摘要
本论文根据比较基因定位信息,并结合公共数据库中的EST资源,分离、定位猪12号染色体上的新基因,并对基因与部分性状进行了初步关联分析,取得了如下结果:
1.以人的17号染色体上的同源基因的cDNA为探针,从GeneBank中搜索猪的同源EST,由EST构成的连接群(contig)设计引物,从湖北白猪基因组中分离克隆了10个基因片段。这10个基因分别为:磷脂酶D2基因(PLD2)、生长因子受体结合蛋白7基因(GRB7)、颗粒蛋白基因(GRN)、Ⅰ型胶原蛋白α1亚基基因(COL1A1)、磷酸甘露糖长醇利用缺陷型基因(MPDU1)、微管蛋白特异的伴侣蛋白基因(TBCD)、组蛋白乙酰转移酶基因(GCN5L2)、MAC30蛋白基因(MAC30)、Tax模体作用蛋白1基因(TIP-1)、KIAA1582蛋白(KIAA1582)基因。
2.采用电脑克隆策略结合RACE技术,获得了MPDU1和GRN基因的全长cDNA,推导出了相应的氨基酸序列。将推导出的氨基酸序列与其它物种的对应序列进行同源性比较,发现猪的MPDU1氨基酸序列与人的该氨基酸序列同源性达91%,猪的GRN氨基酸序列与人的同源性达70%。
3.采用半定量RT-PCR方法检测了MPDU1基因和GRN基因在通城猪的肾脏、心脏、肺、脾脏和肝脏等5个组织中的表达情况,结果表明MPDU1基因在所检测的组织中广泛表达,且表达量无显著性差异,而GRN基因在不同组织中表达量不同,脾脏中表达量最高,而心脏中的表达水平最低。
4.用猪×啮齿类体细胞杂种板将新分离的8个基因进行染色体区域定位,定位结果如下:PLD2基因被定位于SSC12q11-q15(P=0.9862),与该区域标记间的相关系数为1(Cor=1);GRB7基因被定位于SSC12 p11-(2/3 p13),P=0.8901,Cor=1;COL1A1基因被定位于SSC12 p11-(2/3 p13),P=0.8901,Cor=1;MPDU1基因被定位于SSC12q11-q15,P=0.9862,Cor=1;TBCD基因被定位于SSC12p11-(2/3 p13),P=0.4701,Cor=0.9177或12q11-q15,P=0.4701,Cor=0.9117;MAC30基因被定位于SSC12q11-q15,P=0.9862,Cor=1;TIP-1基因被定位于SSC12q11-q15,P=0.9862,Cor=1;KIAA1582基因被定位于12p11-(2/3 p13),P=0.8901,Cor=1。
5.用猪×仓鼠辐射杂种板将新分离的9个基因进行染色体精细定位,表明这些基因均与猪12号染色体上的基因或标记紧密连锁。具体定位结果如下:PLD2基因与Swc23
猪12号染色体上10个新基因的分商、定位及其与部分性状的关联分析
紧密连锁,LOD=6.11;分万尸基因与及记习J紧密连锁,LOD二15.25;份私/基因与介石万7紧
密连锁,LOD二8.81;‘况劲了基因与及叮J夕口紧密连锁,LOD二21.28;留即基因与万心荃夕O
紧密连锁,LOD=4.96;义)朽乙2基因与万帕璧J紧密连锁,LOD二10.03;蒯c3口基因与介‘23
紧密连锁,LOD二巧.58;几甲可基因与万冲了刃占紧密连锁,LOD二10.95消大去心了压貂基因与
及松洲夕乎紧密连锁,LOD=9.47。
6.采用PCR一RFLP方法检测了分咨7、酬脚{‘优IAI、树叨艺口、蒯c3o、了丫尸一了等6个基
因9个位点的多态性和PL刃基因第21内含子万石飞万序列的插入、缺失造成的PCR产物长
度多态性,分析了不同猪品种中的基因型频率和基因频率,以及各等位基因在不同猪品
种中的分布差异。
7.在长白占X(大白X通城)早和大白占X(长白X通城)早的上下代个体中,分
析了PL刃、命公7、以动v、‘况俐了匆77尸一了基因上述多态位点的遗传方式,结果表明均呈
共显性遗传方式。
8.以本室与通城县畜牧局种畜场合作所建立的试验群体(含长白猪22头、通城猪
58头、大白猪23头、长大通23头、大长通21头)为材料,分析了前述7基因的DNA
多态位点与部分生产性状和免疫性状的关联,结果发现①G尺B7基因第163位点不同基因
型个体在屠宰率困P)、红细胞数(RBC)上差异显著(P<0.05),CC基因型的个体与
TT基因型的个体的20周龄时血清中IgG的含量(I gG)差异极显著(P<0.01);在243位
点,不同基因型的个体之间肌肉pH值(MPH)、红细胞数(RBC)、红细胞压积(HCT)
差异显著(P<0.05);②G尺厅基因不同基因型的个体之间剪切力(Shear foree)、眼肌面
积(LMA)、板油率差异显著(P<0.05);③COLIA了基因第170位点GG基因型个体与
AA、AG基因型个体之间红细胞分布宽度(RDw)差异显著(P二0.0146);④MAc30
基因AG基因型个体与AA基因型个体之间第6、7肋间背膘厚(6一7BF)及三点平均背
膘厚(ABF)差异极显著(P<0.01);⑤7了P一了基因282位点TG基因型个体与TT基因型
个体屠宰率(DP%)差异显著(P=0.0404),腿臀比率(Ham)差异极显著(P=0.0087)。
The objective of this thesis was to isolate, characterize and physically map the novel genes on pig chromosome 12 using information derived from comparative mapping and pig EST database. The association between genes and some economic traits is also studied. The main results are as follows:
1. Pig ESTs were identified through standard BLAST analysis by using cDNA sequences
of human homologous genes on chromosome 17. Highly conserved pig ESTs were assembled
to EST contigs. And primers selected from the contigs were used to isolate 10 novel porcine
gene fragments from Hubei White pigs genomic DNA. The 10 novel porcine genes were as
follows: phospholipase D2(PLD2),Growth factor receptor binding protein7(CRB7), granulins
(GRN) , collagen, type I, alpha 1 (COL1A1) , amnnose-p-dolichol utilization defect 1
(MPDU1) ,tubulin-specific chaperone d (TBCD) ,GCN5 general control of amino-acid
