吡咯伯克霍尔德氏菌JK-SH007产嗜铁素的相关基因克隆及条件优化

为了明确吡咯伯克霍尔德氏菌Burkholderia Pyrrocinia JK-SH007是否产嗜铁素，揭示其生防机制，本文通过CAS检测该菌株产嗜铁素的能力，并对其嗜铁素合成相关基因cepR进行克隆及序列分析，同时采用单因素试验及响应面法对菌株JK-SH007产嗜铁素条件进行优化。结果表明，菌株JK-SH007具有明显的产嗜铁素能力，其嗜铁素合成相关基因cepR大小为720 bp，与B.pyrrocinia（EU034001）的亲缘关系最近，同源性为99%，两者序列相似性为97%，13个位点出现SNP；另外菌株JK-SH007 cepR基因编码的氨基酸序列有3个氨基酸发生了替换，两者一致性为97%。该菌产嗜铁素最优培养时间是15 h、pH 8、转速200 r/min、最佳碳源和氮源分别是甘油和氯化铵；PB试验筛选出影响该菌株产嗜铁素的关键性因素分别是pH、加碳量、加氮量；CCD试验结果显示，pH和加氮量的交互作用较明显；最终采用Design-Expert软件分析出菌株JK-SH007产嗜铁素最佳方案为碳源加入量15.00 g/L、氮源加入量10.50 g/L、温度30℃、pH 7.36，优化后菌株产嗜铁素能力由18.59提升到37.86，响应面优化产嗜铁素条件的效果是显著的，嗜铁素产量得到明显提高。

Cloning and Conditions Optimization of the Siderophore Producing Gene for Burkholderia pyrrocinia JK-SH007

Siderophore plays an important role in the plant growth promotion and plant disease control. The effective biocontrol strain Burkholderia pyrrocinia JK-SH007 was isolated from the poplar stem. In order to determine whether JK-SH007 strain can produce siderophore and reveal its biocontrol mechanism, the ability of the strain to produce siderophore was detected by CAS detection, the cepR gene of JK-SH007 was cloned and the sequence was analyzed. On the basis of single factor experiment, response surface method (RSM) was used to optimize the siderophore production conditions of JK-SH007 strain. The results showed that JK-SH007 strain has the capacity to produce siderophore, and the cepR gene size of JK-SH007 was 720 bp. The strain was most closely related to B. pyrrocinia (EU034001), with 99% homology, 97% the sequence similarity was and SNP at 13 sites. Compared with the cepR gene of B. pyrrocinia (EU034001), three amino acids encoded by cepR gene of JK-SH007 were found to be replaced and the consistency was 97%. The experiment of siderophore production showed that the optimal culture time for siderophore producing was 15 h, the pH value of the medium was 8, and the rotating speed was 200 r/min. The optimal carbon and nitrogen sources were glycerin and ammonium chloride. According to PB (Plackett-Burman) experiment, the key factors influencing siderophore producing were pH, carbon and nitrogen. CCD (short for Central Composite Design) experiment showed that the interaction between pH and nitrogen addition was obvious. The optimal solution of siderophore producing for JK-SH007 by Design-Expert was as follows:carbon source at 15.00 g/L, nitrogen source at 10.50 g/L, the culture temperature at 30℃ and the medium starting pH value of 7.36. After optimization, the siderophore production capacity of JK-SH007 increased from 18.59 to 37.86. The culture conditions obtained by RSM were effective and reliable, and the yield of siderophore improved significantly.