Overexpression of Marine Microbial Metagenomic MbCSP Enhanced Drought and Cold Tolerance of Transgenic Arabidopsisthaliana

doi:10.7525/j.issn.1673-5102.2022.02.009

Abstract

Abstract:

Drought and low temperature are important factors affecting the growth and development of crop， genetically modified crop is an effective way to solve this problem. Cold shock proteins（CSPs）are a class of highly conserved nucleic acid binding proteins， which participate in cell physiological activities such as abiotic stress response， and the transfer of CSP gene can enhance the stress tolerance of crop. In this study， MbCSP gene was cloned by anchor PCR with marine microbial metagenomic DNA as template， and ORF was 216bp， encoded 71 amino acids. Homology analysis showed that the amino acid sequence had homology from 60% to 90% with cold shock proteins such as EcCSPG， EcCSPA（Escherichia coli）， BsCSPB（Bacillus subtilis） and BcCSPA（Bacillus cereus）， respectively. The results of multiple sequence alignment and phylogenetic tree analysis showed that MbCSP protein contained the classical conserved domains of CSP proteins such as RNP1（KGFGFI） and RNP2（VFVHF）， and were closely related to the cold shock proteins of EcCSPG（E. coli） and CmCSPA， CmCSPB（compost metagenome）. To further explore the function of MbCSP， a plant expression vector pTF101-MbCSP was constructed， and transfered to Arabidopsis thaliana by floral dip method. Transgenic plants were obtained by herbicide screening and PCR. Semi quantitative RT-PCR analysis was carried out， and the positive strain with the highest expression level was selected for subsequent physiological detection. The results showed that the growth of transgenic Arabidopsis plants were significantly better than that of wild-type， and its biomass was significantly higher than that of wild-type， under drought stress and low temperature stress. The relative water content， chlorophyll content and superoxide dismutase activity of transgenic Arabidopsis plants were higher than those of wild-type， while the content of malondialdehyde was lower than that of wild-type. The results showed that overexpression of marine microbial metagenomic MbCSP could improve drought and cold tolerance of transgenic A.thaliana plant， which laid the foundation for cultivating new varieties of transgenic crops.

Key words: cold shock protein, metagenome, Arabidopsis thaliana, drought, low temperature

CLC Number:

Mengjiao Wang, Yuxue Cao, Yongsheng Xu, Fenge Ding, Qiao Su. Overexpression of Marine Microbial Metagenomic MbCSP Enhanced Drought and Cold Tolerance of Transgenic Arabidopsisthaliana[J]. Bulletin of Botanical Research, 2022, 42(2): 243-251.

Figures/Tables 9

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引物名称 Primer name	引物序列 Primer sequence（5′—3′）
CSPU5	CCCGAATTCGGTAHAGTAAAATGGTTYAACKC
CSPU3	CCCGGATCCGGTTACGTTASCWGCTKSHGGDCC
CSP-1	GCCATTCTCGATAGAGAACTCAACT
CSP-2	CTGCTGATGGTAGCAAAGATGTGTT
CSP-3	GAACTCAACTTTCTGGCCTTCATCC
CSP-4	CAATGACTTCCGTACCCTGGATGAA
CSP-S	TCCCCCGGGATGTCTAACAAAATGACTGGTT
CSP-A	CGAGCTCTTAGCTTAAGGCTACAACGTT
XHNR	TTGCGGGACTCTAATCATAAAAACC
actin-F	CCCTCGTTTGTGGGAATGGA
actin-R	TCTGCTGGAATGTGCTGAGG

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