synthesis 5-like 2 (yeast) (GCN5L2) , MAC30 (.MAC30) , TIP-1 Tax interaction protein 1
(TIP-1) and KIAA1582 (KIAA1582) gene.
2. The full length cDNA of MPDU1 and GRN genes were obtained through in silico cloning together with RACE. The deduced amino acid sequences of MPDU1 and GRN proteins show 91% and 70% similarity to corresponding human sequences, respectively.
3. Expression profiles of MPDU1 and GRN genes of Tongcheng pigs were analysis by semi-quantitative RT-PCR assay. MPDU1 gene is widely expressed in tissues of kidney, heart, lung, spleen and liver and there is no significant difference between each tissue; while GRN gene is highly expressed in the spleen and lowly expressed in heart.
4. The pig rodent somatic cell hybrid panel (SCHP) was used for regional assignment of the 8 genes. The GRB7, COL1A1 and K1AA1582 genes were localized to SSC12pl 1- (2/3) pl3 with error risk <0.1%. The probability of regional localization and correlation for the 3 genes was 0.8901 and 1.0, respectively. The PLD2, MPDU1, MAC30 and TIP-1 genes were physically mapped to SSC12qll-ql5 with error risk <0.1%. The probability of regional localization and correlation for the 4 genes was 0.9862 and 1, respectively. The TBCD gene was physically mapped to SSC12p 11-(2/3 p13) (P=0.4701) or 12qll-ql5 (P= 0. 4701) with error risk <0.1%. The correlation of localization to the two regions of the gene was 0.9177 and 0.9117, respectively.
5. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of nine novel genes. Statistical analysis revealed that all the 9 genes were closely linked to genes or markers on pig chromosome 12. PLD2 and
MAC30 genes were both located closely to marker Swc23, with LOD score threshold 6.11 and 15.58, respectively. The GRB7 and GCN5L2 genes were both closely linked to Sw943, with LOD score threshold of 15.25 and 10.03, respectively. The GRN gene was closely linked to Sw957, with LOD score threshold of 8.81. The COLlAl gene was closely linked to Swr390, with LOD score threshold of 21.28. The TBCD gene was closely linked to Sw2490, with LOD score threshold of 4.96. The TIP-1 gene was closely linked to Swl936, with LOD score threshold of 10.96. The KIAA1582 gene was closely linked to Sw2494, with LOD score threshold of 9.47.
6. Studies on polymorphic sites of GRB7, GRN, COLlAl, MPDU1 > MAC30, TIP-1 genes by PCR-RFLP revealed nine polymorphic sites. Allele frequencies among different pig populations were determined. A SINE indel polymorphism was detected in the intron 21 of PLD2, and allele frequencies among different pig populations was determined, too.
7. Codominant inheritance of the above polymorphic sites were demonstrated in the Landracec (Large WhitexTongcheng) and Large Whitex (LandracexTongcheng) two-generation pedigrees.
8. The above polymorphic sites were genotyped in the experimental pig population constructed under the cooperation of our lab and animal husbandry bureau of Tongcheng county (include 22 Landrace pigs,58 Tongcheng pigs,23 Large White pigs,23 Landrace
(Large WhitexTongcheng) pigs and 21 Large White (LandracexTongcheng) pig
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