草业学报 ›› 2023, Vol. 32 ›› Issue (5): 147-158.DOI: 10.11686/cyxb2022183
• 研究论文 • 上一篇
收稿日期:
2022-04-20
修回日期:
2022-06-20
出版日期:
2023-05-20
发布日期:
2023-03-20
通讯作者:
赵桂琴
作者简介:
E-mail: zhaogq@gsau.edu.cn基金资助:
Ji-kuan CHAI(), Ze-liang JU, Gui-qin ZHAO()
Received:
2022-04-20
Revised:
2022-06-20
Online:
2023-05-20
Published:
2023-03-20
Contact:
Gui-qin ZHAO
摘要:
为了明确低温青贮中筛选出的优异耐低温乳酸菌菌株戊糖片球菌OL77在低温和低pH胁迫下的基因表达情况,探讨其耐低温机理,本研究通过同源克隆获得了OL77的CspP基因,运用GeNorm,NormFinder,BestKeeper和RefFinder程序,基于SYBR-Green的实时荧光定量PCR(RT-qPCR)筛选OL77在低温和低pH胁迫条件下表达稳定的内参基因,并利用其研究了CspP基因的低温表达模式。 结果表明:OL77在受到外界低温和低pH胁迫时表达较稳定的内参基因为Ldh、tufA和6PGDH,而GAPDH和GyrA是表达较不稳定的内参基因。采用同源克隆获得OL77的CspP基因片段,以Ldh和6PGDH为内参基因,对25、15和5 ℃下培养0、1、3、6、12、24 h的OL77中CspP基因的相对表达量进行分析,发现低温条件下CspP基因瞬时表达量激增,在5 ℃下上调86倍,这可能是OL77耐低温的主要原因,OL77对低温的适应性部分来自其较强的冷休克蛋白表达能力。CspP基因可用作OL77低温适应性的指示基因。
柴继宽, 琚泽亮, 赵桂琴. 低温和低pH胁迫下青贮乳酸菌OL77的内参基因筛选及CspP基因的表达模式分析[J]. 草业学报, 2023, 32(5): 147-158.
Ji-kuan CHAI, Ze-liang JU, Gui-qin ZHAO. Screening of internal reference genes of Lactobacillus strain OL77 and determination of CspP expression patterns under low-temperature and low-pH stress[J]. Acta Prataculturae Sinica, 2023, 32(5): 147-158.
基因名 Gene symbol | 引物序列(5′-3′正向/反向) Primer sequence (5′-3′,forward/reverse) | 产物长度 Product length (bp) | 扩增效率 Amplification efficiency (%) |
---|---|---|---|
tufA | GAGAAGCGTCACTATGCCCA/CACCAACTTGACGTGCCAAC | 166 | 101 |
rpoD | GGTGTTGGTGGCTATCCCAA/GGTGTTGGTGGCTATCCCAA | 120 | 98 |
recA | GGTGTTGGTGGCTATCCCAA/GGTGTTGGTGGCTATCCCAA | 114 | 96 |
Ldh | TCGTCCTACACCGACAATGC/AGTAGCCGAAATGGCGCTTA | 151 | 99 |
GyrA | ACCGGAATTGCTGTTGGAATG/CATCAGGTCGGCTGTGGTAG | 114 | 100 |
GyrB | TCTACCCGGAAAGTTGGCAG/TGAAGCTTTCCCCACGTTCA | 166 | 98 |
GAPDH | TTGAATTGACCATGAGCTGTATC/TCGGACGTATTGGTCGTTTA | 142 | 106 |
6PGDH | CTTCTTTGGCGATACAATTCG/CTAATGCGCCTAATTCACCA | 98 | 103 |
CspP1 | TGTAATAGATTACCCCTTGT/TTCTTGTGATTCCCCAGATT | 164 | 99 |
CspP2 | AACGACTGTTGCTTGTTCC/GCAAGTGTCAGGGTTTCTCT | 111 | 98 |
表1 候选内参基因及引物序列
Table 1 Candidate internal reference genes and primer sequences
基因名 Gene symbol | 引物序列(5′-3′正向/反向) Primer sequence (5′-3′,forward/reverse) | 产物长度 Product length (bp) | 扩增效率 Amplification efficiency (%) |
---|---|---|---|
tufA | GAGAAGCGTCACTATGCCCA/CACCAACTTGACGTGCCAAC | 166 | 101 |
rpoD | GGTGTTGGTGGCTATCCCAA/GGTGTTGGTGGCTATCCCAA | 120 | 98 |
recA | GGTGTTGGTGGCTATCCCAA/GGTGTTGGTGGCTATCCCAA | 114 | 96 |
Ldh | TCGTCCTACACCGACAATGC/AGTAGCCGAAATGGCGCTTA | 151 | 99 |
GyrA | ACCGGAATTGCTGTTGGAATG/CATCAGGTCGGCTGTGGTAG | 114 | 100 |
GyrB | TCTACCCGGAAAGTTGGCAG/TGAAGCTTTCCCCACGTTCA | 166 | 98 |
