基于CRISPR/Cas9技术的水稻OsARF12突变体的构建

为研究水稻生长素响应因子OsARF12在水稻生长发育中的作用,通过CRISPR/Cas9基因编辑技术,设计OsARF122个不同外显子突变靶点,化学合成包括突变靶点的特异性序列,并与中间载体sgRNA连接进而与Cas9终载体重组获得OsARF122个不同突变靶点的表达载体。选取测序成功的单一克隆,利用农杆菌介导法导入粳稻品种日本晴,获得转基因植株,并结合测序结果分析突变单株的突变类型,T1突变体KO-ARF12-1有3种纯合突变、2种双等位突变,共5种突变类型;KO-ARF12-2有6种纯合突变、4种双等位突变和1种杂合突变,共11种突变类型。挑选突变类型为缺失1 nt、缺失4 nt、缺失7 nt和缺失2 nt插入1 nt、缺失5 nt的株系作为研究对象,荧光定量PCR鉴定结果表明,OsARF12在突变体中的表达量较野生型极显著下降(P<0.01)。在正常大田生长条件下,2种突变体不同突变类型株系与野生型相比,株高显著降低,降幅为13.15%~18.39%;第1,2茎节间长度无显著差异,第3,4茎节间长度显著降低,降幅为21.91%,23.46%。利用CRISPR/Cas9创制的OsARF12突变体,对水稻节间延伸及株高具有一定调节作用,对于完善水稻生长素响应因子的分子调控机制具有重要意义。

Rice OsIAA6 interacts with OsARF1 and regulates leaf inclination

Leaf inclination,a component of crop architecture,influences photosynthetic efficiency and planting density.Various factors,particularly the phytohormones auxin and brassinosteroids(BRs),function in regulating lamina joint bending,and understanding of the genetic control of leaf inclination will help to elucidate the relevant regulatory network.Screening a rice T-DNA insertion population revealed a mutant that was insensitive to auxin and displayed an enlarged leaf angle due to increased cell length on the adaxial side of the lamina joint.Genetic analysis revealed that the increased leaf inclination was caused by T-DNA insertion in the promoter region of OsIAA6,resulting in elevated OsIAA6 expression.Further study showed that OsIAA6 interacts with OsARF1 to suppress auxin signaling and regulates leaf inclination.OsIAA6 mediates the BR effects on lamina joint development,and OsBZR1,the key transcription factor in BR signaling,binds directly to the promoter of OsIAA6 to stimulate its transcription.These results indicate the roles of the OsIAA6-OsARF1 module in regulating rice leaf inclination and suggest the synergistic effects of the phytohormones auxin and BR.

利用CRISPR/Cas9创建osarf7突变体及其农艺性状调查

【目的】探究OsARF7基因在水稻生长发育中的生物学功能及其对水稻农艺性状的影响。【方法】利用CRISPR/Cas9技术,在粳稻中花11背景下对OsARF7进行定向编辑,获得OsARF7基因突变植株,并考查其农艺性状。【结果】获得22株T0代转基因株系,经PCR扩增潮霉素基因鉴定出20株阳性植株。提取T2代转基因植株的基因组DNA,通过对OsARF7的2个编辑位点区域DNA片段进行PCR扩增及测序分析,筛选到15种突变类型的纯合基因型植株。基因表达分析显示,osarf7突变体的OsARF7和生长素转运关键基因OsPIN1b、Os PIN2和OsLAX2的表达量均显著低于野生型。田间调查发现,与野生型相比,osarf7突变体的分蘖成穗率显著降低,其中,分蘖数比野生型增多的osarf7突变体,有效穗数基本保持不变,而分蘖数与野生型相近的osarf7突变体,有效穗数少于野生型,最终表现为无效分蘖明显增多,分蘖成穗率降低。【结论】OsARF7基因与生长素调控水稻分蘖发生相关,并影响分蘖芽的生长发育及成穗;OsARF7基因突变会导致迟发分蘖芽不能正常发育成穗;粳稻OsARF7突变体的创建对揭示OsARF7基因生理功能和探索生长素促控分蘖发生与成穗机理等具有积极意义。

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin(OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines.Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways.We also suggested rice auxin response factor(OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the ‘best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

The miR167-OsARF12 module regulates rice grain filling and grain size downstream of miR159

Grain weight and quality are always determined by grain filling.Plant microRNAs have drawn attention as key targets for regulation of grain size and yield.However,the mechanisms that underlie grain size regulation remain largely unclear because of the complex networks that control this trait.Our earlier studies demonstrated that suppressed expression of miR167(STTM/MIM167)substantially increased grain weight.In a field test,the yield increased up to 12.90%-21.94% because of a significantly enhanced grain filling rate.Here,biochemical and genetic analyses revealed the regulatory effects of miR159 on miR167 expression.Further analysis indicated that OsARF12 is the major mediator by which miR167 regulates rice grain filling.Overexpression of OsARF12 produced grain weight and grain filling phenotypes resembling those of STTM/MIM167 plants.Upon in-depth analysis,we found that OsARF12 activates OsCDKF;2 expression by directly binding to the TGTCGG motif in its promoter region.Flow cytometry analysis of young panicles from OsARF12-overexpressing plants and examination of cell number in cdkf;2 mutants verified that OsARF12 positively regulates grain filling and grain size by targeting OsCDKF;2.Moreover,RNA sequencing results suggested that the miR167-OsARF12 module is involved in the cell development process and hormone pathways.OsARF12-overexpressing plants and cdkf;2 mutants exhibited enhanced and reduced sensitivity to exogenous auxin and brassinosteroid(BR)treatment,confirming that targeting of OsCDKF;2 by OsARF12 mediates auxin and BR signaling.Our results reveal that the miR167-OsARF12 module works downstream of miR159 to regulate rice grain filling and grain size via OsCDKF;2 by controlling cell division and mediating auxin and BR signals.

