CN103992973A - Agrobacterium tumefaciens SCUEC1 strain as well as screening method and application thereof - Google Patents
Agrobacterium tumefaciens SCUEC1 strain as well as screening method and application thereof Download PDFInfo
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- CN103992973A CN103992973A CN201410216950.8A CN201410216950A CN103992973A CN 103992973 A CN103992973 A CN 103992973A CN 201410216950 A CN201410216950 A CN 201410216950A CN 103992973 A CN103992973 A CN 103992973A
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Abstract
The invention provides an agrobacterium tumefaciens SCUEC1 strain. The collection number of the strain is CCTCC NO:M2014133, and the nucleotide sequence of the strains is as shown in SEQ ID NO. 1. The strains not only can be applied to nicotine degradation, but also can accelerate the maturity of tobacco leaves. Meanwhile, the invention further provides a method for screening and separating the strains. The method comprises the following steps: performing enrichment culture, then performing separation purification, inoculating separated and purified strains to a nicotine culture medium according to a single colony, culturing in a shaking table for 1-3 days at room temperature, measuring the nicotine content of a fermentation broth of the nicotine culture medium, and secondarily screening the strains according to the magnitude of the nicotine degradation rate to obtain the agrobacterium tumefaciens SCUEC1 strains.
Description
Technical field
The present invention relates to a kind of microorganism strains of degrading nicotine, be specifically related to a kind of Agrobacterium tumefaciens SCUEC1 bacterial strain, and the screening method of this bacterial strain and purposes, nicotine degradation technical field belonged to.
Background technology
Nicotine is not only the most important chemical composition being present in tobacco, and the height of nicotine content plays considerable effect to the quality of tobacco product.At present, China's Flue-cured Tobacco alkali content is generally higher, especially upper tobacco leaf is more obvious, as: subregion, Shennongjia surrounding area tobacco leaf visual appearance has approached internal and international advanced level, but some Chan Yan county nicotine content of tobacco leaves are up to 3.5%～4.5%, and burley tobaccos upper smoke alkali content is especially up to 6%.This has not only affected the quality of upper smoke, and affects the operability on top, has brought difficulty to cigarette composition industry, has restricted upper tobacco leaf market management.
The nicotine content effectively reducing in high-nicotine content tobacco leaf is rapidly the realistic problem that tobacco industry faces, thereby, the nicotine content reducing in high-nicotine content tobacco leaf has been made to large quantity research both at home and abroad, these researchs mostly concentrate on ecology, the cultivation factor, physical measure reduction nicotine content, as fertilising, filtration, punching dilution, tobacco sheet etc., and to utilizing biotechnology degrading nicotine research report less.
(application number is Chinese invention patent application: 200710013068.3,200710013069.8,200710070805.3,200910097536.9, Deng), the bacterial strain that these patents relate to has effective degradation property to nicotine, and unique shortcoming is under indoor laboratory condition, to carry out nicotine degradation, whether has effect also without experiment, confirm for the field tobacco leaf of true production.
Summary of the invention
The invention provides a kind of Agrobacterium tumefaciens SCUEC1 (Agrobacterium tumefaciens SCUEC1) bacterial strain, not only can the applying in nicotine degradation of this bacterial strain, but also can accelerate tobacco leaf maturation.
Agrobacterium tumefaciens SCUEC1 bacterial strain provided by the invention, this bacterial strain is preserved in Chinese Typical Representative culture collection center on April 17th, 2014, address is Wuchang District, Wuhan City, Hubei Province Wuhan University, the deposit number of this bacterial strain is: CCTCC NO:M2014133, the nucleotide sequence of this bacterial strain is as shown in SEQ ID NO.1.
