苜蓿中华根瘤菌与耐盐有关DNA片段的亚克隆和测序分析

SUBCLONING AND SEQUENCING OF DNA FRAGMENT RELATED TO SALT TOLERANCE IN SINORHIZOBIUM MELILOTI 042B

将苜蓿中华根瘤菌 (Sinorhizobiummeliloti) 0 4 2B与耐盐有关的 4kbClaⅠDNA片段克隆在 pML1 2 2上 ,用HindⅢ酶切下其 2 4kbDNA片段 ,回收后与 pBBR1 MCS2连接 ,然后转化大肠杆菌 (Escherichiacoli)DH5α,筛选到转化子GS2。将残留在 pML1 2 2上 1 6kbClaⅠ HindⅢDNA片段连同质粒一起回收 ,让其自连 ,转化大肠杆菌S1 7- 1 ,得到转化子GS0。以GS0为供体 ,0 4 2B的盐敏感突变株GZ1 7为受体 ,进行二亲本杂交 ,没有得到接合子。以GS2为供体 ,GZ1 7为受体 ,在辅助质粒pRK2 0 1 3的协助下 ,进行三亲本杂交 ,筛选到接合子GG2 ,获得 2 4kbHindⅢ与耐盐有关的DNA片段。将此片段连接到测序载体 pGEM 7Zf(+)上进行测序。测序结果表明 ,该 2 4kbHindⅢDNA片段含有 3个开放阅读框 (ORF)。在此基础上再一次亚克隆 ,获得 1 9kb与耐盐有关的DNA片段。

A 4kb \%Cla\%I DNA fragment related to salt tolerance from \%S.meliloti\% 042B was digested by \%Hin\%dⅢ down 2.4kb fragment, and a 1.6kb \%Cla\%I\|\%Hin\%dⅢ fragment was retained on plasmid pML122. Then, the 2.4kb DNA fragment was ligated with plasmid pBBR1\|MCS2, and the recombinant plasmid was transformed to \%E.coli\% DH5α, and transformant GS2 was obtained. Three\|parental mating experiments were carried out with transformant GS2 as donor, salt sensitive strains GZ17 as recipient and pRK2013 as helper plasmid, then the transconjugant GG2 was selected on FY plates containing kanamycin and 0 4mol/L \{NaCl.\} The remaining DNA fragment was self ligated with pML122 and then transformed into \%E.coli\% S17\|1 and transformat GS0 was obtained. Two\|parental mating experiment was carried out with transformant GS0 as donor and salt sensitive strain GZ17 as recipient, but no transconjugant was obtained on the FY plates. Then,the 2.4kb \%Hin\%dⅢ DNA fragment was ligated into seguencing vector pGEM\|7Zf(+) for sequencing. The result of sequencing and analysis showed that the 2.4kb DNA fragment contained three ORFs. Accordning to the result of sequencing, further subcloning was conducted and 1.9kb \%Hin\%dⅢ\|\%Sac\%Ⅱ DNA fragment related to salt tolerance was obtained.