Heterologous expression of miR397-5p from Medicago sativa cv. ‘Pianguan’ improves the drought tolerance of tobacco

doi:10.11686/cyxb2023498

Abstract

Abstract:

Medicago sativa cv.‘Pianguan’ is a well-regarded local germplasm in Shanxi Province， known for its strong stress resistance， adaptability， and resistance to barren soils. It is crucial to identify genes related to drought resistance in M. sativa cv. ‘Pianguan’， so that they can be utilized for the genetic enhancement of alfalfa， thereby facilitating the development of new germplasm. In this study， miR397-5p was identified as a potential microRNA responsive to drought stress in M. sativa cv. ‘Pianguan’ through miRNA sequencing analysis. The responsiveness of miR397-5p from M. sativa cv. ‘Pianguan’ to drought stress was validated in transgenic tobacco， and its potential downstream target， and its effects， were identified. After drought stress， miR397-5p-overexpressing tobacco showed elevated levels of osmotic regulators， including soluble sugars， soluble proteins， and proline. Additionally， the activities of the antioxidant enzymes catalase， superoxide dismutase， and peroxidase were increased， while the malondialdehyde content was decreased. Under drought stress， miR397-5p decreased the expression of its target gene LAC4. In miR397-5p-overexpressing tobacco， most genes encoding key enzymes in the lignin biosynthesis pathway （PAL， 4CL， CCR， CAD， HCT， F5H， and PAO）showed decreased transcript levels， while genes such as C4H， CCoAOMT， COMT，and others exhibited increased transcript levels. Additionally， the lignin content was decreased in miR397-5p-overexpressing tobacco. The transcript levels of most of these genes and lignin content exhibited consistent trends.It was inferred that M. sativa cv. ‘Pianguan’ may regulate lignin accumulation in response to drought stress through miR397-5p-LAC4， but the specific underlying mechanism needs to be further investigated. Further research is required to clarify the mechanism of this response. The results of this study may provide a genetic resource for the genetic improvement of alfalfa， which is potentially valuable for the creation of excellent new germplasm.

Key words: Medicago sativa cv.‘Pianguan’, drought stress, miR397-5p, lignin

Yu-xin WANG, Jia-li TAO, Hui-sen ZHU, Tao XU, Yi-fei ZHANG, Hui-fang CEN. Heterologous expression of miR397-5p from Medicago sativa cv. ‘Pianguan’ improves the drought tolerance of tobacco[J]. Acta Prataculturae Sinica, 2024, 33(11): 123-134.

Figures/Tables 11

References 35

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基因Gene	正向引物Forward primer （5'-3'）
conservative_7_25433	TTTCCAATTCCACCCATTCCTA
conservative_8_17329	ATTGGATTGAAGGGAGCTCC
conservative_8_17330	ATTGGATTGAAGGGAGCTCC
miR397-5p	TCATTGAGTGCAGCGTTGATG
miR5205a	CATACAATTTGGGACGGAGGGAG
miR530	TGCATTTGCACCTGCACTTTC
unconservative_6_4438	GCGTTAGCTCAGTTAGTAAGGACAATG
U6	CCTGCGCAAGGATGACACGCAT

基因Gene	正向引物Forward primer （5'-3'）
conservative_7_25433	TTTCCAATTCCACCCATTCCTA
conservative_8_17329	ATTGGATTGAAGGGAGCTCC
conservative_8_17330	ATTGGATTGAAGGGAGCTCC
miR397-5p	TCATTGAGTGCAGCGTTGATG
miR5205a	CATACAATTTGGGACGGAGGGAG
miR530	TGCATTTGCACCTGCACTTTC
unconservative_6_4438	GCGTTAGCTCAGTTAGTAAGGACAATG
U6	CCTGCGCAAGGATGACACGCAT

