Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psb...Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psbA, the gene encoding D1 protein of O. sativa ssp. japonica cv. 9516, and that of O. sativa ssp. indica cv. Shanyou 63 was cloned. As revealed by homology comparison of their sequences, the sequences are identical in the regions of promoter and 5′-UTR; differences are found in individual bases in the coding region all of which, being in the third position of respective codons, however, do not affect the amino acids coded finally; a difference is noted in the length of the oligo-U sequence in the region of 3′-UTR. It is thus apparent that, rather than a result of any difference in the amino acid sequences, the differences in the sensitivity to photoinhibition of D1 proteins between japonica and indica rice may be related to the upstream factors that regulate expression of psbA or to differences of photoprotective mechanisms.展开更多
拟验证天然断裂内含肽Npu Dna E、Ssp Dna E的反式剪接反应对嵌合蛋白形成的影响,探讨依据此方法表达抗菌肽的可行性,规避抗菌生物表达中对宿主的毒害作用.将花蝉属抗菌肽ADP-1(Amblyomma defensin peptide 1)拆分为N端和C端两部分,其N...拟验证天然断裂内含肽Npu Dna E、Ssp Dna E的反式剪接反应对嵌合蛋白形成的影响,探讨依据此方法表达抗菌肽的可行性,规避抗菌生物表达中对宿主的毒害作用.将花蝉属抗菌肽ADP-1(Amblyomma defensin peptide 1)拆分为N端和C端两部分,其N端与断裂型内含肽Npu Dna E的N端融合,C端与Ssp Dna E的C端融合,并分别构建到原核表达载体p ET-23a(Amp+)和p ET-30a(Kan+)上,获得重组表达质粒p ET-23a-ADP-1-N-Npu和p ET-30a-Ssp-ADP-1-C,然后单独或共转化大肠杆菌感受态细胞BL21.实验结果表明,两个重组质粒分别单独或组合转化至Bl21(DE3),经IPTG诱导后,SDS-PAGE检测转化的菌株中都表达了目的蛋白,共转化的菌株诱导表达破菌后其上清液有抑菌活性.该实验验证以两种天然断裂型内含肽Ssp Dna E和Npu Dna E及搭载的ADP-1 N端和C端可在大肠杆菌中获得表达,且共转化的两段内含肽可在离体上清中发生反式剪接,促进融合在内含肽上的ADP-1两部分形成完整具有抑菌活性产物.展开更多
Brassica campestris male fertility 19 （BcMF19; GenBank accession number GQ902048.1）, a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, whi...Brassica campestris male fertility 19 （BcMF19; GenBank accession number GQ902048.1）, a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4＇-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander＇s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension orB. campestris ssp. chinensis.展开更多
文摘Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psbA, the gene encoding D1 protein of O. sativa ssp. japonica cv. 9516, and that of O. sativa ssp. indica cv. Shanyou 63 was cloned. As revealed by homology comparison of their sequences, the sequences are identical in the regions of promoter and 5′-UTR; differences are found in individual bases in the coding region all of which, being in the third position of respective codons, however, do not affect the amino acids coded finally; a difference is noted in the length of the oligo-U sequence in the region of 3′-UTR. It is thus apparent that, rather than a result of any difference in the amino acid sequences, the differences in the sensitivity to photoinhibition of D1 proteins between japonica and indica rice may be related to the upstream factors that regulate expression of psbA or to differences of photoprotective mechanisms.
文摘拟验证天然断裂内含肽Npu Dna E、Ssp Dna E的反式剪接反应对嵌合蛋白形成的影响,探讨依据此方法表达抗菌肽的可行性,规避抗菌生物表达中对宿主的毒害作用.将花蝉属抗菌肽ADP-1(Amblyomma defensin peptide 1)拆分为N端和C端两部分,其N端与断裂型内含肽Npu Dna E的N端融合,C端与Ssp Dna E的C端融合,并分别构建到原核表达载体p ET-23a(Amp+)和p ET-30a(Kan+)上,获得重组表达质粒p ET-23a-ADP-1-N-Npu和p ET-30a-Ssp-ADP-1-C,然后单独或共转化大肠杆菌感受态细胞BL21.实验结果表明,两个重组质粒分别单独或组合转化至Bl21(DE3),经IPTG诱导后,SDS-PAGE检测转化的菌株中都表达了目的蛋白,共转化的菌株诱导表达破菌后其上清液有抑菌活性.该实验验证以两种天然断裂型内含肽Ssp Dna E和Npu Dna E及搭载的ADP-1 N端和C端可在大肠杆菌中获得表达,且共转化的两段内含肽可在离体上清中发生反式剪接,促进融合在内含肽上的ADP-1两部分形成完整具有抑菌活性产物.
基金supported by the National Basic Research Program of China (2012CB113900)the National Natural Science Foundation of China (31071805)the Key Sci-Technology Project of Zhejiang Province, China (2010C12004)
文摘Brassica campestris male fertility 19 （BcMF19; GenBank accession number GQ902048.1）, a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4＇-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander＇s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension orB. campestris ssp. chinensis.
