| Scytosiphon lomentaria is an economic brown macroalgae with high edible and medicinal value in China.In this paper,the effects of different temperatures on the growth and development of filaments of S.lomentaria were studied at the level of cytology and molecular biology.The morphological and structural changes of filaments,seedling emergence rate and related indicators of seedling growth were observed,and the expression of genes related to the growth and development of filaments and transcriptome analysis were carried out,providing a theoretical basis for the temperature regulation of artificial seedling raising of S.lomentaria.The experimental material used in this study were the unisexual filaments of S.lomentaria.The temperature gradients of growth and development and the expression of rbcL gene,psbA gene and rps9 gene were set at 6℃,10℃,12℃,14℃,16℃,20℃,and the experimental period was 30 days,and it was measured once every 5 days.The temperature gradient of transcriptome study was set at 5℃,10℃,15℃,20℃ and 25℃,and the experimental period was 30 days.At the end of the experiment,transcriptome sequencing and analysis were carried out.The main experimental results were reported as follows:(1)Effects of temperature on the growth and development of filaments of S.lomentaria:At 6℃,the filaments grew slowly and sprouted slowly.Seedlings began to emerge after 10 days of culture.After 30 days of culture,the emergence rate reached 95.9%,the seedling development was normal,and the length of seedlings and filaments reached 377 μm.and 292 μm,respectively.At 10℃,the seedlings emerged after 10 days of culture,and the emergence rate reached 100% after 25 days of culture.After 30 days of culture,the morphological development of seedlings were basically normal,and the length of seedlings and filaments reache 6 552 μm and 551 μm,respectively.At 12℃,14℃ and 16℃,seedlings emerged after 5 days of culture.After 25 days of culture,the emergence rate reached 100%,and the morphological development of seedlings began to be abnormal.After 30 days of culture,the maximum length of seedlings reached 1 403 μm at 12℃.The filament length reached a maximum of 615 μm at 16℃.At 20℃,the seedlings formed late and emerged after 20 days of culture.After 30 days of culture,the emergence rate reached 23.4%.The seedling morphology was normal and the seedling length reached 1 199 μm.The length of filaments reached 687 μm.The results showed that the water temperature should be reduced by 2 ℃ every 5 days from 16℃ to 6～10℃ and then continued to cultivate until the seedlings were transplanted to the sea.(2)Effect of temperature on the expression of genes related to the growth and development of filaments of S.lomentaria:At 6℃,the relative expression of rbcL gene reached the peak after 10 days of culture,psbA gene reached the peak after 5 days of culture,and rps9 gene reached the peak after 30 days of culture.After 10 days of culture at 10℃,12℃ and 16℃,the relative expression of rbcL gene,psbA gene and rps9 gene reached the peak;At 14℃,the relative expression of psbA gene and rps9 gene reached the peak after 10 days of culture,and the relative expression of rbcL gene reached the peak after 15 days of culture.After 10 days of culture at10℃,12℃ and 16℃,the relative expression of rbcL gene,psbA gene and rps9 gene reached the peak;At 14℃,the relative expression of psbA gene and rps9 gene reached the peak after 10 days of culture,and the relative expression of rbcL gene reached the peak after15 days of culture.The relative expression of rbcL gene reached the peak at 20℃ for 10 days,and the relative expression of psbA gene and rps9 gene reached the peak at 15 days.The average relative expressions of rbcL gene,psbA gene and rps9 gene in 30 days were the highest at 20℃,the lowest at 14℃,the next low at 6℃,and the medium level at 12℃,16℃and 10℃.The results showed that the expression of rbcL gene,psbA gene and rps9 gene reached or approached the peak value when filaments emerged in large numbers after 10-15days’ culture,indicating that these three chloroplast genes were actively involved in the process of seedling morphogenesis.(3)Effect of temperature on the transcriptome of S.lomentaria:A total of 189 520 unigenes were generated by de novo assembly of S.lomentaria transcriptome data.The base number of all assembled bases was 132 630 870 bp,the average length was 69 9.83 bp,N50 was 1 219 bp,and the average GC content was 63.96%.Compared with the 5℃ group,the maximum number of DEGs detected in the 15℃ group was 18 760,the next at 10℃ was 18 485,the third at 20℃ was 17 926,and the minimum number of DEGs detected at 25℃ was 10 414.In the range of 5～25℃,after the temperature increases gradually,the up-regulated DEGs gradually increased,reached the peak at 15℃,and the down-regulated DEGs gradually decreased with the increase of temperature,and reached the lowest at 25℃.The difference gene go enrichment analysis showed that compared with the 5℃ group,at 10℃ and 15℃,DEGs were mainly enriched in the metabolism of cytoplasm,cytoplasmic components,mitochondria and cell amides;at20℃,DEGs were mainly enriched in the metabolism of organic nitrogen compounds,biosynthesis of organic nitrogen compounds,metabolism of cell amides and ribonucleoprotein complexes;at 25 ℃,DEGs were mainly enriched in membranes,membrane components and inherent components of membranes transferase activity.KEGG enrichment analysis showed that compared with the 5℃ group,DEGs were mainly enriched in ribosomes,carbon metabolism,amino acid biosynthesis and oxidative phosphorylation at10℃;At 15℃,DEGs were mainly concentrated in ribosomes,carbon metabolism,oxidative phosphorylation and proteasome;At 20℃,DEGs were mainly concentrated in ribosomes,carbon metabolism,oxidative phosphorylation and RNA transport;At 25 ℃,DEGs were mainly concentrated in protein processing,ubiquitin mediated protein hydrolysis,RNA degradation and cell cycle.t can be seen that at 20 ℃ and below,DEGs are mainly concentrated in the process of synthesis and metabolism,indicating that seedlings or filaments are growing vigorously.At 25 ℃,DEGs are mainly concentrated in membrane components,protein processing,etc.,indicating that filaments are under heat stress.The up regulation of the expression of these genes is conducive to improving the stress resistance of filaments.The results showed that at 20℃ and below,DEGs were mainly concentrated in the process of synthesis and metabolism,indicating that seedlings or filaments were growing vigorously.At 25℃,DEGs were mainly concentrated in membrane components,protein processing,etc.,indicating that filaments were under heat stress.The up regulation of the expression of these genes was conducive to improving the stress resistance of the filaments. |
