qPCR法构建双菌发酵制氢中两菌动态监测标准曲线

doi:10.19586/j.2095-2341.2024.0133

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 325-332.DOI: 10.19586/j.2095-2341.2024.0133

• 研究论文 • 上一篇

qPCR法构建双菌发酵制氢中两菌动态监测标准曲线

马含笑¹^,²()

^1.延安大学化学与化工学院，陕西延安 716000
^2.北京化工大学生命科学与技术学院，北京 100029

收稿日期:2024-07-21 接受日期:2024-09-27 出版日期:2025-03-25 发布日期:2025-04-29
作者简介:马含笑E-mail: mahanxiaoabc@126.com
基金资助:
延安大学研究项目(2003/2050040322)

Development of qPCR Standard Curves for Monitoring Population Dynamics in a Dual-bacterial Fermentative Hydrogen Production System

HanXiao MA¹^,²()

^1.College of Chemistry and Chemical Engineering，Yan'an University，Shaanxi Yan'an 716000
^2.College of Life Science and Technology，Beijing University of Chemical Technology，Beijing 100029，China

Received:2024-07-21 Accepted:2024-09-27 Online:2025-03-25 Published:2025-04-29

摘要/Abstract

摘要：

为了更好地了解由新的产氢菌株Bacillus cereus ATCC 14 579 (T)和Brevundimonas naejangsanensis BIO-TAS2-2 (T)构成的双菌体系制氢的互作机理,有效控制发酵制氢过程以提高氢气产量, 建立标准曲线以监测该双菌体系中两种细菌的动态变化。采用实时定量PCR (real-time quantitative PCR，qPCR)技术建立了两菌动态监测方法。首先以两株产氢菌的全基因组为模板，设计引物首次扩增出了它们的氢酶基因(Genbank No. CP015727.1和CP015614.1)。随后将扩增得到的两菌的氢酶序列与pMD19-T载体连接构建氢酶质粒，用其做模板进行qPCR扩增，以不同氢酶质粒浓度的对数值和对应的循环阈值（C_t）建立标准曲线,建立的标准曲线方程分别为y=-3.435 36 x+53.5698 7和y=-3.374 62 x+43.079 02,对应的相关系数（R²）分别为0.997和0.998，扩增效率分别为95.5%和97.8%。建立的标准曲线符合要求，在后续研究中可利用它们对发酵体系中的两种细菌进行绝对定量。方法为研究两株产氢菌在以不同底物进行混合发酵制氢过程中的相互作用及控制发酵过程奠定了基础,还为其他酶基因的体外扩增提供了方法学指导。

关键词: 生物制氢, Bacillus cereus ATCC14579(T), Brevundimonas naejangsanensis BIO-TAS2-2(T), qPCR, 氢酶基因

Abstract:

To better understand the interaction mechanism in hydrogen production by a dual-bacterial system comprising novel hydrogen-producing strains Bacillus cereus ATCC 14579 （T） and Brevundimonas naejangsanensis BIO-TAS2-2 （T）， and to effectively control the fermentation process for enhanced hydrogen yield， we established standard curves to monitor population dynamics of both strains. This study employed real-time quantitative PCR （qPCR） to develop a dynamic monitoring method. Hydrogenase genes of both strains were first amplified using specifically designed primers with whole genomes as templates （GenBank No.： CP015727.1 and CP015614.1）. The amplified hydrogenase sequences were then cloned into pMD19-T vectors to construct recombinant plasmids. These plasmid standards served as templates for qPCR amplification， enabling the establishment of standard curves through linear regression between log₁₀-transformed plasmid copy numbers and cycle threshold （C_t） values. The resulting calibration equations were y=-3.435 36x+53.569 87 （R2=0.997） for B. cereus and y=-3.374 62x+43.079 02 （R2=0.998） for B. naejangsanensis， with amplification efficiencies of 95.5% and 97.8%， respectively. The established standard curves meet the requirements， and they could be used to absolutely quantify two kinds of bacteria in fermentation system in subsequent studies. This methodology provides a foundation for investigating strain interactions during the mixed fermentation hydrogen production of both strains with different substrates and offers technical guidance for in vitro amplification of other enzymatic genes.

