Genetic transformation of larch
Embryogenic calli on 1/10-BM proliferation media were selected for genetic transformation of larch. The genetically transformed calli were cocultured for approximately 2 days and then transferred to culture media consisting of 500 mg/L Cef for strict sterilization. After three separate cultures on the screening media, five resistant cell lines were obtained. The resistant cell lines were then cultured in proliferation media for 10 days, after which they then in 1/4-BM transition media for 10 days. Afterward, the calli were placed in somatic embryo induction media for somatic embryo induction to obtain larch transgenic somatic embryos (Fig. 1). Then, the somatic embryos were placed in 1/2-strength MS rooting media to obtain transgenic larch seedlings, as shown in Fig. 2.
Molecular detection of transgenic LoHDZ2 calli
Five transgenic callus cell lines growing on 1/10-BM were randomly selected to extract plant genomic DNA. The DNA was subsequently used as template for PCR detection. p1302-LoHDZ2::GUS plasmids were used as positive controls, and the wild-type was used as a negative control. The PCR detection results of p1302-LoHDZ2::GUS-transformed embryogenic calli are shown in Fig. 3. The results showed that four transgenic calli produced bands at corresponding positions, which preliminarily showed that p1302-LoHDZ2::GUS had been integrated successfully into the larch genome.
The five abovementioned resistant callus cell lines growing on 1/10-BM media and one wild-type callus cell line were collected, and their RNA was extracted and then reverse transcribed into cDNA. The cDNA was used as a template for qRT–PCR-based detection, and wild-type cDNA was used as a control. The results showed that the expression levels of transgenic lines OE1, OE2 and OE4 were higher, approximately 1.9, 2.9 and 3.6 times higher, respectively, than that of the wild-type cell lines(Fig. 4).
To further confirm that p1302-LoHDZ2::GUS was integrated into the resistant calli, GUS staining solution was applied to wild-type larch calli and resistant calli. The results showed that four of the transgenic calli turned blue, while the wild-type callus in the control group exhibited no color change, indicating that the recombinant p1302-LoHDZ2::GUS plasmid had been successfully integrated into the larch calli (Fig. 5, Fig. 6).
According to the detection of the DNA and RNA in the transgenic cell lines and the analysis of the GUS staining results, the OE1, OE2 and OE4 transgenic cell lines were ultimately selected as follow-up research materials.
Unigene functional annotation
The assembled unigenes do not have corresponding functional annotations, so we added corresponding functional annotations using Diamond, a new comparison software similar to BLASTX. Because the sequences of similar functional genes (nucleic acid sequences or protein sequences) are highly conserved among different species, we selected six authoritative databases, namely, the NCBI NR, GO, KEGG, Pfam, SwissProt and eggNOG databases (Tab.2).
To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 downregulated ones) were selected for qRT-PCR-based verification. The results show that these genes have different expression fold changes, as detected by RNA-seq and qRT-PCR (Fig. 7). This may be because the sensitivity of the two detection methods is different. Nonetheless, the expression trends are consistent, indicating that the transcriptome data are reliable and can be used for further gene functional analysis.
Screening of differentially expressed genes
In this study, DESeq2 was used to screen differentially expressed genes. The differentially expressed genes among different varieties were screened on the basis of their expression fold change (|log2(fold change)|>1) and significance level (pvalue < 0.05). The results showed that there were 304 differentially expressed genes in transgenic larch cell lines compared with wild-type cell lines: 167 upregulated differentially expressed genes and 140 downregulated differentially expressed genes (Fig. 8).
Cluster analysis of differentially expressed genes
To compare the clustering patterns of the differentially expressed gene expression profiles of the transgenic and wild-type calli, a clustering heatmap of differentially expressed genes was constructed for the genes with a large variance in expression in different samples (Fig. 9). The results showed that compared with that in the wild-type resistant calli, the expression of many genes in the transgenic resistant calli changed, the number of upregulated genes especially changed, which was approximately 176. There were 140 downregulated genes, indicating that the overexpression of LoHDZ2 has a positive regulatory effect on downstream genes. The heatmap shows that the transcriptome sequencing of the three biological replicates of each sample have good consistency, indicating that the sequencing data are relatively reliable.
