摘要：

为明确陆地棉细胞分裂素受体基因(GhCRE1)在调控植物生长发育中的功能,为研究陆地棉GhCRE1基因调控种子萌发的分子机理提供材料,进一步从分子水平上提高种子萌发率,通过克隆陆地棉GhCRE1基因,利用基因过表达技术将该克隆片段连接至含Ubiquitin启动子的pCUbi1390载体上构建该基因的过表达载体,同时利用CRISPR/Cas9基因编辑技术设计拟南芥CRE1基因的sgRNA片段,合成sgRNA核苷酸序列,从而构建拟南芥CRE1基因靶向敲除载体。将以上2种载体转化农杆菌,使用花序浸染法,以拟南芥为受体材料创建GhCRE1过表达株系及基因编辑功能缺失突变体。接着利用PCR技术和qPCR技术筛选鉴定获得过量表达的GhCRE1拟南芥株系及CRE1功能缺失的拟南芥株系并收获种子,经过对比统计收获的种子与野生型种子在1/2MS培养基上的萌发势进行表型分析,进一步明确陆地棉GhCRE1基因影响种子萌发的功能。结果显示,本研究成功获得了拟南芥GhCRE1过表达株系及CRISPR基因编辑功能缺失突变体株系,且通过数据整理后发现,在种子萌发第2天时,相比野生型,2个过表达转基因株系萌发势分别提高15%和11%左右,而拟南芥CRE1功能缺失型突变体萌发势降低15%左右。 该研究揭示,GhCRE1基因过表达能显著提高拟南芥萌发速度。

关键词: 陆地棉, GhCRE1基因, 过表达, CRISPR/Cas9技术, 种子萌发

Abstract:

To clarify the function of the receptor gene for Gossypium hirsutum L. cytokinin receptor (GhCRE1) in the regulation of plant growth and development, and to provide material for studying the molecular mechanism of the GhCRE1 gene in regulating seed germination in Gossypium hirsutum, ultimately improving seed germination rates at the molecular level. The GhCRE1 gene was cloned and the fragment was ligated to the pCUbi1390 vector containing the Ubiquitin promoter using gene overexpression technology to construct GhCRE1 overexpression vector, while the sgRNA fragment of the Arabidopsis CRE1 gene was designed by CRISPR/Cas9 gene editing technology and the sgRNA nucleotide sequence was synthesized to construct the Arabidopsis CRE1 gene targeting knockout vector. The above two vectors were transformed into Agrobacterium and the inflorescence dip method was used to create GhCRE1 overexpression strains and gene editing loss-of-function mutants using Arabidopsis thaliana as the recipient material. Screening and identification by PCR and qPCR techniques, then, two strains of GhCRE1 Arabidopsis over-expressed and the CRE1-deficient Arabidopsis strains were obtained. After comparing the germination potential of harvested seeds with that of wild-type seeds on 1/2 MS medium, further clarify the function of GhCRE1 gene in affecting seed germination of Gossypium hirsutum. The results showed that the Arabidopsis GhCRE1 overexpression strains and CRISPR gene editing loss-of-function mutant strain were successfully obtained, after compiling the data related to seed germination potential, it was found that on the 2nd day, the germination potential of the two overexpression transgenic strains increased by about 15% and 11%, respectively, compared with the wild type, while the Arabidopsis CRE1 loss-of-function mutant germination potential decreased by about 15%. This study revealed that GhCRE1 gene overexpression significantly increased germination speed in Arabidopsis.

Key words: Gossypium hirsutum L., GhCRE1 gene, Overexpression, CRISPR/Cas9 technology, Seed germination