SSH法结合定量PCR技术研究双肌臀猪肌肉组织的差异表达基因

Isolation of Differentially Expressed Genes in Double-muscling Large White Pig by SSH and Q-PCR Strategy

利用抑制性消减杂交技术(suppression subtractive hybridization,SSH) 成功地构建了双肌臀与非双肌臀大白猪肌肉组织差异表达的消减cDNA文库,获得有效克隆686个. 对整个文库测序分析,共获得587条有效序列. 利用BLAST在线软件与GenBank、GenBank EST和Tigr Porcine EST等数据库进行同源序列比较,发现其中有11个未知新序列,可能代表“双肌臀”大白猪肌肉组织特异表达的新基因. 对文库中所包含的兰尼啶受体基因(RYR1) 、钙依赖性蛋白激酶基因(CAMK2)、人类胰岛素生长因子结合蛋白-7 基因(IGFBP7),以及695号、882号和480号3个新基因进行了实时荧光定量PCR检测,结果显示:RYR1、CAMK2及IGFBP7基因在双肌臀猪肌肉组织RNA池中的表达量,分别为对照的非双肌臀猪肌肉组织RNA池中表达量的1.87、1.90和1.85倍;695号、882号和480号3个新的ESTs在双肌臀猪肌肉组织RNA池中的表达量为非双肌臀猪肌肉组织RNA池中表达量的1.48、1.44和1.78倍. 这提示我们,上述基因的上调表达很可能与猪双肌臀性状的发生有密切的关系,可以作为研究该性状的候选基因.

Double-muscling pig is good at meat yielding, but the genetic reason for this trait was not clear till now. A SSH (suppression subtractive hybridization) library was built with cDNA from LD (longissimus dorsi) muscle of double-muscling pigs as tester, and that of non-double-muscling as driver. 686 clones in this library were single insertion. And they were all sequenced. The sequences were BLAST on line with the GenBank dbase, GenBank EST dbase and also the Tigr Porcine EST dbase, 11 of them were not matched in any of the databases which might represent new genes related to porcine double-muscling trait. 3 of the functional genes, which are RYR1, CAMK2 and IGFBP 7, were chosen firstly to do quantitative PCR to confirm the expression differentiation between double-muscling LD tissue and non-double-muscling pigs LD tissue. The genes expressed in the former tissue were 1.87, 1.90, 1.85 times higher, respectively, than in the later tissue. 3 clones of the new ESTs were also detected with quantitative PCR, they were 1.48, 1.44 and 1.78 times higher in the former, respectively. These results implied that new candidate genes could be selected from the SSH library constructed in this research, and this could be a way to make the genetic base of double-muscling pig more clearly.