基因Gene	正向引物Forward primer （5'-3'）	反向引物Reverse primer （5'-3'）
MTR_4g015120	AACTACCTGTGGAGAGTGGC ACTAGGCCATGGATCCGATG	CATCGGATCCATGGCCTAGT GCCACTCTCCACAGGTAGTT
MsActin	TCGAGACCTTCAATGTGCCT	ACTCACACCGTCACCAGAAT
PAL	NATWGACTTGAGGCAYTTGG	TTYTGCATYARTGGGTAGTT
C4H	GGCAATCCCTCTTTTAGTCCC	CTCCTACCAACACCAAATGGA
F5H	CCAGCGACCGTAGCCATAAGTTAC	TGCCGCCAGAAGAGTCCATAGTC
4CL	CTCTGGKACTACRGGKCTGC	AYTCCARGAACGGAGCAATG
CCR	TGGCAAACAGAGCAGGTGAAGTAG	CGGTGGCGTGAACAGTGTAGC
HCT	CTCAACCCACTCCCAACCAT	GCCTCCTTTAGCACTTTTCCG
CCoAOMT	AATGGTTCTGTGGTGGCTCC	CGGCGGCACAAGGTAATG
COMT	GATGTTGGAGGTGGTCTTGGA	CTGGTTTCACTGGTAAAATGGC
CAD	GAGGGTATGGCACCAGAACAA	GATGTCCCATTGCCTTTGCTAT
PAO	GTCGCTGCTCTGTCGTCATAGTC	CGCCGAATTCCTCCTTCCTTATCC
NtL25	GCTTTCTTCGTCCCATCA	CCCCAAGTACCCTCGTAT

基因Gene	正向引物Forward primer （5'-3'）	反向引物Reverse primer （5'-3'）
MTR_4g015120	AACTACCTGTGGAGAGTGGC ACTAGGCCATGGATCCGATG	CATCGGATCCATGGCCTAGT GCCACTCTCCACAGGTAGTT
MsActin	TCGAGACCTTCAATGTGCCT	ACTCACACCGTCACCAGAAT
PAL	NATWGACTTGAGGCAYTTGG	TTYTGCATYARTGGGTAGTT
C4H	GGCAATCCCTCTTTTAGTCCC	CTCCTACCAACACCAAATGGA
F5H	CCAGCGACCGTAGCCATAAGTTAC	TGCCGCCAGAAGAGTCCATAGTC
4CL	CTCTGGKACTACRGGKCTGC	AYTCCARGAACGGAGCAATG
CCR	TGGCAAACAGAGCAGGTGAAGTAG	CGGTGGCGTGAACAGTGTAGC
HCT	CTCAACCCACTCCCAACCAT	GCCTCCTTTAGCACTTTTCCG
CCoAOMT	AATGGTTCTGTGGTGGCTCC	CGGCGGCACAAGGTAATG
COMT	GATGTTGGAGGTGGTCTTGGA	CTGGTTTCACTGGTAAAATGGC
CAD	GAGGGTATGGCACCAGAACAA	GATGTCCCATTGCCTTTGCTAT
PAO	GTCGCTGCTCTGTCGTCATAGTC	CGCCGAATTCCTCCTTCCTTATCC
NtL25	GCTTTCTTCGTCCCATCA	CCCCAAGTACCCTCGTAT

miRNA	序列Sequence	log₂（FC）（72 h VS 0 h）	靶基因Target gene
conservative_7_25433	TTTCCAATTCCACCCATTCCTA	-1.69	MTR_0038s0060
conservative_8_17329	ATTGGATTGAAGGGAGCTCC	2.19	MTR_3g011610
conservative_8_17330	ATTGGATTGAAGGGAGCTCC	2.19	MTR_8g042410
miR397-5p	TCATTGAGTGCAGCGTTGATG	2.02	MTR_4g015120
miR5205a	CATACAATTTGGGACGGAGGGAG	-1.25	MTR_7g100190
miR530	TGCATTTGCACCTGCACTTTC	0.32	MTR_1g107575
unconservative_6_4438	TTAGCTCAGTTAGTAAGGACAATG	-1.54	MTR_2g020900