烟草甲体内烟碱降解菌的筛选、鉴定及降解特性

doi:10.13560/j.cnki.biotech.bull.1985.2022-1198

摘要/Abstract

摘要：

从烟草甲体内筛选鉴定烟碱降解菌，并对其降解特性进行探索研究。通过梯度稀释法从烟草甲体内分离筛选出烟碱降解菌，并利用4种烟叶培养基进行复筛。通过16S rRNA基因测序以及生理生化实验进行菌种鉴定；通过全基因组序列分析和验证探究菌株的生物特性。再通过分子对接及RT-qRCR探究其生物学特性和应用潜力。从烟草甲中分离到1株显著降解烟碱的优势菌株J1，16S rRNA基因同源性和生化特征分析鉴定为Mixta，不同酶活性检测结果显示J1不产β-葡萄糖苷酶、纤维素酶、蛋白酶、淀粉酶。烟碱降解菌通过数据库进行功能注释，发现了菌株上存在的烟碱代谢通路以及烟碱代谢关键基因或酶，选择吡啶途径中J1具有的烟碱脱氢酶（NDH）进行分子对接和功能验证。通过RT-qPCR验证，1 g/L烟碱诱导J1 24 h，与对照相比，ndh基因相对表达量上调15.978倍。0.8%烟叶浸提液诱导J1 24 h，与对照相比，ndh基因相对表达量上调1.117倍。在烟草甲体内分离筛选高效降解烟碱细菌J1，验证了降解相关基因的功能，为烟碱降解菌的筛选提供了新思路，同时，丰富了降解烟碱微生物资源。

关键词: 烟草甲内生菌, Mixta属, 烟碱耐受, 烟碱降解

Abstract:

The nicotine-degrading bacteria were screened and identified from Lasioderma serricorne, and their degradation characteristics were explored. The nicotine-degrading bacteria were primarily isolated and screened from L. serricorne by gradient dilution method, and re-screening was carried out using the four kinds of medium containing tobacco leaves. The strains were identified based on 16S rRNA gene sequencing combined with physiological and biochemical analysis. The whole genome sequence of the bacteria with degradation capabilities was analyzed to explore the degradation characteristics. Then, the potential degradation genes were explored through molecular docking and RT-qRCR. One bacterial strain J1 was isolated from L. serricorne, which significantly degraded nicotine. The strain J1 was identified as Mixta by 16S rRNA gene homology combined with physiological and biochemical characteristics. The results on the enzyme activities analysis showed that J1 did not produce β-glucosidase, fiber Enzymes, proteases, and amylases. The metabolism pathway and the key genes or enzymes involved in degrading nicotine in J1 were explored through functional annotation in the database. The nicotine dehydrogenase（NDH）possessed by J1 in the pyridine pathway was selected for molecular docking and functional verification. It was verified by RT-qPCR that the relative expression of ndh gene was up-regulated by 15.978 times using 1 g/L nicotine induction for 24 h compared with the control group. Additionally, the relative expression of ndh gene was up-regulated by 1.117 times when J1 was induced by 0.8% tobacco leaf extract for 24 h. The high-efficiency nicotine-degrading bacteria J1 was isolated and screened in L. serricorne. The degradation-related genes were verified. The results provided a new idea for the screening of nicotine-degrading bacteria and simultaneously enriched the nicotine-degrading microbial resources.

Key words: endophytic bacteria of Lasioderma serricorne, genus Mixta, nicotine tolerance, nicotine degradation

王羽, 尹铭绅, 尹晓燕, 奚家勤, 杨建伟, 牛秋红. 烟草甲体内烟碱降解菌的筛选、鉴定及降解特性[J]. 生物技术通报, 2023, 39(6): 308-315.

WANG Yu, YIN Ming-shen, YIN Xiao-yan, XI Jia-qin, YANG Jian-wei, NIU Qiu-hong. Screening, Identification and Degradation Characteristics of Nicotine-degrading Bacteria in Lasioderma serricorne[J]. Biotechnology Bulletin, 2023, 39(6): 308-315.

图/表 12

图1 烟碱降解菌的分离与筛选结果 A：烟草甲研磨液涂布在烟草平板上；B：烟草甲体表无菌化处理后ddH2O冲洗液涂布在烟草平板上

Fig. 1 Isolation and screening results of nicotine-degrading bacteria A: The tobacco plate spread with Lasioderma serricorne abrasive solution. B: The tobacco plate spread with ddH2O rinse solution after aseptic treatment of Lasioderma serricorne

表1 烟碱降解菌的形态特征

Table 1 Morphological characteristics of high nicotine-degrading bacteria

菌株编号No. of strain	形态特征Morphological characteristics
J1	菌落小，圆形，表面光滑，白色
J2	菌落小，圆形，表面光滑，白色
YC1	菌落小，圆形，表面光滑，浅黄色
YC2	菌落大，圆形，表面黏稠，黄色
YC3	菌落小，圆形，表面光滑，乳白色
YC4	菌落大，圆形，表面褶皱，白色
YC5	菌落小，圆形，表面光滑，白色不透明
YC6	菌落小，圆形，表面黏稠，黄色
YC7	菌落大，圆形，表面光滑，乳白色
YC8	菌落小，圆形，表面黏稠，乳白色
YC9	菌落小，圆形，表面褶皱，浅黄色
YC10	菌落大，圆形，表面褶皱，浅黄色

图2 烟碱降解菌在不同烟叶培养基平板上的复筛结果

Fig. 2 Rescreening results of nicotine-degrading bacteria on different tobacco medium plates

图3 J1的酶活性检测

Fig. 3 Detection of enzyme activities in J1

图4 基于16S rRNA基因序列J1系统发育树

Fig. 4 Phylogenetic tree of J1 based on 16S rRNA gene sequence

图5 烟碱紫外吸收标准曲线

Fig. 5 Standard curve of nicotine UV absorption

图6 J1菌液处理烟叶5 d后烟碱降解率变化 *P<0.05，下同

Fig. 6 Changes of nicotine degradation rates of tobacco leaves treated with J1 bacterial solutions for 5 d *P<0.05, the same below

图7 J1蛋白样品SDS-PAGE电泳结果 M为marker，泳道1-5为硫酸铵饱和度：100%、80%、50%、30%、0%

Fig. 7 Results of SDS-PAGE electrophoresis for J1 protein samples M is marker; lane 1-5 are ammonium sulfate saturation: 100%, 80%, 50%, 30%, and 0%

图8 BCA法检测蛋白浓度标准曲线

Fig. 8 Standard curve of protein concentration detected by BCA method

表2 BCA法检测粗酶液浓度结果

Table 2 Results of crude enzyme solution concentration detected by BCA method

样品Sample	蛋白浓度Protein concentration/（μg·μL^-1）
J1（0%）	2.317
J1（50%）	3.250
J1（80%）	4.360
J1（100%）	2.978

图9 J1粗酶液处理烟叶2 d后烟碱降解率变化

Fig. 9 Changes of nicotine degradation rates of tobacco leaves treated with crude enzyme solutions of J1 for 2 d

图10 J1的烟碱脱氢酶（NDH）与烟碱对接结果

Fig. 10 Docking results of nicotine dehydrogenase（NDH）of J1 with nicotine

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