The present invention also provides a kind of method of screening and separating above-mentioned bacterial strains simultaneously, comprises the following steps:
(1), enrichment culture: the soil in tobacco planting field is put into nicotine substratum, shaking table is cultivated at ambient temperature, cultivate after 1-3 days, pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, more than repeating to cultivate switching step 3 time, to reach the object of enrichment, collect enrichment culture liquid standby;
(2), separation and purification: adopt physiological saline by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivate, then choose fast growth, the good bacterial strain of the growth impetus carries out separation and purification;
(3), the bacterial strain after separation and purification is pressed to single colony inoculation in nicotine substratum, at room temperature shaking table was cultivated after 1-3 days, nicotine content in the fermented liquid of mensuration nicotine substratum, according to the big or small reflex sieve bacterial strain of nicotine degradation rate, thereby obtain Agrobacterium tumefaciens (Agrobacterium tumefaciens) SCUEC1 bacterial strain.
Described culturing step is specially: under 30 ℃ of conditions, adopt 180rpm shaking table to cultivate, incubation time is 48h.
Described physiological saline is the physiological saline of mass concentration 0.9%.
Agrobacterium tumefaciens SCUEC1 bacterial strain provided by the invention has the following advantages: 1, the degradation capability of this bacterial strain is strong, after tested, in laboratory, the degradation rate of this bacterial strain can reach 96.63%, and in field test, the nicotine degradation rate of this bacterial strain reaches as high as 26.07%.2, this bacterial strain can accelerate the maturation of tobacco leaf.
Accompanying drawing explanation
Fig. 1 is nicotine degradation rate curve table in the embodiment of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to detailed specific description, but protection scope of the present invention is not limited to following examples.
The Agrobacterium tumefaciens that provide in the present embodiment (Agrobacterium tumefaciens) SCUEC1 bacterial strain is preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M2014133, the 16S nucleotide sequence of this bacterial strain, as shown in SEQ ID NO.1, comprises 1189 bases.
The method for screening and separating of the Agrobacterium tumefaciens that provide in the present embodiment (Agrobacterium tumefaciens) SCUEC1 is as follows: (1), enrichment culture: the soil 2g in tobacco planting field is put into nicotine substratum 20mL, under 30 ℃ of conditions, adopt 180rpm shaking table to cultivate, cultivate after 48h, 1mL pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, repeat to cultivate switching step 3 time, to reach the object of enrichment, collect enrichment culture liquid standby;
(2), separation and purification: adopt the physiological saline of mass concentration 0.9% by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivate, then choose fast growth, the growth impetus good bacterial strain carries out separation and purification;
In this step, because nicotine isolation medium is usingd nicotine as sole carbon source, nitrogenous source is inorganic salt, and the microorganism that therefore can grow on this isolation medium is exactly the microorganism may with the ability of degrading nicotine.
(3), the bacterial strain after separation and purification is pressed to single colony inoculation in nicotine substratum, at room temperature shaking table was cultivated after 1-3 days, nicotine content in the fermented liquid of mensuration nicotine substratum, according to the big or small reflex sieve bacterial strain of nicotine degradation rate, thereby obtain Agrobacterium tumefaciens (Agrobacterium tumefaciens) SCUEC1 bacterial strain.
Although the bacterial strain that can grow on above nicotine isolation medium likely has the ability of degrading nicotine, be likely also that resistance to nicotine is not degraded it, so will carry out multiple sieve to the bacterium colony of purifying in this step.
Agrobacterium tumefaciens (Agrobacterium tumefaciens) the SCUEC1 bacterial strain that screening in the present embodiment is obtained carries out the experiment of laboratory smoke alkaline degradation, determine afterwards that by analysis optimum degradation condition is: nicotine starting point concentration is that 2g/L, temperature are that 30 ℃, initial pH are 5.5-6.0, to take peptone and each 0.5% concentration as compound nitrogen source, trace element of extractum carnis be 5mL/L, and nicotine degradation rate can reach 96.63% under this optimal conditions.Experimental result as shown in Figure 1, can find out in Fig. 1, and along with the growth of incubation time, nicotinic density reduces gradually, and absorbancy constantly raises.