Nuclear translocation of OsMADS25 facilitated by OsNAR2.1 in reponse to nitrate signals promotes rice root growth by targeting OsMADS27 and OsARF7

Nitrate is an important nitrogen source and signaling molecule that regulates plant growth and development.Although several components of the nitrate signaling pathway have been identified,the detailed mechanisms are still unclear.Our previous results showed that OsMADS25 can regulate root development in response to nitrate signals,but the mechanism is still unknown.Here,we try to answer two key questions:how does OsMADS25 move from the cytoplasm to the nucleus,and what are the direct target genes activated by OsMADS25 to regulate root growth after it moves to the nucleus in response to nitrate?Our results demonstrated that OsMADS25 moves from the cytoplasm to the nucleus in the presence of nitrate in an OsNAR2.1-dependentmanner.Chromatin immunoprecipitation sequencing,chromatin immunoprecipitation qPCR,yeast one-hybrid,and luciferase experiments showed that OsMADS25 directly activates the expression of OsMADS27 and OsARF7,which are reported to be associated with root growth.Finally,OsMADS25-RNAi lines,the Osnar2.1 mutant,and OsMADS25-RNAi Osnar2.1 lines exhibited significantly reduced root growth compared with the wild type in response to nitrate supply,and expression of OsMADS27 and OsARF7 was significantly suppressed in these lines.Collectively,these results reveal a new mechanismby which OsMADS25 interacts with OsNAR2.1.This interaction is required for nuclear accumulation of OsMADS25,which promotes OsMADS27 and OsARF7 expression and root growth in a nitratedependent manner.

利用CRISPR-Cas9基因编辑技术获得水稻osarf8-1杂合突变体

为了研究生长素是如何参与调控植物的生长发育过程,我们利用CRISPR-Cas9基因编辑技术,设计水稻生长素响应因子OsARF8的两个靶点,对水稻中的该基因进行敲除。构建基因编辑载体pBIN-sg R-Cas-Os,通过农杆菌介导转化水稻品种‘9522’。我们从潮霉素抗性筛选出的28株转基因植株中鉴定得到针对OsARF8的一种杂合突变类型,共计6棵在外显子第174号碱基处缺失一个C碱基的杂合突变类型osarf8-1,该处碱基突变所对应的是第7个氨基酸的变化,导致亮氨酸(Leu)变成色氨酸(Trp),自此处开始随后的氨基酸序列发生移码突变,并且提前终止于第123个氨基酸,正好位于Os ARF蛋白的DBD功能域的最始端(DBD为120~222个氨基酸),说明DBD功能域在该突变体中完全不能表达,ARF8蛋白的功能将受到极大的影响。本研究成功利用CRISPR-Cas9基因编辑技术精确地敲除了水稻OsARF8基因,为CRISPR技术在水稻中的应用提供了一个可供参考的范例,同时也为进一步研究OsARF8基因功能提供了重要的参考依据。

A Novel QTL qTGW3 Encodes the GSK3/ SHAGGY-Like Kinase OsGSK5/OsSK41 that Interacts with OsARF4 to Negatively Regulate Grain Size and Weight in Rice

Grain size and shape are important determinants of grain weight and yield in rice. Here, we report a new major quantitative trait locus （QTL）, qTGW3, that controls grain size and weight in rice. This locus, qTGW3, encodes OsSK41 （also known as OsGSK5）, a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like family. Rice near-isogenic lines carrying the loss-of-function allele of OsSK41 have increased grain length and weight. We demonstrate that OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 （OsARF4）. Co-expression of OsSK41 with OsARF4 increases the accumulation of OsARF4 in rice protoplasts. Loss of function of OsARF4 results in larger rice grains. RNA-sequencing analysis suggests that OsARF4 and OsSK41 repress the expression of a common set of downstream genes, including some auxin-responsive genes, during rice grain development. The loss-of-function form of OsSK41 at qTGW3 represents a rare allele that has not been extensively utilized in rice breeding. Suppression of OsSK41 function by either targeted gene editing or QTL pyramiding enhances rice grain size and weight. Thus, our study reveals the important role of OsSK41 in rice grain development and provides new candidate genes for genetic improvement of grain yield in rice and perhaps in other cereal crops.

A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice

Grain size is one of the most important factors that control rice yield,as it is associated with grain weight(GW).To date,dozens of rice genes that regulate grain size have been isolated;however,the regulatory mechanism underlying GW control is not fully understood.Here,the quantitative trait locus qGL5 for grain length(GL)and GW was identified in recombinant inbred lines of 9311 and Nipponbare(NPB)and fine mapped to a candidate gene,OsAUX3.Sequence variations between 9311 and NPB in the OsAUX3 promoter and loss of function of OsAUX3 led to higher GL and GW.RNA sequencing,gene expression quantification,dual-luciferase reporter assays,chromatin immunoprecipitation-quantitative PCR,and yeast one-hybrid assays demonstrated that OsARF6 is an upstream transcription factor regulating the expression of OsAUX3.OsARF6 binds directly to the auxin response elements of the OsAUX3 promoter,covering a single-nucleotide polymorphism site between 9311 and NPB/Dongjin/Hwayoung,and thereby controls GL by altering longitudinal expansion and auxin distribution/content in glume cells.Furthermore,we showed that miR167a positively regulate GL and GW by directing OsARF6 mRNA silencing.Taken together,our study reveals that a novel miR167a-OsARF6-OsAUX3 module regulates GL and GW in rice,providing a potential target for the improvement of rice yield.