Chalkiness is one of the most important agronomic traits in rice breeding,which directly affects the quality of rice seed.In this study,we identified a chalkiness endosperm mutant,chalk-h,from N-methyl-N-nitrosourea(M...Chalkiness is one of the most important agronomic traits in rice breeding,which directly affects the quality of rice seed.In this study,we identified a chalkiness endosperm mutant,chalk-h,from N-methyl-N-nitrosourea(MNU)-induced japonica rice cultivar Hwacheong(HC).Compared with wild type(WT)-HC,chalk-h showed severe chalkiness in the endosperm,yellowish green leaves,as well as reduced plant height.Scanning electron microscopy(SEM)analysis showed that starch grains in the chalk-h mutant were irregular in size and loosely arranged,with large gaps between granules,forming ovoid or orbicular shapes.MutMap analysis revealed that the phenotype of chalk-h is controlled by a single recessive gene LOC_Os11g39670 encoding seryl-tRNA synthetase,which is renamed as CHALK-H.A point mutation occurs in chalk-h on the sixth exon(at nucleotide 791)of CHALK-H,in which adenine(A)is replaced by thymidine(T),resulting in an amino acid codon change from glutamine(Glu)to valine(Val).The chalk-h mutant exhibited a heat-sensitive phenotype from the 3-leaf stage,including yellow-green leaves and reduced pigment content.The transcriptional expression of starch synthesis-related genes was down-regulated in the chalk-h mutants compared to WT-HC at different grain-filling stages.With an increase in temperature,the expression of photosynthesis-related genes was down-regulated in the chalk-h mutant compared to WT-HC.Overexpression of CHALK-H rescued the phenotype of chalk-h,with endosperm and leaf color similar to those of WT-HC.Our findings reveal that CHALK-H is a causative gene controlling chalkiness and leaf color of the chalk-h mutant.CHALK-H is the same gene locus as TSCD11,which was reported to be involved in chloroplast development under high temperature.We suggest that CHALK-H/TSCD11 plays important roles not only in chloroplast development,but also in photosynthesis and starch synthesis during rice growth and development,so it has great application potential in rice breeding for high quality and yield.展开更多
[目的]构建敲除Toll Like Receptor 4(TLR4)基因的Ha Ca T细胞株,为后续利用该细胞株进行过敏原性研究提供材料。[方法]利用CRISPR/Cas9系统,根据靶向原理设计并合成4条特异性识别TLR4基因的向导RNA(single-molecule guide RNAs,sgRNAs)...[目的]构建敲除Toll Like Receptor 4(TLR4)基因的Ha Ca T细胞株,为后续利用该细胞株进行过敏原性研究提供材料。[方法]利用CRISPR/Cas9系统,根据靶向原理设计并合成4条特异性识别TLR4基因的向导RNA(single-molecule guide RNAs,sgRNAs),构建p X459-sgRNAh TLR4重组质粒,并转入Ha Ca T中,用嘌呤霉素筛选出单克隆阳性细胞。测序确认突变位点,然后利用脂多糖(lipopolysaccharide,LPS)刺激对TLR4进行功能验证来进一步确认敲除效果。[结果]测序结果表明#26单克隆细胞株在靶点附近缺失1 bp,造成TLR4编码基因的移码突变,蛋白翻译提前终止。功能性验证结果表明,在LPS的刺激下,IL-8和CCL20的mRNA水平分别下降约85%和90%,且IL-8的蛋白分泌水平也显著性下调(87%)。[结论]成功构建了敲除TLR4的稳定细胞株,并且验证TLR4的功能缺损。展开更多
基金the Administration of Central Funds Guiding the Local Science and Technology Development,China(202002069JC)the earmarked fund for the China Agriculture Research System(CARS-01-10)。
文摘Chalkiness is one of the most important agronomic traits in rice breeding,which directly affects the quality of rice seed.In this study,we identified a chalkiness endosperm mutant,chalk-h,from N-methyl-N-nitrosourea(MNU)-induced japonica rice cultivar Hwacheong(HC).Compared with wild type(WT)-HC,chalk-h showed severe chalkiness in the endosperm,yellowish green leaves,as well as reduced plant height.Scanning electron microscopy(SEM)analysis showed that starch grains in the chalk-h mutant were irregular in size and loosely arranged,with large gaps between granules,forming ovoid or orbicular shapes.MutMap analysis revealed that the phenotype of chalk-h is controlled by a single recessive gene LOC_Os11g39670 encoding seryl-tRNA synthetase,which is renamed as CHALK-H.A point mutation occurs in chalk-h on the sixth exon(at nucleotide 791)of CHALK-H,in which adenine(A)is replaced by thymidine(T),resulting in an amino acid codon change from glutamine(Glu)to valine(Val).The chalk-h mutant exhibited a heat-sensitive phenotype from the 3-leaf stage,including yellow-green leaves and reduced pigment content.The transcriptional expression of starch synthesis-related genes was down-regulated in the chalk-h mutants compared to WT-HC at different grain-filling stages.With an increase in temperature,the expression of photosynthesis-related genes was down-regulated in the chalk-h mutant compared to WT-HC.Overexpression of CHALK-H rescued the phenotype of chalk-h,with endosperm and leaf color similar to those of WT-HC.Our findings reveal that CHALK-H is a causative gene controlling chalkiness and leaf color of the chalk-h mutant.CHALK-H is the same gene locus as TSCD11,which was reported to be involved in chloroplast development under high temperature.We suggest that CHALK-H/TSCD11 plays important roles not only in chloroplast development,but also in photosynthesis and starch synthesis during rice growth and development,so it has great application potential in rice breeding for high quality and yield.
文摘[目的]构建敲除Toll Like Receptor 4(TLR4)基因的Ha Ca T细胞株,为后续利用该细胞株进行过敏原性研究提供材料。[方法]利用CRISPR/Cas9系统,根据靶向原理设计并合成4条特异性识别TLR4基因的向导RNA(single-molecule guide RNAs,sgRNAs),构建p X459-sgRNAh TLR4重组质粒,并转入Ha Ca T中,用嘌呤霉素筛选出单克隆阳性细胞。测序确认突变位点,然后利用脂多糖(lipopolysaccharide,LPS)刺激对TLR4进行功能验证来进一步确认敲除效果。[结果]测序结果表明#26单克隆细胞株在靶点附近缺失1 bp,造成TLR4编码基因的移码突变,蛋白翻译提前终止。功能性验证结果表明,在LPS的刺激下,IL-8和CCL20的mRNA水平分别下降约85%和90%,且IL-8的蛋白分泌水平也显著性下调(87%)。[结论]成功构建了敲除TLR4的稳定细胞株,并且验证TLR4的功能缺损。
