苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

doi:10.19586/j.2095-2341.2021.0040

生物技术进展 ›› 2022, Vol. 12 ›› Issue (1): 90-98.DOI: 10.19586/j.2095-2341.2021.0040

苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

张卡(), 王泳浩, 吴云峰, 许雷(), 王海胜()

中国农业科学院研究生院，北京 100081

收稿日期:2021-03-25 接受日期:2021-06-16 出版日期:2022-01-25 发布日期:2022-01-26
通讯作者: 许雷,王海胜
作者简介:张卡 E-mail：2235805046@qq.com
基金资助:
中国农业科学院基本科研业务费项目(1610042018005)

The Non⁃coding RNA Regulated Nitrogen Metabolism in Sinorhizobium meliloti

Ka ZHANG(), Yonghao WANG, Yunfeng WU, Lei XU(), Haisheng WANG()

Graduate School of Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-03-25 Accepted:2021-06-16 Online:2022-01-25 Published:2022-01-26
Contact: Lei XU,Haisheng WANG

摘要/Abstract

摘要：

为了探究非编码RNA（non-coding RNA，ncRNA）在氮代谢调控过程的作用，以苜蓿中华根瘤菌（Sinorhizobium meliloti）为出发菌株，鉴定并获得了与ntrC基因表达相关的ncRNA，利用lncPRO软件分析苜蓿中华根瘤菌中ncRNA与调控蛋白NtrC相互作用的可能性，最终确定了评分较高的4个基因，即SM2011_c06191、SM2011_c06248、SM2011_c07102和SM2011_c07132，并对其中得分最高的SM2011_c06248基因进行研究。利用Northern blot验证ncRNA的表达，发现富氮处理后其表达水平呈先升高后降低的趋势。实验构建了SM2011_c06248基因敲除菌株，通过qRT-PCR发现SM2011_c06248在自然生长条件下表达无明显规律，富氮处理后，野生型菌株ncRNA表达水平呈先降低后升高再降低的趋势，与自然生长相比，ncRNA在20 min后表达水平明显上调；野生型菌株ntrC、nar基因表达水平呈先降低后升高再降低的趋势；与野生型相比，SM2011_c06248基因敲除菌株SM∷248中ntrC、nar基因表达水平明显下调。实验中构建SM2011_c06248、NtrC双突变菌株SM∷248-NtrC和SM2011_c06248过表达菌株SM:dld-248，qRT-PCR分析表明，与野生型相比，富氮处理后，SM∷248-NtrC菌株nar基因表达量无明显变化，SM:dld-248菌株ntrC、nar基因表达水平显著上调。ncRNA SM2011_c06248可响应环境中氮元素信号变化，对ntrC、nar基因的表达有正调控作用，且ntrC对nar有上位基因效应。

关键词: 苜蓿中华根瘤菌, 氮代谢调控, 非编码RNA, ntrC基因, nar基因

Abstract:

To explore the function of non-coding RNA （ncRNA） in the progress of nitrogen metabolism， we used Sinorhizobium meliloti as starting strain， and identified and obtained ncRNA related to ntrC gene expression. We used lncPRO software to analyze the possibility of interaction between ncRNA and regulatory protein NtrC in Sinorhizobium meliloti， the four genes with higher scores were finally determined， namely SM2011_c06191， SM2011_c06248， SM2011_c07102 and SM2011_c07132， and the SM2011_c06248 gene with the highest score was studied. Northern blot was used to verify the expression of ncRNA， and it was found that the expression level of ncRNA increased first and then decreased after nitrogen-enrichment treatment. The SM2011_c06248 gene knockout strain was constructed in the experiment， and the expression of SM2011_c06248 under natural growth conditions was found to have no obvious regularity by qRT-PCR. After nitrogen enrichment treatment， the ncRNA expression level of wild-type strains first decreased， then increased and then decreased. Compared with natural growth， the ncRNA expression level was significantly increased after 20 min. The expression levels of ntrC and nar genes in wild-type strains first decreased， then increased and then decreased. Compared with the wild type， the expression levels of ntrC and nar genes in SM2011_c06248 knockout strain SM∷248 were significantly down-regulated. The SM2011_c06248， NtrC double mutant strain SM∷248-NtrC and SM2011_c06248 overexpression strain SM：dld-248 were constructed in the experiment. qRT-PCR analysis showed that， compared with the wild type， after nitrogen-enriched treatment， there was no significant change for nar gene expression in the SM∷248-NtrC strain， and the expression levels of ntrC and nar genes in SM：dld-248 strain were significantly up-regulated. ncRNA SM2011_c06248 can respond to the change of nitrogen signal in the environment， and has a positive regulatory effect on the expression of ntrC and nar genes， and ntrC has epistatic effect on nar.

