3.1 Transcriptome sequencing, assembly, and annotation
A total of 43,290,792,180 nt of data were generated by sequencing on the Illumina Hiseq2000 platform. A total of 92,790 Unigenes were obtained by the assembly, with a total length of 158,854,805nt, an average length of 1,712nt, and an N50 of 2,940nt. Annotating Unigene functions to NR, NT, Swiss-Prot, KEGG, COG, and GO libraries are 51,884, 9,200, 35,580, 38,453, 32,981, 21,772, and the Unigenes on all annotations are 52,679 individual. A total of 5,519 SSRs. The statistical results of data processing are shown in Table 2.
Table 2 The yield statistics of orthogonal transcriptome sequencing of U. compressa under temperature and light intensity stress | |
Samples | Total Raw reads | Total clean Reads | Total clean Nucleotides(nt) | Q20 percentage(%) | GC percentage(%) |
B1 | 57,959,152 | 53,893,110 | 4,850,379,900 | 97.84 | 58.11 |
B2 | 59,233,936 | 55,278,410 | 4,975,056,900 | 97.82 | 57.97 |
B3 | 58,968,570 | 55,062,386 | 4,955,614,740 | 97.91 | 57.76 |
B4 | 56,196,420 | 52,125,072 | 4,691,256,480 | 97.71 | 56.87 |
B5 | 55,631,066 | 51,649,190 | 4,648,427,100 | 97.70 | 56.71 |
B6 | 57,876,244 | 54,744,178 | 4,926,976,020 | 98.31 | 58.11 |
B7 | 56,074,990 | 52,650,014 | 4,738,501,260 | 97.95 | 57.46 |
B8 | 58,358,300 | 54,159,594 | 4,874,363,460 | 98.20 | 57.60 |
B9 | 55,563,802 | 51,446,848 | 4,630,216,320 | 98.17 | 57.98 |
Note: 1. Total Reads and Total Nucleotides represent the number of filtered reads and bases; |
-
Q20 percentage indicates the proportion of bases with quality of not less than 20 after filtering;
-
GC percentage indicates the ratio of bases G and C to the total number of bases after filtering.
-
Total Clean Nucleotides = Total Clean Reads1ⅹRead1 size + Total Clean Reads2ⅹRead2 size
3.2 Transcriptome expression analysis
Through the analysis of the expression of the transcriptome of U. compressa, it was found that some of the unigene genes were not expressed under B5 (normal conditions), but were only expressed under special conditions, which were called condition-specific expression genes. In previous studies, the analysis of differential genes was mostly performed, that is, under normal conditions, the expression of genes of key enzymes of the organism was up-regulated or down-regulated in the treatment group, to find out that there may be emerging biological processes. In previous studies, through transcriptome sequencing analysis, the up-regulation or down-regulation of carotenoid-related genes in U. prolifera, it was found that high temperature and low temperature can activate the synthesis of carotenoids, which provides a reference for the analysis of other metabolic pathways(Yuan, et al. 2018); the up-regulation and down-regulation of key enzyme genes in photosynthesis of U. line, and found the significance of C4 pathway in CO2 assimilation(JianFang, et al. 2013); the up-regulation of genes in photosynthesis and glycolysis in U. compressa. It was concluded that it may provide sufficient energy for long-term low-salt tolerance(Xing, et al. 2021). In the analysis of transcriptome data in this paper, an interesting phenomenon was found. Among the many condition-specifically expressed genes, the expression levels of some genes showed regular changes with temperature or light intensity. Through further functional analysis of these condition-specific genes, it is possible to infer the biological processes that are taking place in each group, which provides direction for understanding the molecular tolerance mechanism of U. compressa under extreme conditions.
3.2.1 Genes that change with light intensity under extreme temperature conditions
Such condition-specific expression genes can be expressed under extreme light intensity conditions, and the expression level is always the largest in B1, the smallest in B3, and B7 > B9. The genes with known functions are shown in Table 3. These genes are shown in Table 3. The functions expressed are mostly anti-stress and signal transduction. For example, unigene17011_All is heat shock 70 kDa protein 6 (chloroplastic), unigene27131_All has the function of an uncharacterized protein, and unigene34085_All is annotated as profilin-1, which can bind to actin, and it can be determined by the concentration of high and low. Affects the structure of the cytoskeleton. unigene30740_All has the function of ran-binding protein 1 homolog a, and unigene23679_All and unigene33974_All function as formin-like protein 14 and formin-like protein 3, which can regulate cell activity. Others include short-chain dehydrogenase TIC 32 (chloroplastic), chlorophyll ab binding protein L1818 (chloroplastic), 3-isopropyl malate dehydratase, serine/threonine-protein kinase SAPK10, 26S proteasome regulatory subunit 4 homolog B, etc. The specific expression of these genes, by affecting the cytoskeleton and regulating activity, plays an important role in the growth process of U. compressa, enabling it to temporarily adapt to extreme conditions to survive for some time. This is also consistent with the mechanism of the adaptation to salinity stress in the related literature(Xing, et al. 2021), but there is no report on condition-specific genes for its tolerance.
