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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 plays a crucial regulatory role in plant growth and development. To study the simple and rapid cloning of soluble acid invertase gene, genotyping of breeding material and variety resources It is of great significance. In this study, primers were designed based on the known sorghum soluble acid invertase gene sequence and sorghum genome sequence, and the effect of cloning sorghum SAI-1 gene by fragment cloning, full-length cloning and nested PCR cloning were compared , The results showed that the direct amplification of the entire length, the amplified product is extremely unstable and the amplified product was purified, ligation, transformation positive clones can not be obtained; using equally segmented cloning, the first half of the amplified product was purified, To the positive clones, but the latter half clones were successfully cloned. The primers for the unknown sequence upstream of the SAI-1 gene in sorghum genome were designed and cloned separately (635 bp), and the remaining sequences were cloned separately. The two sequences were stitched to obtain SAI -1 gene full length. Sequence analysis revealed that the GC content of the 635 bp fragment of SAI-1 was as high as 69.6% and the content of GC decreased sharply to below 30%. Therefore, it may be speculated that the reasons for the failure of full-length cloning, equal fragment cloning and nested PCR cloning Is a non-uniform distribution of GC in SAI-1 gene. Cloning of SAI-1 gene in sorghum is more suitable for amplification of clones using two pairs of primers. The temperature of the first PCR annealing is 1 ℃ higher than the latter. This method will provide other researchers with useful reference.