Key words: biohydrogen production, Bacillus cereus ATCC 14579 （T）, Brevundimonas naejangsanensis BIO-TAS2-2 （T）, qPCR, hydrogenase genes

中图分类号:

Q815

马含笑. qPCR法构建双菌发酵制氢中两菌动态监测标准曲线[J]. 生物技术进展, 2025, 15(2): 325-332.

HanXiao MA. Development of qPCR Standard Curves for Monitoring Population Dynamics in a Dual-bacterial Fermentative Hydrogen Production System[J]. Current Biotechnology, 2025, 15(2): 325-332.

图/表 8

表1 PCR和qPCR中使用的引物

Table 1 Primers used in PCR and qPCR

PCR

qPCR

Ah-F

Ah-R

Bh-F-2

Bh-R-2

Ah-F-1-y

Ah-R-1-y

Bh-F-y

Bh-R-y

5'-ATGATCCGCACGGAAGAGA-3'

5'-AGCCATCAACCTCCCCAAG-3'

5'-ATGACCGAGAGGCCCAACCG-3'

5'-TCACCCCGCCCGCTCGGCTTTT-3'

5'-CGGGCTTACCGCTCCTATAT-3'

5'-TTCACAGCCAACTCTCATA-3'

5'-CGCCGCCTTCTACGCCCT-3'

5'-GATGAAAGGCAGGTGCGGAA-3'

Strain A

Strain B

Strain A

Strain B

图1 两株产氢菌总基因组DNA和氢酶的PCR扩增结果注：1—DNA marker 1 kb ladder； 2—Strain A总基因组DNA； 3—Strain B总基因组DNA； 4—DNA marker DL 2 000； 5—Strain A氢酶基因； 6—Strain B氢酶基因。

Fig. 1 PCR amplication results of total genomic DNA and hydrogenase of two H2-producing strains

图2 含有两株细菌氢酶基因的阳性转化子的菌落PCR扩增结果注：1—DNA marker 1kb ladder； 2、 3、 4、5、6—Strain A氢酶基因阳性转化子菌落PCR； 7、 8—Strain B氢酶基因阳性转化子菌落PCR。

Fig. 2 Amplication results of colony PCR of positive transformants containing hydrogenase coding genes of two strains

图3 氢酶基因的同源性比对A：菌株A氢酶基因的同源性比对； B：菌株B氢酶基因的同源性比对

Fig. 3 Blast results of homology between hydrogenase genes

图4 两株细菌的氢酶质粒1—DNA marker 1kb ladder； 2、 3—Strain A氢酶质粒；4—DNA marker DL 5000； 5、 6—Strain B氢酶质粒

Fig. 4 Hydrogenase plasmids of two strains

图5 两株细菌氢酶质粒在qPCR扩增过程中的溶解曲线A：菌株A； B：菌株B

Fig. 5 Dissolution curves of hydrogenase plasmids of two strains during qPCR amplification

图6 两株细菌氢酶质粒在qPCR扩增过程中的扩增曲线A：菌株A； B：菌株B

Fig. 6 Amplification curves of hydrogenase plasmids of two strains during qPCR amplification

图7 qPCR扩增过程中不同氢酶质粒浓度的对数值与对应的Ct 值之间的标准曲线A：菌株A； B：菌株B

Fig. 7 Standard curves between the log10 (arithmic value) of the different hydrogenase plasmid concentration and the corresponding Ct value during qPCR amplification

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qPCR法构建双菌发酵制氢中两菌动态监测标准曲线

Development of qPCR Standard Curves for Monitoring Population Dynamics in a Dual-bacterial Fermentative Hydrogen Production System

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