Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
叶绿体相关突变体是研究光合作用、叶绿素生物合成、叶绿体结构发育等生理途径的优良遗传资源,同时作为标记性状在育种中具有一定的应用价值。本课题组发现一个来自恢复系轮回选择群体的子叶黄化致死突变体ytl(yellowing to lethal),突...叶绿体相关突变体是研究光合作用、叶绿素生物合成、叶绿体结构发育等生理途径的优良遗传资源,同时作为标记性状在育种中具有一定的应用价值。本课题组发现一个来自恢复系轮回选择群体的子叶黄化致死突变体ytl(yellowing to lethal),突变体发芽出土后子叶一直处于黄化状态,播种9~15天后死亡,前期的研究将一个控制子叶黄化致死性状的位点定位到C09染色体上。表型观测显示,出苗7天后突变体与野生型相比株高与根长存在显著差异,明显偏短。遗传分析表明,该突变体由两对隐性核基因控制。利用单位点分离群体将BnaC02.YTL定位到对应ZS11参考基因组418 kb的物理区间内,结合定量分析与基因序列比对,BnaC02G0055700ZS作为候选基因的可能性较大。本研究为进一步精细定位基因BnaC02.YTL及后续突变体的功能研究奠定了基础。展开更多
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
文摘叶绿体相关突变体是研究光合作用、叶绿素生物合成、叶绿体结构发育等生理途径的优良遗传资源,同时作为标记性状在育种中具有一定的应用价值。本课题组发现一个来自恢复系轮回选择群体的子叶黄化致死突变体ytl(yellowing to lethal),突变体发芽出土后子叶一直处于黄化状态,播种9~15天后死亡,前期的研究将一个控制子叶黄化致死性状的位点定位到C09染色体上。表型观测显示,出苗7天后突变体与野生型相比株高与根长存在显著差异,明显偏短。遗传分析表明,该突变体由两对隐性核基因控制。利用单位点分离群体将BnaC02.YTL定位到对应ZS11参考基因组418 kb的物理区间内,结合定量分析与基因序列比对,BnaC02G0055700ZS作为候选基因的可能性较大。本研究为进一步精细定位基因BnaC02.YTL及后续突变体的功能研究奠定了基础。
