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Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

ABSTRACT: Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7-13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

SUBMITTER: Song S

PROVIDER: S-EPMC7554901 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Song Shengli S Yan Rui R Wang Chunxia C Wang Jinxia J Sun Hongmei H

International journal of molecular sciences 20200916 18

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic tran ...[more]

PMID: 32947885

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Project description:Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.

Dataset Information

Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Publications

Improvement of a Genetic Transformation System and Preliminary Study on the Function of <i>LpABCB21</i> and <i>LpPILS7</i> Based on Somatic Embryogenesis in <i>Lilium pumilum</i> DC. Fisch.

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