To further explore the function of LoHDZ2, the differentially expressed genes with the largest variance in the transgenic and wild-type samples were used to construct a cluster heatmap of differentially expressed genes, and 46 upregulated genes and 30 downregulated genes with large expression fold changes were selected for NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). When the results were compared with the information in the various aforementioned databases, relevant annotation and species information was obtained .
Among the 46 differentially expressed genes, including 22.0 kDa class IV, heat-shock protein-like 22.0 kDa IV, heat stress transcription factor A-1-like, heat-shock proteins, and heat-shock cognate 70 kDa protein, most have been annotated in broad-leaved tree species . The annotated LRR receptors, such as the serine/threonine protein kinase FLS2 LRR receptor and serine/threonine protein kinase FLS2, may inhibit cell proliferation and stimulate extracellular matrix synthesis depending on the cell type. In addition, the xyloglucan galactosyltransferase GT11, which affects the internal mechanisms of plant growth and development, was also annotated.
Among the 30 downregulated differentially expressed genes, poly[ADP ribose] polymerase 3 isoform X2, a late-embryogenesis abundant protein, was identified. Moreover, this protein plays an important role in plant growth and development, in disease resistance, and in response to hormones and stress and is involved with E3 ubiquitin-protein ligase SINAT2-like protein.
GO functional annotation and enrichment analysis
GO functional annotation was carried out for the differentially expressed genes in the different samples. The differentially expressed genes were annotated to the three different GO classification categories: cellular components, biological processes and molecular functions. The number of differentially expressed genes between the different samples and the wild-type is annotated to the three branches. After GO functional classification annotation, 769 genes were annotated. Among them, 243 gene pathways were enriched in cellular components, 194 molecular function pathways, and 334 genes were annotated to biological process pathways. The enriched differentially expressed genes and their information are shown in Table 3.
In the cellular component pathway, the most abundant genes were associated with terms such as nucleus (39), cytoplasm (31), chloroplast (17), cytosol (15), plasma membrane and integral components of membrane (16), mitochondria (8), and cell wall and plasmodesmata (6). In the molecular function pathway, most (43) genes were related to binding proteins, followed by catalytic activity and molecular function, so binding and catalytic activity are the main molecular pathway processes. Among the pathways involved in biological processes, the largest number of enriched genes was associated with the response to various self-processes (40), followed by transcriptional regulatory processes (23) and then metabolic processes (12). These differentially expressed genes screened by GO enrichment analysis are involved in biological processes such as larch metabolism and response to stimuli. Moreover, these genes may be closely involved in regulating the response of larch to external stimuli and larch growth processes.
KEGG functional annotation and enrichment analysis
The annotation results of the differentially expressed genes were classified according to type of KEGG pathway. The results showed that the differentially expressed genes were enriched in 20 metabolic pathways (Fig. 10). The metabolic pathways were divided into five pathway types, namely, organic systems, environmental information processing, metabolism and genetic information processing and cellular processes. Among them, the pathways enriched in metabolism were the most enriched, including 10 metabolic types, accounting for 50% of the whole enrichment pathway, these pathways mainly included those associated with carbohydrate synthesis, nucleotides, amino acid metabolism, lipid metabolism and energy metabolism. This shows that, on the basis of the premise of normal biological growth, different genes may participate in adaptations associated with plant growth and development via various metabolic pathways.
The genetic information processing pathway had the largest number of genes, including genes related to genetic information processes involving DNA transcription, protein translation, protein folding, classification and degradation. There are two types of environmental information processing pathways: signal transduction and membrane transport. The other two pathways have only one type, namely, environmental adaptation, transportation and catabolism. A variety of metabolic pathways involved in the growth, development and stress resistance of larch, and these metabolic pathways are coordinated by a variety of transcription factor-encoding genes. However, the specific cooperation needs to be further studied and verified.