GAPDH | TTGAATTGACCATGAGCTGTATC/TCGGACGTATTGGTCGTTTA | 142 | 106 |
6PGDH | CTTCTTTGGCGATACAATTCG/CTAATGCGCCTAATTCACCA | 98 | 103 |
CspP1 | TGTAATAGATTACCCCTTGT/TTCTTGTGATTCCCCAGATT | 164 | 99 |
CspP2 | AACGACTGTTGCTTGTTCC/GCAAGTGTCAGGGTTTCTCT | 111 | 98 |
项目Item | tufA | rpoD | recA | Ldh | GyrA | GyrB | GAPDH | 6PGDH |
---|---|---|---|---|---|---|---|---|
pH | ||||||||
6.0 | 24.4 | 27.8 | 21.2 | 25.6 | 21.7 | 21.4 | 24.4 | 24.4 |
5.0 | 24.4 | 27.7 | 22.2 | 26.9 | 21.3 | 20.4 | 23.3 | 24.4 |
4.0 | 24.5 | 28.8 | 22.1 | 26.8 | 21.1 | 21.1 | 23.4 | 24.5 |
温度Temperature (℃) | ||||||||
25 | 24.7 | 27.3 | 22.3 | 24.4 | 20.5 | 20.8 | 21.3 | 24.7 |
15 | 24.8 | 24.5 | 22.1 | 24.9 | 21.1 | 21.0 | 20.7 | 24.8 |
5 | 24.6 | 22.0 | 22.5 | 24.8 | 20.5 | 21.3 | 21.9 | 24.6 |
均值Average | 24.6 | 26.4 | 22.1 | 25.6 | 21.0 | 21.0 | 22.5 | 24.6 |
标准差Standard deviation (SD) | 0.6 | 2.6 | 0.8 | 1.7 | 1.2 | 0.8 | 2.5 | 0.6 |
变异系数Coefficient of variation (CV, %) | 2.4 | 10.0 | 3.4 | 6.5 | 5.9 | 3.7 | 11.3 | 2.4 |
表2 8个候选内参基因的表达水平
Table 2 Expression levels of 8 candidate internal reference genes (Cq)
项目Item | tufA | rpoD | recA | Ldh | GyrA | GyrB | GAPDH | 6PGDH |
---|---|---|---|---|---|---|---|---|
pH | ||||||||
6.0 | 24.4 | 27.8 | 21.2 | 25.6 | 21.7 | 21.4 | 24.4 | 24.4 |
5.0 | 24.4 | 27.7 | 22.2 | 26.9 | 21.3 | 20.4 | 23.3 | 24.4 |
4.0 | 24.5 | 28.8 | 22.1 | 26.8 | 21.1 | 21.1 | 23.4 | 24.5 |
温度Temperature (℃) | ||||||||
25 | 24.7 | 27.3 | 22.3 | 24.4 | 20.5 | 20.8 | 21.3 | 24.7 |
15 | 24.8 | 24.5 | 22.1 | 24.9 | 21.1 | 21.0 | 20.7 | 24.8 |
5 | 24.6 | 22.0 | 22.5 | 24.8 | 20.5 | 21.3 | 21.9 | 24.6 |
均值Average | 24.6 | 26.4 | 22.1 | 25.6 | 21.0 | 21.0 | 22.5 | 24.6 |
标准差Standard deviation (SD) | 0.6 | 2.6 | 0.8 | 1.7 | 1.2 | 0.8 | 2.5 | 0.6 |
变异系数Coefficient of variation (CV, %) | 2.4 | 10.0 | 3.4 | 6.5 | 5.9 | 3.7 | 11.3 | 2.4 |
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(0.06) | tufA(0.13) | GyrA(0.23) | 6PGDH(0.33) | recA(0.54) | GAPDH(1.34) | GyrB(1.34) | rpoD(2.78) |
5.0 | GyrA(0.31) | rpoD(0.42) | GyrB(0.51) | Ldh(0.62) | 6PGDH(0.70) | tufA(1.14) | recA(1.53) | GAPDH(1.80) |
4.0 | Ldh(0.03) | GyrA(0.03) | tufA(0.08) | recA(0.48) | GyrB(1.11) | 6PGDH(1.30) | rpoD(1.69) | GAPDH(2.13) |
温度Temperature (℃) | ||||||||
25 | tufA(0.22) | 6PGDH(0.42) | Ldh(0.62) | rpoD(0.64) | GyrB(0.73) | recA(0.77) | GyrA(0.94) | GAPDH(1.57) |
15 | GyrB(0.11) | Ldh(0.69) | tufA(0.90) | recA(1.00) | 6PGDH(1.05) | GAPDH(1.43) | rpoD(1.67) | GyrA(1.88) |
