| 摘要: |
| 【目的】从DNA分子水平研究葡糖醋杆菌及其经高静水压诱变后所得突变株的差异，优化建立了适合葡糖醋杆菌的AFLP技术体系，为细菌纤维素产生菌的高静水压诱变育种提供技术支撑。【方法】以1株从自制醋中分离出来的细菌纤维素产生菌J-2及其经高静水压诱变后得到的高产细菌纤维素的突变株M438为材料，对选择性扩增程序的影响因素进行分析，优化建立葡糖醋杆菌的AFLP酶切体系，并对适合葡糖醋杆菌高压诱变前后差异分析的引物组合进行筛选。【结果】酶切模板DNA用量600 ng，酶切时间8 h，过夜连接 (反应时间大于10 h)，预扩增产物最适稀释倍数为500倍，从24对AFLP选择性扩增引物组合中筛选出了E-T/M-G为适合葡糖醋杆菌高静水压诱变前后差异分析的引物组合。供试2株菌的选择性扩增差异位点只有1处，经测序扩增片段长度为213 bp，提交Genbank比对，其与Gluconacetobacter hansenii ATCC23769 GXY0064基因组序列中24 917～24 723 bp片段相似度为99%。【结论】所建立的AFLP反应体系，适合葡糖醋杆菌J-2及其突变株M438的AFLP分析。 |
| 关键词: 葡糖醋杆菌 突变株 AFLP 引物对筛选 |
| DOI： |
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| 基金项目:中央高校基本科研业务费专项资金资助项目（QN2009072） |
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| Establishment of AFLP reaction in a mutated Gluconacetobacter sp.induced by high hydrostatic pressure treatment |
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| Abstract: |
| 【Objective】The study intended to research the diversity for Gluconacetobacter and the mutated strain induced by high hydrostatic pressure treatment on molecular level.An AFLP reaction system suitable for Gluconacetobacter was optimized and established in the paper,which could provide a technical support for mutation breeding of bacterial cellulose producing strain by high hydrostatic pressure treatment.【Method】With a bacterial cellulose producing strain isolated from homemade vinegar and its mutated strain with higher yield bacterial cellulose induced by high hydrostatic pressure treatment as materials,the main factors of selective amplification processes were analyzed.The reaction system of digestion was optimized and established.Then the screening of primer pairs suitable for Gluconacetobacter sp.and its mutated strain were carried out.【Result】600 ng genomic DNA was served as template in the digestion system with 8 hours reaction,overnight ligation (the ligation time was more than 10 hours),500 times of dilution for the products of pre-amplification for selective amplification,and 1 pair of primers (E-T/M-G) was selected from 24 pairs of primer combination for the AFLP reaction system of bacteria.There was only 1 diversity locus between the two strains,which was 213 bp and had 99% identity with 24 917-24 723 bp in the genome sequence of Gluconacetobacter hansenii ATCC23769 GXY0064.【Conclusion】Results showed that it was feasible to use the reaction system constructed by the study to conduct the AFLP analysis of Gluconacetobacter sp.and its mutated strain. |
| Key words: Gluconacetobacter mutated strain AFLP screening of primer pairs |