Table 3
Genes that changed with light intensity under extreme temperature conditions
gene | function | annotation number | Swissprot-E-value |
unigene5844_All | wd repeat-containing protein LWD2 | sp|Q38960|LWD2_ARATH | 1.00E-18 |
unigene34958_All | 4.00E-11 |
unigene20376_All | vegetative cell wall protein gp1 | sp|Q9FPQ6|GP1_CHLRE | 1.00E-16 |
unigene20405_All | 2.00E-06 |
unigene9924_All | 3.00E-29 |
unigene13685_All | 2.00E-19 |
unigene33653_All | 9.00E-14 |
unigene27164_All | 4.00E-12 |
unigene17011_All | heat shock 70 kDa protein 6, chloroplastic | sp|Q9STW6|HSP7F_ARATH | 3.00E-82 |
unigene30541_All | chlorophyll a-b binding protein L1818, chloroplastic | sp|Q03965|L181_CHLMO | 3.00E-51 |
unigene225_All | 1.00E-49 |
unigene27131_All | uncharacterized protein | sp|P0C8Z0|Y8359_ORYSI | 1.00E-06 |
unigene6266_All | 40S ribosomal protein S12 | sp|Q9XHS0|RS12_HORVU | 1.00E-33 |
unigene34085_All | profilin-1 | sp|P49231|PROF1_PHAVU | 8.00E-35 |
unigene30740_All | ran-binding protein 1 homolog a | sp|Q9LMK7|RBP1A_ARATH | 8.00E-40 |
unigene23723_All | dynein 8 kDa light chain, flagellar outer arm | sp|Q39580|DYL1_CHLRE | 5.00E-42 |
unigene13240_All | 20 kDa chaperonin, chloroplastic | sp|O65282|CH10C_ARATH | 1.00E-41 |
unigene27075_All | peptide methionine sulfoxide reductase B2, chloroplastic | sp|Q9C5C8|MSRB2_ARATH | 2.00E-39 |
unigene13846_All | small ubiquitin-related modifier 1 | sp|P55857|SUMO1_ORYSJ | 2.00E-17 |
unigene34046_All | branched-chain-amino-acid aminotransferase 5, chloroplastic | sp|Q9FYA6|BCAT5_ARATH | 4.00E-86 |
unigene27210_All | glutathione S-transferase Z2 | sp|Q9ZVQ4|GSTZ2_ARATH | 1.00E-12 |
unigene23679_All | formin-like protein 14 | sp|Q9C6S1|FH14_ARATH | 4.00E-10 |
unigene10207_All | short-chain dehydrogenase TIC 32, chloroplastic | sp|A2RVM0|TIC32_ARATH | 3.00E-49 |
unigene20031_All | 3-isopropylmalate dehydratase | sp|Q94AR8|LEUC_ARATH | 0 |
unigene30563_All | serine/threonine-protein kinase SAPK10 | sp|Q75H77|SAPKA_ORYSJ | 2.00E-50 |
unigene20380_All | 26S proteasome regulatory subunit 4 homolog B | sp|Q9SL67|PRS4B_ARATH | 0 |
unigene33974_All | formin-like protein 3 | sp|Q7G6K7|FH3_ORYSJ | 5.00E-08 |
Among the genes that change with light intensity under extreme temperature conditions, there are more genes related to the function of the Vegetative cell wall protein gp1, and there are 6 genes. Further analysis of their homology found that the consistency was 30.71% in DNAMAN, and the homology was 30.71%. The sex is not high. unigene5844_All and unigene34958_All express the same function: wd repeat-containing protein LWD2. The homology comparison found that the consistency is 50.95%. These genes do not belong to one class, and may have formed different Categories; unigene30541_All and unigene225_All are annotated as chlorophyll ab binding protein L1818 (chloroplastic), and the homology comparison found that the consistency is 84.65%, the homology is high, and it may be a class of genes.
In addition to the above genes, there are still some genes with unknown functions that enable the growth of E. prolifera under extreme conditions, including unigene30667_All, unigene27474_All, unigene26919_All, unigene10018_All, unigene23770_All, unigene34014_All, unigene13559_All, etc.
The expression levels of these genes under high light intensity are higher than those under low light intensity, which may be because the stress of high light intensity is more extreme for U. compressa, which leads to the need for more gene expression levels to adapt to high light intensity stress, but it still needs to be further proved by follow-up experiments.