5 | Ldh(0.10) | recA(0.43) | tufA(0.45) | 6PGDH(0.48) | GyrB(0.48) | rpoD(0.90) | GyrA(1.29) | GAPDH(2.32) |
总体Total | GyrB(0.26) | GyrA(0.34) | 6PGDH(0.57) | Ldh(0.60) | tufA(0.73) | GAPDH(0.73) | recA(0.87) | rpoD(0.92) |
表3 基于NormFinder分析候选内参基因的稳定性
Table 3 Stability analysis of candidate internal reference genes based on NormFinder
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(0.06) | tufA(0.13) | GyrA(0.23) | 6PGDH(0.33) | recA(0.54) | GAPDH(1.34) | GyrB(1.34) | rpoD(2.78) |
5.0 | GyrA(0.31) | rpoD(0.42) | GyrB(0.51) | Ldh(0.62) | 6PGDH(0.70) | tufA(1.14) | recA(1.53) | GAPDH(1.80) |
4.0 | Ldh(0.03) | GyrA(0.03) | tufA(0.08) | recA(0.48) | GyrB(1.11) | 6PGDH(1.30) | rpoD(1.69) | GAPDH(2.13) |
温度Temperature (℃) | ||||||||
25 | tufA(0.22) | 6PGDH(0.42) | Ldh(0.62) | rpoD(0.64) | GyrB(0.73) | recA(0.77) | GyrA(0.94) | GAPDH(1.57) |
15 | GyrB(0.11) | Ldh(0.69) | tufA(0.90) | recA(1.00) | 6PGDH(1.05) | GAPDH(1.43) | rpoD(1.67) | GyrA(1.88) |
5 | Ldh(0.10) | recA(0.43) | tufA(0.45) | 6PGDH(0.48) | GyrB(0.48) | rpoD(0.90) | GyrA(1.29) | GAPDH(2.32) |
总体Total | GyrB(0.26) | GyrA(0.34) | 6PGDH(0.57) | Ldh(0.60) | tufA(0.73) | GAPDH(0.73) | recA(0.87) | rpoD(0.92) |
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(0.28) | 6PGDH(0.35) | tufA(0.40) | GyrA(0.49) | recA(0.58) | GAPDH(1.04) | GyrB(1.44) | rpoD(2.89) |
5.0 | rpoD(0.32) | 6PGDH(0.42) | Ldh(0.50) | GyrA(0.52) | GyrB(0.65) | tufA(0.76) | recA(1.65) | GAPDH(1.91) |
4.0 | tufA(0.23) | Ldh(0.36) | GyrA(0.41) | recA(0.50) | 6PGDH(0.87) | rpoD(1.51) | GyrB(1.69) | GAPDH(2.34) |
温度Temperature (℃) | ||||||||
25 | tufA(0.37) | 6PGDH(0.50) | rpoD(0.67) | Ldh(0.74) | GyrB(0.87) | recA(0.98) | GAPDH(1.19) | GyrA(1.20) |
15 | GyrB(0.26) | tufA(0.53) | recA(0.63) | 6PGDH(0.75) | Ldh(0.92) | GAPDH(1.62) | rpoD(2.06) | GyrA(2.22) |
5 | Ldh(0.27) | recA(0.47) | tufA(0.56) | 6PGDH(0.58) | GyrB(0.62) | rpoD(0.97) | GyrA(1.22) | GAPDH(2.20) |
总体Total | tufA(0.46) | Ldh(0.60) | 6PGDH(0.62) | GyrB(0.95) | GyrA(1.24) | GAPDH(1.98) | recA(2.08) | rpoD(2.19) |
表4 候选内参基因的SD值
Table 4 SD value of candidate internal reference genes
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(0.28) | 6PGDH(0.35) | tufA(0.40) | GyrA(0.49) | recA(0.58) | GAPDH(1.04) | GyrB(1.44) | rpoD(2.89) |
5.0 | rpoD(0.32) | 6PGDH(0.42) | Ldh(0.50) | GyrA(0.52) | GyrB(0.65) | tufA(0.76) | recA(1.65) | GAPDH(1.91) |
4.0 | tufA(0.23) | Ldh(0.36) | GyrA(0.41) | recA(0.50) | 6PGDH(0.87) | rpoD(1.51) | GyrB(1.69) | GAPDH(2.34) |
温度Temperature (℃) | ||||||||
