Analysis of oxidase activity and transcriptomic changes related to cutting propagation of hybrid larch.

Hybrid larch is the main timber and afforestation tree species in Northeast China. To solve the problem of rooting difficulties in larch cutting propagation, enzyme activity determination and transcriptome sequencing were carried out on the rooting tissues at five timepoints after cutting. peroxidase (POD), indole acetic acid oxidase (IAAO) and polyphenol oxidase (PPO) play important roles in the larch rooting process after cutting. A total of 101.20 Gb of clean data was obtained by transcriptome sequencing, and 43,246 unigenes were obtained after further screening and assembly. According to GO analysis and KEGG enrichment analysis, we think that plant hormones play an important role in the rooting process of larch stem cuttings. in the plant hormone signal transduction pathway, a larch gene c141104.graph_c0 that is homologous to the Arabidopsis AUX1 was found to be significantly up-regulated. We suggest that AUX1 may promote IAA transport in larch, thus affecting adventitious root development. According to the results of POD, PPO IAAO indexes and GO analysis, we think s1 and s2 periods may be important periods in the rooting process of larch stem cuttings, so we built a gene regulatory network, a total of 14genes, including LBD, NAC, AP2/ERF, bHLH and etc., may be important in different stages of cutting propagation. As the rooting rate after cutting inhibits the development of larch clone propagation, identifying the genes that regulate rooting could help us to preliminarily understand the molecular mechanism of adventitious root formation and select a better treatment method for cutting propagation.

Genetic transformation of LoHDZ2 and analysis of its function to enhance stress resistance in Larix olgensis.

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.

Genetic transformation and growth index determination of the Larix olgensis LoHDZ2 transcription factor gene in tobacco.

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis. To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.