PCR步行法克隆油菜自交不亲和基因

Cloning of S-locus gene by PCR walking in Brassica campestris L.

同源序列法与PCR步行法相结合,在油菜中克隆自交不亲和基因SLG和eSRK(extra-cellularSRK)。所克隆的SLG序列长为882bp,克隆的eSRK序列长度分别为1495bp(青海大黄)、1494bp(黄籽沙逊)、1727bp(关中油白菜);其GeneBank登陆号分别为AY448025、AY448026、AY448027、AY448028、AY448029、AY448030。缺失突变可能是导致青海大黄和黄籽沙逊自交亲和的原因。克隆的SLG基因与甘蓝和白菜型油菜的SLG基因有很高的同源性,而eSRK基因与甘蓝和白菜型油菜的SRK基因的同源性较高。同源序列法与PCRwalking法相结合,可以有效地克隆基因;但在克隆SLG基因的两翼序列时遇到了困难,说明PCR步行法仍有待改进。

PCR walking and homologue-sequence method were used to clone the S-locus genes: SLG and eSRK in B. campestris. SLGs cloned were 882bp, while eSRKs cloned were 1 495bp (Qinghai dahuang), 1 494bp (Yellow sarson), 1 727bp (Guanzhong youbaicai). GeneBank accessions of these genes were AY448025, AY448026, AY448027, AY448028, AY448029 and AY448030. Deletion might result in self-compatibility of Qinghai dahuang and Yellow sarson. The SLGs cloned showed high sequence identities with SLGs of B.oleracea and B.rapa while eSRKs cloned displayed high sequence similarities with SRKs of B.oleracea and B.rapa. These results supported that combination of PCR walking and homologue-sequence method could be well used to clone genes. But, there were difficulties in cloning SLG-flanking sequences indicating that PCR walking needed to be still modified.