25 | tufA(0.37) | 6PGDH(0.50) | rpoD(0.67) | Ldh(0.74) | GyrB(0.87) | recA(0.98) | GAPDH(1.19) | GyrA(1.20) |
15 | GyrB(0.26) | tufA(0.53) | recA(0.63) | 6PGDH(0.75) | Ldh(0.92) | GAPDH(1.62) | rpoD(2.06) | GyrA(2.22) |
5 | Ldh(0.27) | recA(0.47) | tufA(0.56) | 6PGDH(0.58) | GyrB(0.62) | rpoD(0.97) | GyrA(1.22) | GAPDH(2.20) |
总体Total | tufA(0.46) | Ldh(0.60) | 6PGDH(0.62) | GyrB(0.95) | GyrA(1.24) | GAPDH(1.98) | recA(2.08) | rpoD(2.19) |
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(1.00) | tufA(2.21) | 6PGDH(2.83) | GyrA(3.22) | recA(5.00) | GAPDH(6.00) | GyrB(7.00) | rpoD(8.00) |
5.0 | rpoD(2.00) | GyrA(2.11) | Ldh(2.45) | 6PGDH(2.51) | GyrB(3.87) | tufA(6.00) | recA(7.00) | GAPDH(8.00) |
4.0 | Ldh(1.19) | tufA(2.06) | GyrA(2.06) | recA(4.00) | 6PGDH(5.48) | GyrB(5.69) | rpoD(6.74) | GAPDH(8.00) |
温度Temperature (℃) | ||||||||
25 | tufA(1.41) | 6PGDH(1.68) | Ldh(2.45) | rpoD(3.94) | recA(4.82) | GyrB(5.48) | GyrA(7.24) | GAPDH(7.74) |
15 | GyrB(1.32) | tufA(2.06) | Ldh(3.16) | 6PGDH(3.16) | recA(3.72) | GAPDH(6.00) | rpoD(7.00) | GyrA(8.00) |
5 | Ldh(1.00) | recA(1.68) | 6PGDH(3.46) | tufA(4.05) | GyrB(4.47) | rpoD(5.73) | GyrA(7.00) | GAPDH(8.00) |
总体Total | GyrB(2.00) | 6PGDH(2.06) | Ldh(2.21) | tufA(2.94) | GyrA(3.76) | GAPDH(6.00) | recA(7.00) | rpoD(8.00) |
表5 基于RefFinder分析候选内参基因的稳定性
Table 5 RefFinder based candidate internal reference gene stability
项目 Item | 排序Rank | |||||||
---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
pH | ||||||||
6.0 | Ldh(1.00) | tufA(2.21) | 6PGDH(2.83) | GyrA(3.22) | recA(5.00) | GAPDH(6.00) | GyrB(7.00) | rpoD(8.00) |
5.0 | rpoD(2.00) | GyrA(2.11) | Ldh(2.45) | 6PGDH(2.51) | GyrB(3.87) | tufA(6.00) | recA(7.00) | GAPDH(8.00) |
4.0 | Ldh(1.19) | tufA(2.06) | GyrA(2.06) | recA(4.00) | 6PGDH(5.48) | GyrB(5.69) | rpoD(6.74) | GAPDH(8.00) |
温度Temperature (℃) | ||||||||
25 | tufA(1.41) | 6PGDH(1.68) | Ldh(2.45) | rpoD(3.94) | recA(4.82) | GyrB(5.48) | GyrA(7.24) | GAPDH(7.74) |
15 | GyrB(1.32) | tufA(2.06) | Ldh(3.16) | 6PGDH(3.16) | recA(3.72) | GAPDH(6.00) | rpoD(7.00) | GyrA(8.00) |
5 | Ldh(1.00) | recA(1.68) | 6PGDH(3.46) | tufA(4.05) | GyrB(4.47) | rpoD(5.73) | GyrA(7.00) | GAPDH(8.00) |
总体Total | GyrB(2.00) | 6PGDH(2.06) | Ldh(2.21) | tufA(2.94) | GyrA(3.76) | GAPDH(6.00) | recA(7.00) | rpoD(8.00) |
图5 不同温度下CspP基因的相对表达量同一时间不同字母表示不同温度处理间差异显著(P<0.05)。Different letters at the same time indicate significant differences among different temperature treatments (P<0.05).
Fig.5 The relative expression of CspP gene at different temperatures
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