水稻SAUR23基因的生物信息学分析及其CRISPR/Cas9基因编辑载体的构建

Bioinformatics Analysis and Construction of CRISPR/Cas9 Gene Editing Vector of OsSA UR23 in Oryza sativa

为探索水稻SAUR23基因在水稻生长发育过程中发挥的作用,本研究利用生物信息学分析发现,OsSAUR23编码区长462 bp,编码153个氨基酸,相对分子量为16.89 kD,理论等电点为10.04,是一种不稳定的、亲水性的、非跨膜不分泌蛋白,属于水稻SAUR基因家族的一员;二级结构预测发现Os SAUR23蛋白以无规则卷曲和α-螺旋为主。此外,利用CRISPR-GE在线网站设计一对sg RNA序列,采用自然降温的方法获得双链sg RNA,并在T4连接酶的作用下与线性化载体p HSN401相连,经过菌落PCR以及测序验证表明,针对水稻SAUR23基因的CRISPR/Cas9基因编辑载体构建成功。随后,将其转入农杆菌,为遗传转化水稻获得水稻OsSAUR23基因的基因编辑突变体提供实验材料。

In order to explore the role of rice SA UR23 gene in the growth and development of rice,bioinformatics analysis was used in this study.The results showed that the length of OsSA UR23 coding region was 462 bp,encoded 153 amino acids,the theoretical relative molecular weight was 16.89 kD,and the theoretical isoelectric point was 10.04.It was an unstable,hydrophilic,non-transmembrane nonsecretory protein,which belongs to the rice SAUR gene family.The secondary structure of OsSAUR23 protein was predicted to be haphazard coil and alα-helix.In addition,the CRISPR-GE online website was used to design a pair of sgRNA sequences,and the natural cooling method was used to obtain double-stranded sgRNA,which was linked to the linearized vector PHSN401 under the action of T4 ligase.The colony PCR and sequencing verification showed that the CRISPR/Cas9 gene editing vector targeting rice SA UR23 gene was successfully constructed.Then,it was transfected into A grobacterium tumefida to provide experimental materials for gene editing mutants of Os SA UR23 gene in rice.