OsSP1基因编辑和过表达对水稻生长和干旱抗性的影响

Effects of Editing and Overexpression of OsSP1 Gene on Rice Growth and Drought Tolerance

OsSP1基因是水稻叶绿体定位基因,并且具有明显的干旱响应性。为确定OsSP1基因对水稻生长和干旱胁迫抗性的影响,分别采用CRISPR/Cas9基因编辑技术和基因过表达技术创建了水稻品种中花11 OsSP1基因编辑株系和过表达株系,并通过对靶序列进行片段扩增和测序确定基因编辑植株中OsSP1基因的突变方式,通过hpt序列扩增和潮霉素筛选获得单拷贝插入的纯合过表达植株。表型对比、叶绿素含量测定、过氧化氢测定和干旱抗性分析的结果表明,过表达OsSP1基因对水稻的生长和干旱胁迫抗性均无明显影响,但通过基因编辑引起OsSP1基因突变后,水稻的生长和结实受到了严重影响,在正常生长条件下,T_(0)和T_(1)双等位杂合突变和纯合突变的基因编辑植株均出现矮小、黄化、早衰、不分蘖等生长抑制表型。T_(0)植株结实数很低,T_(1)植株均未抽穗结实,叶片的叶绿素含量也显著或极显著低于野生型和过表达植株,上述结果显示了OsSP1基因在维持水稻正常生长和产量中的关键性作用,为进一步揭示OsSP1基因的作用机制提供了理论和材料基础。

The OsSP1 gene encodes a chloroplast localized protein in rice,and its expression clearly responds to drought stress.In order to elucidate the effect of OsSP1 gene on rice growth and drought stress tolerance,OsSP1 gene-edited and overexpressed rice plants were created by using CRISPR/Cas9 gene editing technology and gene overexpression technology,respectively.The mutation pattern of OsSP1 gene in the gene-edited plants was determined by sequencing analysis,and homozygous overexpression plants were selected by hpt sequence detection and hygromycin B screening assay.The results of phenotypic comparison,chlorophyll content and reactive oxygen species(ROS)determination and drought tolerance analysis showed that overexpression of OsSP1 gene in rice had almost no effect on rice growth and drought stress tolerance.However,after the OsSP1 gene was mutated through gene editing,the growth and fruiting of rice T_(0) and T_(1) gene-edited plants were severely affected with biallelic heterozygous or homozygous mutations,and all showed severe growth inhibition such as dwarf,etiolation,premature aging,and non-tillering,T_(0) plants produced very few seeds,while the T_(1) plants did not head at all,and the chlorophyll content in leaf of the gene-edited plants was significantly or extremely significantly lower than that of the wild-type and overexpressed plants.These results indicated the essential role of OsSP1 gene in rice growth and yield,and provided a theoretical and material basis for elucidating the underlying mechanism of OsSP1 gene in rice.