Construction of Rhizobacteria with High-efficiency ACC Deaminase Using Promoter Replacement Technology
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摘要:
目的 探究细菌对代谢型趋化物的代谢速率对其趋化作用强弱的影响，同时为选育高效植物根际促生菌（Plant growth promoting rhizobacteria，PGPR）菌株开辟新路径。 方法 采用基因克隆得到4种含不同强弱启动子序列的基因片段，将其连至表达载体pBBR1MCS-2，通过三菌杂交接合转移成功构建出生长速率基本一致的4种目标菌株。 结果 ACC脱氨酶活性及AcdS基因表达量测定结果说明AcdS基因表达量、ACC脱氨酶活与启动子强弱之间呈现正相关关系；定性趋化结果表明菌株的ACC代谢速率越高，其对ACC的趋化能力也越强；各菌株在小麦根际定殖数量及对小麦生物量影响结果显示：UW4△AcdS+Bra20A菌株定殖数量最多，UW4△AcdS和UW4△AcdS +Bra1A菌株定殖数量较少；UW4△AcdS+Bra20A菌株处理后小麦茎干及根部重量均最重，UW4△AcdS+Bra1A和UW4△AcdS菌株处理后的小麦茎干较轻，UW4△AcdS菌株处理后的小麦根部重量也最轻。 结论 ACC脱氨酶活性基本与启动子序列强弱呈正相关。菌株的ACC脱氨酶活性越高，其对ACC代谢速率越高，ACC代谢速率越高，其趋化作用越强，对植株的促生效果也越好。 Abstract:Objective Strains of plant growth promoting rhizobacteria (PGPR) with varied ACC metabolic strengths were constructed by promoter replacement technology, and the resulting chemotaxis and effects on the growth of wheat seedlings analyzed. Methods Four gene fragments containing different promoter sequences were cloned and connected to the expression vector pBBR1MCS-2. Using the 3-parent hybridization method, strains of a similar growth rate was constructed. Results Four strains with basically a same growth rate were obtained by the hybridization and transformation method. The AcdS gene expression and ACC deaminase activity were found positively correlated to the strength of the promoter transferred to the strain. The higher the ACC metabolism rate, the greater the chemotactic ability of the strain. In the experiment on wheat seedling root growth and plant biomass with or without the PGPR strains, the colonization number in rhizosphere of UW4△AcdS+Bra20A was the highest, while those of UW4△AcdS and UW4△AcdS+Bra1A lower; the stem and root weight of wheat treated with UW4△AcdS+Bra20A the highest, the stem weight of wheat treated with UW4△AcdS+Bra1A and UW4△AcdS lower, and the root weight of wheat treated with UW4△AcdS lowest. Conclusion The promoter-replaced PGPRs with high enzyme activity displayed high ACC metabolic rate, which enhanced the chemotaxis and the plant-growth promotion ability. -
Key words:
- promoter /
- 3-parent hybridization /
- ACC deaminase /
- chemotaxis /
- metabolic rate /
- colonization
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图 1 4种目标菌株菌液RCR凝胶电泳图
注：M：Trans5k DNA Marker；1：Bra20A片段；2：Bra10A片段；3：Bra1A片段；4；PAA片段。
Figure 1. Gel electrophoresis of 4 target strains
Notes: M: Trans5k DNA marker; 1: Bra20A fragment; 2: Bra10A fragment; 3: Bra1A fragment; 4: PAA fragment.
图 3 各菌株ACC脱氨酶活性对比
注：柱形图上所标小写字母不同表示不同处理间差异显著（P<0.05）。图4、6、7同。
Figure 3. ACC deaminase activity of strains
Notes: Different lowercase letters indicate significant differences (P＜0.05). Same for Figs. 4, 6, and 7.
表 1 含不同强度启动子片段的引物设计
Table 1. Primers with promoters of different strength
引物名称
Name引物序列
Sequence扩增产物
PCR productBra20A-F CGGGATCCAATACTTGACATATCACTGTGATTA Bra20A CATATAATATGCGAAATCTGTAAGGCTAGCCAG GCTACACAGGGAATGAACCTGAATCGTTTTG Bra10A-F CGGGATCCACCTATTGACAATTAAAGGCTAAAA Bra10A TGCTATAATTCCACAAATCTGTAAGGCTAGCCA GGCTACACAGGGAATGAACCTGAATCGTTTTG Bra1A-F CGGGATCCTCCCTTTGATATTGCATCCCGCGTAT Bra1A ATAATATGTCAAATCTGTAAGGCTAGCCAGGCT ACACAGGGAATGAACCTGAATCGTTTTG A-R CCAAGCTTGTCAATCACGTATTTGGGTAAC 各片段下游引物 PAA-F CGGGATCCGGTTGAAACTCTGG PAA PAA-R CCAAGCTTTTTGACCCAGAC 注：上游酶切位点为Bam H Ⅰ，下游酶切位点为Hind III，分别以下划线标注。
Note: Upstream restriction sites Bam H I and downstream restriction sites Hind III are underlined.
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