应用CRISPR/Cas9技术研究水稻OsPIN10a基因功能

Function analysis of OsPIN10a gene in rice by CRISPR/Cas9 technology

生长素输出载体蛋白(PIN-FORMED,PIN)是控制生长素极性运输的关键蛋白。OsPIN10a是单子叶植物特异的生长素输出载体蛋白,但其生物学功能报道甚少。本研究在OsPIN10a基因的第1个外显子上设计编辑位点,构建了OsPIN10a的CRISPR/Cas9基因编辑载体。通过遗传转化水稻粳稻品种‘日本晴’获得19株转基因植株。测序分析表明14个转基因株系中有7种不同的突变类型,总突变率为73.68%。进一步鉴定获得4种不同类型的纯合突变体。序列比对分析表明,这4种类型的突变均造成移码突变和蛋白翻译提前终止,其中2种突变蛋白的长度为7和9个氨基酸,将其命名为ospin10a-1和ospin10a-2;2种突变蛋白的长度为235和232个氨基酸,命名为ospin10a-3和ospin10a-4。跨膜螺旋结构域分析表明这四种突变体中OsPIN10a蛋白的跨膜结构全部消失。幼苗期表型分析表明,ospin10a-1和ospin10a-2突变体植株地上部的生长和不定根的发育都受到显著影响。ospin10a-1和ospin10a-2突变体根的向重性受到显著抑制,说明OsPIN10a参与调控水稻根的向重性。

Auxin efflux carriers PIN-FORMED(PIN)are the crucial proteins controlling polar auxin transport(PAT).OsPIN10a is a monocot-specific PIN gene in rice,while its biological function remains elusive.Here,the target site was designed at the first exon of OsPIN10a,the OsPIN10a recombinant CRISPR/Cas9 vector was constructed,and 19 independent transgenic lines were obtained.Sequencing results showed seven different mutation types were detected in 14 transgenic lines,and the mutation rate was 73.68%.Subsequent analysis identified four different homozygous mutations.Sequence blast showed that all of the mutations resulted in frame-shift mutation and premature translation termination.The mutants that possess OsPIN10a-1 and OsPIN10a-2 mutation proteins,which contain 7 and 9 amino acids,respectively,were named ospin10a-1 and ospin10a-2,and the other two mutants,in which the mutation proteins containing 232 and 235 amino acids,respectively,were named ospin10a-3 and ospin10a-4.Bioinformatics analysis showed that the transmembrane helices in mutation proteins disappeared completely.Phenotype analysis displayed that the shoot height and adventitious root number were significantly decreased in ospin10a-1and ospin10a-2 mutants compared to the wild-type(WT)plants at the seedling stage.Root gravitropism response in ospin10a mutants was significantly reduced compared to WT after gravity stimulus,indicating that OsPIN10a is involved in modulating root gravitropism in rice.