基于CRISPR/Cas9技术的水稻miR1866突变体构建

microRNA(miRNA)是长度为18~25个碱基的非编码单链RNA,在植物生长发育过程中起着重要的调节作用。水稻是世界上重要的粮食作物之一,近年来调控水稻生长发育的miRNA不断被发掘,但目前关于miR1866的研究还比较少。本研究通过构建miR1866的CRISPR/Cas9表达载体,创制miR1866的突变体,挖掘水稻miR1866的功能。依据CRISPR/Cas9基因编辑技术,设计了miR1866的突变靶点,通过人工合成的方法得到包含突变靶点的特异序列,经过磷酸化修饰和退火后与载体Sg RNA连接进而与Cas9重组得到miR1866的CRISPR/Cas9表达载体,利用农杆菌介导法导入粳稻品种日本晴,获得转基因植株,结合测序结果判断转基因植株突变单株的突变基因型。本研究比较了野生型和突变体(KO1866-1-2和KO1866-2-4)在相同培养条件下苗期的根长、株高、茎基粗、鲜重和干重,结果表明:在营养液培养条件下,突变体的株高、根长、茎基粗、鲜重和干重均显著低于野生型。据此推测,miR1866对水稻的生长发育有一定的调节作用。本研究利用CRISPR/Cas9创制水稻miR1866的突变体,对研究miRNA调控途径,完善水稻miRNA调控的分子机制具有重要的意义。

Construction of Rice miR1866 Mutant Based on CRISPR/Cas9 Technology

microRNA is a class of non-coding single-stranded RNA,which plays an important regulatory role in the growth and development of plants.Rice is one of the most important food crops in the world.microRNA that regulate growth and development of rice have been continuously discovering in recent years.However,there are few studies about miR1866 function.In this study,the mutant of miR1866 created by constructing the CRISPR/Cas9 expression vector to explore the function of miR1866 in rice.The mutant target sites of miR1866 was designed according to CRISPR/Cas9 gene editing techniques.The specific sequence of mutant target sites of miR-1866 was obtained by artificial synthesis and modified by phosphorylation.And the annealed mutant target sites were cloned into vector Sg RNA.The intermediate vector was cloned into Cas9 by LR reaction to get the final miR1866 CRISPR/Cas9 expressing vector of miR1866.The construct was transformed into Agrobacterium tumefaciens and then into cultivated varieties of Japonica rice(Nipponbare)by Agrobacterium-mediated transformation to obtain transgenic plants.The mutant genotype of transgenic plants was confirmed by sequencing results.This study compared the seedlings root length,plant height,stem diameter,fresh weight and dry weight of wild type and mutant(KO1866-1-2 and KO1866-2-4)under the same culture condition.The results showed that the plant height,root length,stem diameter,fresh weight and dry weight of miR1866 mutants were significantly lower than those of wild type under the same nutrient solution condition.Therefore,it is implied that miR1866 could control rice seedling plant height and biomass to a certain degree.In this study,CRISPR/Cas9 was used to create mutants of miR1866 in rice,which is very important for studying the regulation pathway of miRNA and perfecting the molecular mechanism of rice miRNA regulation.