高粱可溶性酸性转化酶基因(SAI-1)PCR克隆方法

Modified Method for PCR Cloning of Soluble Acid Invertase Gene in Sorghum bicolor

可溶性酸性转化酶(SAI)是蔗糖代谢途径中的关键酶,对植物生长发育起着至关重要的调节作用,研究简捷快速克隆可溶性酸性转化酶基因方法,对于育种材料和品种资源的基因分型具有重要意义。本研究通过已知的高粱可溶性酸性转化酶基因序列及高粱基因组中该基因序列片段,设计引物,比较了分段克隆、基因全长克隆、巢式PCR克隆等方法克隆高粱SAI-1基因的效果,结果表明,直接扩增全长,扩增产物极其不稳定且扩增产物纯化、连接,转化后得不到阳性克隆;采用均等分段克隆,前半段扩增产物纯化、连接转化后得不到阳性克隆,但后半段克隆成功;针对高粱基因组信息中SAI-1基因上游的未知序列部分设计引物,进行单独克隆(635 bp),再单独克隆其其余序列,两段序列拼接后得到SAI-1基因全长。序列分析发现,SAI-1前段635 bp的扩增片段GC含量高达69.6%,而其后GC含量急剧下降至30%以下,所以推测全长克隆、均等片段克隆以及巢式PCR克隆失败的原因可能是SAI-1基因中GC分布不均匀,克隆高粱SAI-1基因较为适宜的方法为利用2对引物进行不均等分段扩增克隆,前段PCR退火温度较后段高1℃。该方法将为其他研究人员提供有益参考。

Soluble acid invertase（ SAI） is a key enzyme in sucrose metabolism which takes a pivotal role in sugar sensing and plant development. It is necessary to find a simple method of cloning soluble acid invertase gene for genotyping a large quantity of breeding materials or germplasm. Based on known full-length sequence of SAI gene obtained by 5 segments PCRs and assembled in our lab,in order to further find a simpler one,we tried several strategies,i. e. one time cloning for full-length sequence,nested PCR,two even fragments PCR,and two uneven fragments PCR. And only was success achieved in two uneven fragments PCRs with a set of special PCR parameters. Sequence analysis showed bases of GC was strongly unevenly distributed in SAI-1 gene sequence,with a high GC content（ 69. 6%） in 635 bp PCR product of the first fragment,with highest over 80% in some region of this segment.Except for high GC content,steep change of GC content may be another factor that affects PCR result. The suitable strategy for PCR cloning SAI-1 gene is through an approach of two uneven segments PCR with an annealing temperature difference.