Key words: Sinorhizobium meliloti, regulation of nitrogen metabolism, non-coding RNA, ntrC gene, nar gene

中图分类号:

S541

张卡, 王泳浩, 吴云峰, 许雷, 王海胜. 苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究[J]. 生物技术进展, 2022, 12(1): 90-98.

Ka ZHANG, Yonghao WANG, Yunfeng WU, Lei XU, Haisheng WANG. The Non⁃coding RNA Regulated Nitrogen Metabolism in Sinorhizobium meliloti[J]. Current Biotechnology, 2022, 12(1): 90-98.

图/表 9

表1 实验中使用的引物

Table 1 Primers used in the experiment

引物名称	引物序列（5'→3'）	产物长度/bp	用途
248a	5'⁃CGCGGATCCGCGTCTGAGCGTCGATCCCCAC⁃3'	103	获得248s片段
248b	5'⁃CAATGCGAAACAATAACAAGCGCCGCG⁃3'	103
248c	5'⁃ACAATAACACAATGCGAACAACGCCAAG⁃3'	104
248d	5'⁃ACTGCAGTGCAGAAAGCATTGATTAACCAAG⁃3'	104
TcF	5'⁃CAAGGATCCAACCGCACTCCTA⁃3'	2 223	克隆抗性基因
TcR	5'⁃CATGGATCCGCTGTAGTGAGTGG⁃3'	2 223	克隆抗性基因
248F	5'⁃CGGACTAGTTCTGAGCGTCGATCCCCAC⁃3'	302	菌株鉴定
248R	5'⁃TGCACTGCAGGAAAGCATTGATTAACCAAGAGAA⁃3'	302	菌株鉴定
dldF	5'⁃CTTGATATCGAATTCCTGCAGGTCCAAAGCGCCCGAGAC⁃3'	214	构建载体PBBR1MCS⁃3⁃dld⁃248
dldR	5'⁃CTTCCCGGAAATTCTTTCGTTATCCATGAAAATTCTCCTTCTCA⁃3'	214
248F1	5'⁃ACGAAAGAATTTCCGGGAAGACAA⁃3'	334
248R1	5'⁃CTATAGGGCGAATTGGAGCTCTCGCCAAAACGCCGATTG⁃3'	334
NtrCq⁃F	5'⁃ACCTCCAATGCCGCAACCC⁃3'	1 026	获得NtrC片段
NtrCq⁃R	5'⁃AATGACATCCTGTGGATAAAGTGCC⁃3'	1 026	获得NtrC片段
16⁃F	5'⁃GTAAACTGCCCGACGGCTAAC⁃3'	179	qRT⁃PCR
16⁃R	5'⁃GTGAGTGGAATTCCGAGTGTAG⁃3'	179	qRT⁃PCR
NtrC⁃F	5'⁃CGACGAACAAGGACCTGAAACAG⁃3'	145	qRT⁃PCR
NtrC⁃R	5'⁃TGGACGAAATGCCGAACGAG⁃3'	145	qRT⁃PCR
NR⁃F	5'⁃CGGCGAGCAGGACTTCTACGT⁃3'	216	qRT⁃PCR
NR⁃R	5'⁃CGGTTGGCTTGCGAATGGAC⁃3'	216	qRT⁃PCR
248qF	5'⁃GGAAGACCGTAAACGAGGCC⁃3'	174	qRT⁃PCR
248qR	5'⁃GTTCGGCACGTTCCAGAGTT⁃3'	174	qRT⁃PCR

图1 载体构建示意图A：SM2011_c06248基因敲除载体pJQ200SK?248s?Tc；B：ntrC基因敲除载体pJQ200SK?ntrC?Tc；C：SM2011_c06248基因过表达载体pBBR1MCS?3?dld?248

