作　　者： ; ; ; ; ; ; ; ; ;

机构地区： 华中农业大学动物科学技术学院农业部猪遗传育种重点开放实验室

出　　处： 《农业生物技术学报》 2005年第5期601-607,共7页

摘　　要： 实验选取中国地方品种梅山猪和外国品种大白猪进行正反向杂交产生杂种梅大和大梅猪,用mRNA差异显示技术,分析了亲本和杂种猪背部皮下脂肪组织间基因差异表达。结果发现,在10条5'端随机引物和10条3'端锚定引物共100对引物组合的差异显示扩增下,共获得1500多条可重复的扩增条带,选取其中能够重复出现的40条差异表达带(在4组样本中不能同时出现的条带)进行重扩增、克隆并测序。通过GenBank/BLAST比对发现,其中有4条EST分别与GenBank序列同源性较高,其余36条属于新的EST,将这36条EST提交GenBank获得注册号,并选取其中的3条进行半定量RT-PCR验证并检测其相对表达,其中2条表现不同程度的差异,1条没有明显差异。为提高DDRT-PCR真阳性,实验中的4种组合各屠宰6头以建立RNA池,采取低高严谨结合的退火温度进行差异显示扩增以及只回收在2次PCR扩增均能重复出现的条带。结果表明,所获得的36条新的EST在亲子代之间的差异表达方式不尽相同,这些差异表达的基因可能与脂肪组织杂种优势的形成有关。 In order to reveal molecular basis of heterosis, Large White （LW）, an introduced European pig breed, and Meishan （MS）, a Chinese indigenous pig breed, were selected to hybridize directly and reciprocally in the present experiment, mRNA differential display （DD） technique was performed to identify genes that were differentially expressed among backfat tissues of hybrids （LW×MS, MS×LW） and purebred （LW×LW, MS×MS） pigs. The 10 anchor primers in combination with ten arbitrary primers （100 sets in total） were used and nearly 1 500 reproducible bands were observed in polyacrylamide gel. The 40 differentially displayed bands were selected to clone and sequence. The 36 out of 40 ESTs were proved to be novel and the sequences were submitted to GenBank （accession No. CV507051～CV507087）, and the other 4 showed similarity to known genes published in GenBank. Three among 36 novel ESTs were chosen for further identification with semi-quantitative RT-PCR. The result showed that 2 ESTs were differentially expressed, and the 3rd showed no obvious difference between hybrids and purebreds. In order to reduce the percentage of false positive ofDD, RNA pools of four types of pigs were constructed respectively by mixing 6 samples from 6 pigs of the same genotype, and subjected to DD. Stringent annealing temperature was applied and only bands that could be repeated in duplicate PCR were used for further study. The results showed that the expression pattern of these 36 ESTs differed among the 4 genotypes of pigs, suggesting that the genes corresponding to these differentially expressed ESTs might be related to the formation of heterosis of fat tissue.

关 键 词： 杂种优势 差异显示 半定量 猪

领　　域： [农业科学] [农业科学] [生物学]