GH基因部分序列的PCR-SSCP分析 在四个扩增片段上均发现了突变，其中在第1对引物P1上出现6种基因型(AA，AB，BB，BC，CC，AC)；第2对引物P2上出现3种基因型(DD，DE，EE)；第3对引物出现2种基因型(GG，GJ)；第4对引物出现6种基因型(MM，MN，NN，KK，MK，NK)。测序结果表明：第1个位点上存在2处突变，分别是60位CT和211位C碱基的缺失；第2个位点上存在3处突变，分别是264位TC，292位TA，372

3、位CT；第2外显子的782位发生了GA的突变，并且该处突变导致了丙氨酸苏氨酸的改变；第3外显子的存在两处突变，分别是1116位AG，1148位CT，这两处突变并未造成氨基酸的改变，属于无义突变。 2 GH基因群体遗传学分析及与山羊生产性能的关系 群体遗传学分析表明：在第1个位点，黄淮山羊和长江三角洲白山羊均以AB型个体最多，B等位基因频率最高，达到0.55左右；第2个位点，三个群体均以DD型个体最多，以等位基因D为主，基因频率在0.750.91之间；外显子2只出现GG、GJ两种基因型，并未发现JJ型个体，其中GJ基因型个体在三个山羊群体中出现频率最高，在0.7160.909之间；外显子3在三个

4、群体中均以MM型个体居多，黄淮山羊和长江三角洲白山羊都出现了MM、MN、KK、MK、NK等5种基因型个体，但是并未发现NN型个体；而波尔山羊则仅出现MM、MN、NN基因型个体，K等位基因未出现。群体平衡检测发现，山羊GH基因P2扩增片段在三个品种中的x2值都未达到显著水平，处于Hardy-Weinberg平衡状态(P0.05)，而外显子2的扩增片段E2的x2值则都达到显著水平，三个群体在该位点的基因型分布极显著偏离Hardy-Weinberg平衡(P0.01)，说明该位点有经过人工选择的可能，此外，在P1位点，波尔山羊的基因型分布处于平衡状态，而黄淮山羊和长江三角洲白山羊则都偏离平衡状态，在E

5、3位点，波尔山羊和长江三角洲白山羊处于平衡状态，而黄淮山羊则偏离平衡状态，估计原因是这两个位点分别受到不同的选育方向或是遗传进化环境不同造成的。 各多态位点与体重性状的最小二乘分析表明：启动子区域第一个位点P1上黄淮山羊AA、CC基因型个体的出生重和周龄重均略高于BB型，但不同基因型之间差异并不显著；而波尔山羊AA型个体在出生重、6月龄重以及周龄重上都显著高于BB型个体(P0.05)，并且存在AACCBB的趋势。 3山羊GH基因的生物信息学分析 运用生物信息学软件对GH基因的氨基酸组成、蛋白理化性质、5侧翼序列的转录因子结合位点以及序列的特征进行了分析，并进行了结构和功能的预测。Growth

6、hormone (GH) is a breed-specific hormone, secreted by the anteriorpituitary, which is necessary to growth and lactation and plays an important role inpromoting protein synthesis, regulation of glucose and fat metabolism. In this study,The polymorphisms of GH gene in the 5" flanking region from

7、168 Huanghuai goats、Changjiangsanjiaozhou white goats and Boer goats were analyzed by PCR-SSCP, atthe same time, the two sites of GH gene in the 5" flanking region and thepolymorphisms of the second and third exon were analyzed, moreover, therelationship between polymorphic sites and weight cha

8、racteristics was also analyzedin order to study the gene sites controlling goat growth, which will provide theoreticalbasis for selection and preservation of Huanghuai goats、Changjiangsanjiaozhou whitegoats. Furthermore, the amino acid composition, protein properties, transcription factorbinding sit

9、es in 5 "flanking region and the sequence characteristics of GH gene wereanalyzed by bioinformatics software assay. In additon, the structure and function werepredicted. Results are as follows: 1 PCR-SSCP analysis of goat GH gene partial sequence Mutations were found in all four fragments ampli

10、fied, There were six genotypes(AA, AB, BB, BC, CC, AC) with the first pair primers; and two genotypes (GG, GJ)with the second pair primers; two genotypes (GG, GJ) with the third pair primers; sixgenotypes (MM, MN, NN, KK, MK, NK) with the fourth pair primers. Thesequencing results demonstrate: Two m

11、utations were existed in the first site, whichwere 60 CT and C(211) deletion; Three mutations were existed in the second site,which were 264 TC,292 TA,372 TC; The mutation of GA(782)in exon 2results in the change of AlaThr; Two mutations were existed in exon 3, which were1116 AG, 1148 CT. 2 Group Ge

12、netics analysis of goat GH gene and its relation with goat productionperformance Genetic analysis shows :In site1, the Huanghuai goat and Changjiangsanjiaozhouwhite goats were mostly AB genotype, B allele frequency was highest at 0.55; Insite 2, Most of three groups were DD genotype, D allele freque

13、ncy were between0.750.91; Only two genotypes without JJ genotype existed in exon 2, and the highest frequency in the three goat populations was GJ genotype, which was between0.7160.909; Exon 3 of the three groups were mostly MM genotype, MM, MN, KK,MK, NK five genotypes without NN genotype were foun

14、d in Huanghuai goat andChangjiangsanjiaozhou white goats. But only MM, MN, NN genotype without Kallele existed in Boer goats. Group-equilibrium tests show: the x2 value was notsignificant difference in goat GH gene amplifying with P2 fragment in the threebreeds, which was in Hardy-Weinberg equilibri

15、um (P0.05), but there was significantdifference in amplifying fragment E2 of exon 2. In the three groups, there wassignificant difference in genotype distribution of this site, which deviated from Hardy-Weinberg equilibrium (P0.01), which suggests that artificial selection might bepossible in this s

16、ite. In addition, genotype distribution in P1 site of Boer goat was in astate of equilibrium, but Huanghuai goat and Changjiangsanjiaozhou white goats bothdeviated from a state of equilibrium. In E3 site, Boer goat and Changjiangsanjiaozhouwhite goats were both in a state of equilibrium, but Huanghu

17、ai goat deviated from astate of equilibrium. The possible reason is the two sites result from differentBreeding direction and genetic evolution of the environment. Least squares analysis of the polymorphicsits and weight traits show: AA gentype had significant higher birth weight, weight of six months and weight of one yearold than BB and AB genotyps in Boer goats (P0.05). 3 Bioinformatics Analysi