Fig.1 The construction of vectors

图2 苜蓿中华根瘤菌基因发生双交换的筛选路线

Fig.2 Screening route for double exchange of genes in Sinorhizobium meliloti

图3 SM2011_c06248基因敲除菌株和SM2011_c06248、ntrC双突变菌株鉴定A：pJQ200SK?248s?Tc载体酶切验证；M—DL5 000 marker；1~3—pJQ200SK?248s?Tc载体PstⅠ、SpeⅠ双酶切。B：SM2011_c06248基因敲除菌株进行PCR鉴定；M—DL500 marker；1~3—SM2011_c06248基因敲除菌株；4~6—野生菌。C：pJQ200SK?ntrC?Tc载体酶切验证；M—DL5 000 marker；1—pJQ200SK?ntrC?Tc载体；2～3—pJQ200SK?ntrC?Tc载体BamHⅠ、EcoRⅠ双酶切。D：SM2011_c06248、ntrC基因双突变菌株进行PCR鉴定；M—DL2 000 marker；1~3：SM2011_c06248基因敲除菌株；4～6：SM2011_c06248、ntrC基因双突变菌株。

Fig.3 SM2011_c06248 gene knockout strain and identification of SM2011_c06248,ntrC double mutant strain

图4 SM2011_c06248基因过表达载体及过表达菌株的鉴定A：pBBR1MCS?3?dld?248载体酶切验证；M—DL5000 marker；1—pBBR1MCS?3?dld?248载体；2~3—pBBR1MCS?3?dld?248载体PstⅠ、SacⅠ双酶切。B：SM2011_c06248基因过表达菌株PCR鉴定；M—DL500 marker；1~3：SM2011_c06248基因过表达菌株。

Fig.4 SM2011_c06248 gene overexpression vector and identification of overexpression strain

图5 Nortern blot图结果图A：自然生长条件下野生型菌株ncRNA丰度；M—DL500 Marker；1~6—培养0、10、20、30、60和120 min后ncRNA丰度。B：30 ℃富氮培养条件下野生型菌株ncRNA丰度；M—DL500 Marker；1~6—富氮培养0、10、20、30、60和120 min后的ncRNA丰度。

Fig.5 Nortern blot results

图6 基因敲除菌株SM∷248氮代谢相关基因表达量A：自然生长条件下野生型菌株ncRNA表达水平；B：30 ℃富氮培养条件下野生型菌株SM2011_c06248表达水平；C：野生型菌株在自然生长条件下和富氮处理条件下ncRNA表达水平；D：30 ℃富氮培养条件下基因敲除菌株SM∷did?248 ntrC基因表达水平与野生型比较；E：30 ℃富氮培养条件下基因敲除菌株SM∷248?NtrCnar基因表达水平与野生型比较。

Fig.6 Gene expression levels related to nitrogen metabolism in SM∷248

图7 基因过表达菌株SM∷dld?248氮代谢相关基因表达量A：30 ℃富氮培养条件下基因过表达菌株SM∷dld?248与野生型ntrC基因表达量比较；B：30 ℃富氮培养条件下基因过表达菌株SM∷dld?248与野生型nar基因表达量比较。

Fig.7 Gene overexpression strain SM∷dld?248 nitrogen metabolism?related gene expression

