The \%E.coli\% trehase synthalose gene(\%otsA\%) was transferred into \%Nicotiana tabacum\% mediated by \%Agrobacterium\%, but the method for detecting low concentration of trehalose in transgenic plant was not availa...The \%E.coli\% trehase synthalose gene(\%otsA\%) was transferred into \%Nicotiana tabacum\% mediated by \%Agrobacterium\%, but the method for detecting low concentration of trehalose in transgenic plant was not available.The high performance liquid chromatograph(HPLC) with evaporative light\|scatting detector (ELSD) using water:methyl cyanide(1∶2\^6 v/v) as mobile phase was established in this work. An ODS column Zorbax RX\|SIL was employed. the trehalose detection limits of ELSD was 5mg/L. From the linear relationship between the logarithm of trehalose concentration and the logarithm of peak area, it was shown there was 14\^7μg\5(g FW)\+\{-1\} in transgenic plant. The data strongly confirmed that trehalose was responsible for the improved stress tolerance of the tobacco.展开更多
Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the reverta...Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the revertant has been obtained by PCR amplification,and the DNA fragment is 1022 base pair in size.By the analysis of sequencing result and compared with the AOX2 gene sequences recorded in Gene Bank,two point mutations which at positions of 529 and 255(relative to the translation initiation codon)respectively,have been found.Since the positions are located in the AOX2 upstream respression sequences,the mutations may act interfere the repressor to combine with the functional sequences,and increase transcriptional activity.The above result implicated that the P.pastoris’ ability to utilize methanol could be incresed through modification the upper stream transcriptional regulation region in the AOX2 gene.展开更多
文摘The \%E.coli\% trehase synthalose gene(\%otsA\%) was transferred into \%Nicotiana tabacum\% mediated by \%Agrobacterium\%, but the method for detecting low concentration of trehalose in transgenic plant was not available.The high performance liquid chromatograph(HPLC) with evaporative light\|scatting detector (ELSD) using water:methyl cyanide(1∶2\^6 v/v) as mobile phase was established in this work. An ODS column Zorbax RX\|SIL was employed. the trehalose detection limits of ELSD was 5mg/L. From the linear relationship between the logarithm of trehalose concentration and the logarithm of peak area, it was shown there was 14\^7μg\5(g FW)\+\{-1\} in transgenic plant. The data strongly confirmed that trehalose was responsible for the improved stress tolerance of the tobacco.
文摘Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the revertant has been obtained by PCR amplification,and the DNA fragment is 1022 base pair in size.By the analysis of sequencing result and compared with the AOX2 gene sequences recorded in Gene Bank,two point mutations which at positions of 529 and 255(relative to the translation initiation codon)respectively,have been found.Since the positions are located in the AOX2 upstream respression sequences,the mutations may act interfere the repressor to combine with the functional sequences,and increase transcriptional activity.The above result implicated that the P.pastoris’ ability to utilize methanol could be incresed through modification the upper stream transcriptional regulation region in the AOX2 gene.