Then carry out field nicotine degradation experiment, Agrobacterium tumefaciens (Agrobacterium tumefaciens) the SCUEC1 bacterial strain that just in the present embodiment, screening obtains is made bacteria suspension, bacteria suspension is processed to the new fresh tobacco leaf before gathering, after having tested, can effectively reduce nicotine content in tobacco leaf after measured, nicotine degradation rate is 26.07%.Meanwhile, through test, find that Agrobacterium tumefaciens (Agrobacterium tumefaciens) SCUEC1 can also accelerate tobacco leaf maturation.
Claims (6)
1. for Agrobacterium tumefaciens (Agrobacterium tumefaciens) SCUEC1 bacterial strain for nicotine degradation, the deposit number of this bacterial strain is: CCTCC NO:M2014133, the nucleotide sequence of this bacterial strain is as shown in SEQ ID NO.1.
2. a method for screening and separating for bacterial strain described in claim 1, is characterized in that comprising the following steps:
(1), enrichment culture: the soil in tobacco planting field is put into nicotine substratum, shaking table is cultivated at ambient temperature, cultivate after 1-3 days, pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, more than repeating to cultivate switching step 3 time, to reach the object of enrichment, collect enrichment culture liquid standby;
(2), separation and purification: adopt physiological saline by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivate, then choose fast growth, the good bacterial strain of the growth impetus carries out separation and purification;
(3), the bacterial strain after separation and purification is pressed to single colony inoculation in nicotine substratum, at room temperature shaking table was cultivated after 1-3 days, nicotine content in the fermented liquid of mensuration nicotine substratum, according to the big or small reflex sieve bacterial strain of nicotine degradation rate, thereby obtain Agrobacterium tumefaciens (Agrobacterium tumefaciens) SCUEC1 bacterial strain.
3. method for screening and separating according to claim 2, is characterized in that: described culturing step is specially: under 30 ℃ of conditions, adopt 180rpm shaking table to cultivate, incubation time is 48h.
4. method for screening and separating according to claim 2, is characterized in that: described physiological saline is the physiological saline of mass concentration 0.9%.
5. the purposes of bacterial strain claimed in claim 1 in nicotine degradation.
6. the purposes of bacterial strain claimed in claim 1 in accelerating tobacco leaf maturation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104328067A (en) * | 2014-09-30 | 2015-02-04 | 中南民族大学 | Ochrobactrum intermedium SCUEC4 strain, screening method and uses thereof |
| CN105648036A (en) * | 2014-11-17 | 2016-06-08 | 山东国际生物科技园发展有限公司 | A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain |
| CN113774001A (en) * | 2021-11-02 | 2021-12-10 | 盐城师范学院 | High-yield C21Steroid glycoside agrobacterium tumefaciens F-45 and application thereof |
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| CN101525579A (en) * | 2009-04-09 | 2009-09-09 | 浙江中烟工业有限责任公司 | Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof |
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| CN101525579A (en) * | 2009-04-09 | 2009-09-09 | 浙江中烟工业有限责任公司 | Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof |
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| 孔雯等: "烟碱降解菌的分离鉴定、降解特性及其降解途径的初步研究", 《中国优秀硕士论文全文数据库》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328067A (en) * | 2014-09-30 | 2015-02-04 | 中南民族大学 | Ochrobactrum intermedium SCUEC4 strain, screening method and uses thereof |
| CN105648036A (en) * | 2014-11-17 | 2016-06-08 | 山东国际生物科技园发展有限公司 | A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain |
| CN113774001A (en) * | 2021-11-02 | 2021-12-10 | 盐城师范学院 | High-yield C21Steroid glycoside agrobacterium tumefaciens F-45 and application thereof |
| CN113774001B (en) * | 2021-11-02 | 2023-03-14 | 盐城师范学院 | High-yield C 21 Steroid glycoside agrobacterium tumefaciens F-45 and application thereof |
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