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Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

ABSTRACT: BACKGROUND:MiRNAs (microRNA) are 18-24?nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. RESULTS:This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60?days, miR160, miR166 and miR396-2 exhibited the highest expression in leaves. At 120?days, most miRNAs in roots and stems decreased significantly. At 180?days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396-2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166-1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950-1, first decreased and then increased. Similarly, in the roots, miR950-2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. CONCLUSIONS:The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.

SUBMITTER: Zhang S

PROVIDER: S-EPMC6558743 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

Zhang Sufang S Yan Shanshan S Zhao Jiali J Xiong Huanhuan H An Peiqi P Wang Junhui J Zhang Hanguo H Zhang Lei L

BMC plant biology 20190611 1

<h4>Background</h4>MiRNAs (microRNA) are 18-24 nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression.<h4>Results</h4>This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miR ...[more]

PMID: 31185902

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Project description:Hybrids of larch (Larix kaempferi × Larix olgensis) are important afforestation species in northeastern China. They are routinely propagated via rooted stem cuttings. Despite the importance of rooting, little is known about the regulation of adventitious root development in larch hybrids. 454 GS FLX Titanium technology represents a new method for characterizing the transcriptomes of non-model species. This method can be used to identify differentially expressed genes, and then two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses can be used to analyze their corresponding proteins. In this study, we analyzed semi-lignified cuttings of two clones of L. kaempferi × L. olgensis with different rooting capacities to study the molecular basis of adventitious root development.We analyzed two clones; clone 25-5, with strong rooting capacity, and clone 23-12, with weak rooting capacity. We constructed four cDNA libraries from 25-5 and 23-12 at two development stages. Sequencing was conducted using the 454 pyrosequencing platform. A total of 957832 raw reads was produced; 95.07% were high-quality reads, and were assembled into 45137 contigs and 61647 singletons. The functions of the unigenes, as indicated by their Gene Ontology annotation, included diverse roles in the molecular functions, biological processes, and cellular component categories. We analyzed 75 protein spots (-fold change ? 2, P ? 0.05) by 2D-DIGE, and identified the differentially expressed proteins using MALDI-TOF/TOF MS. A joint analysis of transcriptome and proteome showed genes related to two pathways, polyamine synthesis and stress response, might play an important role on adventitious root development.These results provide fundamental and important information for research on the molecular mechanism of adventitious root development. We also demonstrated for the first time the combined use of two important technologies as a powerful approach to advance research on non-model, but otherwise important, larch species.

Project description:To study the function of LoHDZ2 in larch, we first constructed a VB191103-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured. The results are summarized below: (1) When the specific fragment of the target gene in the genome of the resistant cell line was amplified. At the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines. (2) To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 down regulated genes) were subjected to qRT-PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-Seq, indicating that the transcriptome data were reliable. (3) The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were down regulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process. (4) After 20% PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced. In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2 may participate in the response of plants to the external environment, and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.

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Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

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Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

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