图8 SM∷248?ntrC菌株氮代谢相关基因表达量

Fig.8 SM∷248?ntrC strain nitrogen metabolism?related gene expression

参考文献 23

1	WAGNER E G, ROMBY P. Small RNAs in bacteria and archaea: who they are, what they do, and how they do it[J]. Adv. Genet., 2015, 90: 133-208.
2	PAPENFORT K, VANDERPOOL C K. Target activation by regulatory RNAs in bacteria[J]. FEMS Microbiol., 2015, 39: 362-378.
3	SCHLUTER J P, REINKENSMEIER J, DASCHKEY S, et al.. A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti[J/OL]. BMC Genomics, 2010, 11: 245[2021-11-02]. .
4	SCHLVTER J P, REINKENSMEIER J, BARNETT M J, et al.. Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021[J/OL]. BMC Genomics, 2013, 14: 156[2021-11-02]. .
5	SALLET E, ROUX B, SAUVIAC L, et al.. Next-generation annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011[J]. DNA Res., 2013, 20(4): 339-354.
6	GIBSON K E, KOBAYASHI H, WALKER G C. Molecular determinants of a symbiotic chronic infection [J]. Annu. Rev. Genet., 2008, 42: 413-441.
7	JONES K M, KOBAYASHI H, DAVIES B W, et al.. How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model[J]. Nat. Rev. Microbiol., 2007, 5: 619-633.
8	乔森,刘雪洁,周集体.异养硝化⁃好氧反硝化在生物脱氮方面的研究进展[J].安全与环境学报,2014,14(2):128-135.
9	PAHEL G, TYLER B. A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli[J]. Proc. Natl. Acad. Sci. USA, 1979, 76(9): 4544-4548.
10	MCFARLAND N, MCCARTER L, ARTZ S, et al.. Nitrogen regulatory locus "glnR" of enteric bacteria is composed of cistrons ntrB and ntrC: identification of their protein products [J]. Proc. Natl. Acad. Sci. USA, 1981, 78(4): 2135-2139.
11	张宇.异养硝化菌NP1中NtrC蛋白对脱氮作用的调控研究[D].北京:中国农业科学院, 2018.
12	ZHAN Y, YAN Y, DENG Z, et al.. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501[J]. Proc. Natl. Acad. Sci. USA, 2016, 113(30): 4348-4356.
13	Michael R Green主编,贺福初主译.分子克隆实验指南[M].第4版.北京:科学出版社, 2013.
14	DUELLI D M, TOBIN A, BOX J M. Genetic locus required for antigenic maturation of Rhizobium etli CE3 lipopolysaccharide [J]. J. Bacteriol., 2001, 183(20): 6054-6064.
15	PELICIC V, REYRAT J M, GICQUEL B. Expression of the Bacillus subtilisSacB gene confers sucrose sensitivity on mycobacteria[J]. J. Bacteriol., 1996, 178(4): 1197-1199
16	QUANDT J, HYNES M F. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria[J]. Gene, 1993, 127(1): 15-21.
17	唐永凯,贾永义.荧光定量 PCR 数据处理方法的探讨[J].生物技术,2008,18(3): 89-91.
18	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
19	ROBELDO M, FRAGE B, WRIGHT P R, et al.. A stress-induced small RNA modulates alpha-rhizobial cell cycle progression[J/OL]. PLoS Genet., 2015, 11(4): e1005153[2021-11-02]. .
20	YAN B, WANG Z H, GUO J T. The research strategies for probing the function of long noncoding RNAs[J]. Genomics, 2012, 99(2): 76-80.
21	SURESH V, LIU L, ADIEROH D, et al.. RPI-Pred: predicting ncRNA-protein interaction using sequence and structureal information [J]. Nucl. Acids Res., 2015, 43: 1370-1379.
22	LU Q, REN S, LU M, et al.. Computational prediction of associations between long non-coding RNAs and proteins[J/OL]. BMC Genomics, 2013, 14:651[2021-11-02]. .
23	WANG K C, CHANG H Y. Molecular mechanisms of long noncoding RNAs[J]. Mol. Cell, 2011, 43(6): 904-914.

[1]	张月明, 罗雅琴, 郑伟, 徐杰, 董雪燕, 李善琛, 王敬毅. 急性髓系白血病mRNA与非编码RNA差异表达谱及CeRNA调控网络分析[J]. 生物技术进展, 2023, 13(1): 146-153.
[2]	薛姗, 唐铎, 赵子杰, 洪启浩, 王谦, 刘紫佳, 周志祥. 长链非编码RNA LINC00885在人食管癌细胞中的作用研究[J]. 生物技术进展, 2022, 12(3): 419-426.
[3]	武丽娜, 彭小忠, 鲁重美. 长链非编码RNA ZFAS1在结直肠癌中的表达及生物学功能[J]. 生物技术进展, 2022, 12(3): 427-435.
[4]	雍嘉欣, 韦权峰, 刘尚辉. 基于TCGA和GEO数据库的胃癌lncRNA筛选与ceRNA网络构建[J]. 生物技术进展, 2022, 12(1): 149-157.
[5]	张在宝,,赵海,胡梦辉,邓丽君,王琦,李九丽,袁红雨,. 组学在花药发育研究中的应用进展Ⅰ:转录组学[J]. 生物技术进展, 2019, 9(5): 433-439.
[6]	黄义,张维,燕永亮. 细菌RNA稳定性调控相关元件的研究进展[J]. 生物技术进展, 2018, 8(5): 376-382.
[7]	江湖, ,田健,应碧,伍宁丰,徐波. GlnR介导的代谢调控研究进展[J]. 生物技术进展, 2014, 4(2): 90-95.
[8]	战嵛华,马尧,邓志平,时朝,张云华,燕永亮. 非编码RNAs在细菌代谢网络调控中的研究进展[J]. 生物技术进展, 2011, 1(6): 413-420.

苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

The Non⁃coding RNA Regulated Nitrogen Metabolism in Sinorhizobium meliloti

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 23

相关文章 8

编辑推荐

Metrics

本文评价