Determination of physiological and biochemical indices under different treatments
Effects of different stress treatments on the POD activity of transgenic calli
POD activity reflects the ability of plants to scavenge H2O2 and other reactive oxygen species. POD is an enzyme closely related to energy and respiratory metabolism, and POD activity has effects of antioxidation and delaying aging . The higher the activity of POD is, the stronger the physiological metabolism and antioxidant capacity of plant tissue, which can accelerate the removal of active oxygen such as H2O2 and lead to better adaptability to adverse conditions.
It can be seen in Figure 11, Under PEG6000 simulated drought stress, the POD activity of wild-type calli was very weak with increased stress duration and was always lower than that of the transgenic cell line. The transgenic cell lines showed a trend of "first decreasing, then increasing and then decreasing again", and the change trend of the three transgenic cell lines was very consistent. When the calli were treated for 96 h, the values of OE2 and OE4 were the same as those of the wild-type calli, but the value of OE1 was higher than that of the wild-type and the other two transgenic cell lines. These results show that under drought stress, the POD enzyme activity of the transgenic cell line is stronger than that of the wild type, which means that POD can effectively remove the peroxide produced by stress, increase drought resistance.
Under NaCl stress, the POD activity of the wild-type calli was stable with increasing stress duration, but it was lower than that of transgenic cell line. The transgenic cell lines generally showed the trend of "increasing first and then decreasing", and the value was the highest at 48 hours of stress treatment. These results indicated that 48 hours of treatment is a node, and the expression trend of the three transgenic cell lines was very consistent, indicating that the transgenic cell lines have a certain degree of salt tolerance.
Effects of different stress treatments on the MDA content of transgenic calli
It can be seen in Figure 12, MDA is the main product of membrane lipid peroxidation in plants under stress , and its amount can indirectly reflect the degree of oxidative damage to plants caused by stress. The higher the MDA content is, the greater the degree of oxidative damage . At the same time, the protective enzyme system composed of SOD (superoxide dismutase) and POD can not only remove excess reactive oxygen species over time but also remove excessive amounts of MDA, which can reduce membrane lipid peroxidation and protect membrane structure .
Under PEG6000 simulated drought stress, the MDA content of the wild-type calli increased first and then decreased with the prolonging of stress and reached the maximum at 48 h. The transgenic cell lines also showed a similar trend, but the MDA content reached the highest after 48 hours of stress. Moreover, the MDA content of the transgenic cell lines was lower than that of the wild-type cell lines after 24 hours and 48 hours of stress, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was less than that of the wild-type cell lines.
Under NaCl stress, the MDA content of wild-type and transgenic cell lines decreased with the extension of stress time. In general, the MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines.
Effects of different stress treatments on the content of superoxide dismutase (SOD) in calli
It can be seen in Figure 13, As an important antioxidant enzyme, SOD catalyzes the disproportionation of superoxide anion radicals to produce hydrogen peroxide and oxygen to remove reactive oxygen species and ultimately improve the ability of plants to resist drought stress.
Under PEG6000 simulated drought stress, the SOD content of the wild-type calli showed a trend of "increasing first and then decreasing" with the extension of stress duration, peaking at 24 hours. The transgenic cell lines also expressed a similar trend, in which the SOD content of OE1 and OE2 reached the maximum after 48 hours of treatment, while that of OE4 reached the maximum after 24 hours of treatment. Nonetheless, both of these maximum contents were higher than the those of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced.
Under NaCl stress, the SOD content of wild-type cell lines decreased linearly after 24 hours of stress treatment and decreased continuously until 96 hours of treatment. The SOD content of the three transgenic cell lines was higher than that of the wild-type, and the SOD content of the three transgenic cell lines was relatively high, indicating that the transgenic cell lines had greater salt stress resistance than the wild-type cell lines did.