3.2.2 Genes only expressed at high temperature
These genes were only expressed at high temperatures, that is, the samples were only expressed in B1, B2, and B3. Most of these genes are related to stress resistance and signal transduction, and a few are related to cell division and cell components. It can be seen that under high-temperature conditions, to maintain the growth of U. compressa level. The genes with known functions are shown in Table 4. Among them, the genes such as unigene35167_All and unigene36939_All are respectively formin-like protein 5 and formin-like protein 20 according to the annotations, and they all have the function of regulating cell activity; unigene26553_All, unigene28750_All, unigene46434_All, unigene9437_All, etc. All genes have the function of vegetative cell wall protein gp1, thus constituting cell components. According to the homology comparison, the consistency of these genes is only 22.96%, and the homology is not high.
There are also some genes directly related to resistance to external adverse environments. Among them, unigene17608_All has the function of lectin OS = Ulva pertusa according to the annotation. The activity of the protein was not affected within 30min at 30–70℃, which could protect U. compressa from temporarily adapting to high-temperature environment; unigene19720_All and unigene19717_All both have the function of proline-rich receptor-like protein kinase PERK2, which acts as a protein kinase to temporarily resist the influence of certain adverse environments. These genes all have different expression levels under high temperatures, and they all help U. compressa to temporarily adapt to the high-temperature environment from different angles. This may be a special gene formed in the long evolutionary process, but in terms of growth rate, the growth rate of U. compressa was much lower than that of other U. prolifera at high temperatures(Jianjun, et al. 2015). It is speculated that the condition-specific gene expression mechanism of U. compressa may be different from the differential gene mechanism of other U. prolifera, resulting in a slow growth rate. Therefore, U. compressa mostly appeared in the early green tide of the Yellow Sea, and then with the increase of temperature, resulting in a slow growth rate. U. compressa gradually lost its status of the dominant species(Wang, et al. 2018).
Table 4
Genes that were only expressed at high temperatures
gene | function | annotation number | Swissprot-E-value |
unigene26553_All | vegetative cell wall protein gp1 | sp|Q9FPQ6|GP1_CHLRE | 5.00E-20 |
unigene28750_All | 5.00E-07 |
unigene46434_All | 1.00E-14 |
unigene9437_All | 4.00E-22 |
unigene35167_All | formin-like protein 5 | sp|Q84ZL0|FH5_ORYSJ | 4.00E-10 |
unigene36939_All | formin-like protein 20 | sp|Q9FLQ7|FH20_ARATH | 2.00E-08 |
unigene17608_All | lectin OS = Ulva pertusa | sp|Q6T6H8|LEC_ULVPE | 6.00E-15 |
unigene19720_All | proline-rich receptor-like protein kinase PERK2 | sp|Q9LK03|PERK2_ARATH | 2.00E-07 |
unigene19717_All | 5.00E-07 |
3.2.3 Genes that change with temperature under constant light intensity
The expression level of such genes is not expressed in samples B4 and B5 but is expressed in B1, B2, B3, B6, B7, B8, and B9. When the light intensity is constant, the expression level gradually decreases with the decrease of temperature, that is, Under the same light intensity, to adapt to the change of temperature, this kind of specific genes of U. compressa began to express and B1 > B7, B2 > B8, B3 > B6 > B9. Genes with known functions are shown in Table 5. Such genes are mainly related to cellular components and signal transduction, including unigene27211_All, unigene23849_All, unigene23949_All, unigene6286_All, unigene888_All, unigene26935_All, unigene6099_All, unigene17243_All and unigene229_All. The functions of these genes are related to vegetative cell wall protein gp1, which is involved in the formation of the cell wall. According to the homology comparison, it is found that the consistency is 34.39%, and the homology is not high.
At the same time, the unigene30603_All and unigene10500_All genes are xyluloses 5-phosphate/phosphate translocator, chloroplastic and secretory carrier-associated membrane protein 1 respectively according to their annotation functions, and both have the role of transport.
U. compressa can grow at low temperatures, and the growth rate is higher than other U. prolifera, but the growth rate is lower than other U. prolifera at high temperatures. Therefore, the high expression level of these genes may inhibit the growth of U. compressa, which makes U. compressa withdraw from the early stage of the green tide.
Table 5
Genes that changed with temperature under constant light intensity
gene | function | annotation number | Swissprot-E-value |
unigene27211_All | vegetative cell wall protein gp1 | sp|Q9FPQ6|GP1_CHLRE | 1.00E-10 |
unigene23849_All | 4.00E-20 |
unigene23949_All | 2.00E-12 |
unigene6286_All | 7.00E-06 |
unigene888_All | 3.00E-10 |
unigene26935_All | 4.00E-15 |
unigene6099_All | 1.00E-07 |
unigene17243_All | 2.00E-09 |
unigene229_All | 2.00E-24 |
unigene30603_All | xylulose 5-phosphate/phosphate translocator, chloroplastic | sp|Q9LF61|XPT_ARATH | 1.00E-83 |
unigene10500_All | secretory carrier-associated membrane protein 1 | sp|Q8H5X5|SCAM1_ORYSJ | 7.00E-39 |