【目的】明确PLC4×2基因在蓖麻(Ricinus communis L.)叶片中的瞬时表达效果及亚细胞定位特征,为基因功能解析奠定基础。【方法】采用农杆菌介导法,将构建的瞬时过表达载体pBI121-EGFPPLC4×2导入蓖麻叶片;通过琼脂糖凝胶电泳...【目的】明确PLC4×2基因在蓖麻(Ricinus communis L.)叶片中的瞬时表达效果及亚细胞定位特征,为基因功能解析奠定基础。【方法】采用农杆菌介导法,将构建的瞬时过表达载体pBI121-EGFPPLC4×2导入蓖麻叶片;通过琼脂糖凝胶电泳检测总RNA完整性,利用实时荧光定量PCR(RT-qPCR)验证基因表达量;采用酶解、过滤、离心法提取蓖麻原生质体,结合荧光倒置显微镜观察进行亚细胞定位分析。【结果】总RNA提取结果显示,28S rRNA与18S rRNA条带清晰,完整性良好;RT-qPCR检测证实,PLC4×2瞬时过表达植株的基因相对表达量显著高于野生型对照(P<0.001),瞬时过表达成功;亚细胞定位结果表明,PLC4×2基因编码的蛋白质主要定位于蓖麻原生质体的叶绿体上。【结论】采用农杆菌介导法,将构建的瞬时过表达载体pBI121-EGFP-PLC4×2导入蓖麻叶片,实现了PLC4×2基因在蓖麻叶片中的瞬时过表达,明确其主要定位于蓖麻原生质体的叶绿体上。展开更多
Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries.Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig ...Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries.Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig production.R2R3-MYB transcription factors have promotive or repressive regulatory roles in plant anthocyanin biosynthesis.In this study,113 R2R3-MYB genes were identified in Ficus carica,3 of which were assigned to the S4 subfamily of flavonoid-biosynthesis repressors.FcMYB57 was further recruited as a candidate anthocyaninbiosynthesis repressor based on its sequence features and expression,which was significantly negatively correlated with that of anthocyanin-biosynthesis structural genes.Transient overexpression of FcMYB57 in strawberry totally inhibited fruit pigmentation and significantly increased fruit firmness.The metabolomic analysis confirmed a significant reduction in the contents of cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside,as well as other flavonoids,and transmission electron microscopy revealed an increment in cell-wall thickness.Transcriptome analysis showed downregulation of anthocyanin-biosynthesis structural genes and upregulation of genes related to xylan synthesis.Yeast one-hybrid and dual luciferase assays demonstrated a negative regulatory effect of FcMYB57 on the promoter of FcUFGT3(UDP glucose-flavonoid 3-O-glcosyl-transferase).Yeast two-hybrid assay showed that FcMYB57 does not interact with FcbHLH42,3,14,MYC2,or FcTTG1,all of which have a previously identified or predicted role in flavonoid biosynthesis,however,interaction was detected with FcTPL(Topless),suggesting that FcMYB57 serves as an active repressor of anthocyanin biosynthesis.This is the first identification of an anthocyaninbiosynthesis repressor in fig,with a possible role in fig fruit quality.展开更多
文摘【目的】明确PLC4×2基因在蓖麻(Ricinus communis L.)叶片中的瞬时表达效果及亚细胞定位特征,为基因功能解析奠定基础。【方法】采用农杆菌介导法,将构建的瞬时过表达载体pBI121-EGFPPLC4×2导入蓖麻叶片;通过琼脂糖凝胶电泳检测总RNA完整性,利用实时荧光定量PCR(RT-qPCR)验证基因表达量;采用酶解、过滤、离心法提取蓖麻原生质体,结合荧光倒置显微镜观察进行亚细胞定位分析。【结果】总RNA提取结果显示,28S rRNA与18S rRNA条带清晰,完整性良好;RT-qPCR检测证实,PLC4×2瞬时过表达植株的基因相对表达量显著高于野生型对照(P<0.001),瞬时过表达成功;亚细胞定位结果表明,PLC4×2基因编码的蛋白质主要定位于蓖麻原生质体的叶绿体上。【结论】采用农杆菌介导法,将构建的瞬时过表达载体pBI121-EGFP-PLC4×2导入蓖麻叶片,实现了PLC4×2基因在蓖麻叶片中的瞬时过表达,明确其主要定位于蓖麻原生质体的叶绿体上。
基金supported by the key research project for fig development of Weiyuan County(Grant No.1002-69199007),China.
文摘Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries.Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig production.R2R3-MYB transcription factors have promotive or repressive regulatory roles in plant anthocyanin biosynthesis.In this study,113 R2R3-MYB genes were identified in Ficus carica,3 of which were assigned to the S4 subfamily of flavonoid-biosynthesis repressors.FcMYB57 was further recruited as a candidate anthocyaninbiosynthesis repressor based on its sequence features and expression,which was significantly negatively correlated with that of anthocyanin-biosynthesis structural genes.Transient overexpression of FcMYB57 in strawberry totally inhibited fruit pigmentation and significantly increased fruit firmness.The metabolomic analysis confirmed a significant reduction in the contents of cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside,as well as other flavonoids,and transmission electron microscopy revealed an increment in cell-wall thickness.Transcriptome analysis showed downregulation of anthocyanin-biosynthesis structural genes and upregulation of genes related to xylan synthesis.Yeast one-hybrid and dual luciferase assays demonstrated a negative regulatory effect of FcMYB57 on the promoter of FcUFGT3(UDP glucose-flavonoid 3-O-glcosyl-transferase).Yeast two-hybrid assay showed that FcMYB57 does not interact with FcbHLH42,3,14,MYC2,or FcTTG1,all of which have a previously identified or predicted role in flavonoid biosynthesis,however,interaction was detected with FcTPL(Topless),suggesting that FcMYB57 serves as an active repressor of anthocyanin biosynthesis.This is the first identification of an anthocyaninbiosynthesis repressor in fig,with a possible role in fig fruit quality.
