Preliminary Study On Molecular Mechanism Of Brassica Napus Seed Thermotolerance Based On Transcriptome Sequencing

The research on thermotolerance of Brassica napus seeds is helpful to explore new heat tolerance genes,which can improve the thermotolerance and multiple stress tolerance of crops in complex adversity,and lay the foundation for perfecting the cross stress regulation network of Brassica napus.In this study,14 Brassica napus cultivars were treated at 100℃ to screen out seed thermotolerant and seed thermosensitive strains.By means of activity detection,ultrastructural observation and transcriptome sequencing,the seed thermotolerant of Brassica napus seeds was evaluated and its molecular mechanism was preliminarily studied.The main results are as below:(1)Evaluation of thermotolerance of Brassica napus seeds.Fourteen Brassica napus strains were treated at 100℃ for 0,4,8 and 12 hours,and the germination percentage was calculated after 7 days germination.The results indicated that:germination percentage differences between different strains and heat treatment time was significant,different strains seed germination percentage gradually decreased with high temperature processing time extended.The seed thermotolerance of 6-1 was significantly higher than other strains,the average germination percentage of 6-1 was above 99% after 12 hours heat treatment.6-14 has worst seed thermotolerance,the average germination percentage fell to 0.00% when treated for only 4 hours.Then,four strains with the best thermotolerance were treated at 100℃ for 12,16,20 and 24 hours.The results indicated that 6-1 was the most thermotolerant strain,and the germination percentage was still 45.00 ± 2.00% after 24 hours of heat treatment.(2)Identification of seed vigor of B.napus before and after heat treatment.TTC staining was carried out on the highly seed thermotolerant material 6-1 and thermosensitive material 6-14,which were treated at 100℃ for 8 hours.The results indicated that both the embryo and endosperm of 6-1 seeds could be dyed dark red after heat treatment,manifesting that the seed vigor was normal.After heat treatment,the embryos and endosperm of seeds 6-14 could not be dyed dark red,indicating the loss of seed vigor.(3)Observation on seed ultrastructure of Brassica napus before and after heat treatment.Transmission electron microscopy(TEM)was performed to survey untreated 6-1 and 6-14 seeds and seeds treated at 100℃ for 8 hours.The results indicated that the structure of rapeseed seeds was intact and the ultrastructure was orderly arranged before heat treatment.After heat treatment,the arrangement of oil body and protein body of 6-1 seeds lost order,and oil body interdissolved in a small range,while some cell contents of 6-14 seeds interdissolved and tore and formed cavities,resulting in decreased oil body density and great damage to cell structure.(4)Screening of seed thermotolerant genes in Brassica napus based on transcriptome sequencing.The seeds of 6-1 and 6-14 were treated at 100℃ for 8hours and sampled after 48 hours germination for transcriptome sequencing.3036 differentially expressed genes(DEGs)were screened as seed thermotolerant strain 6-1after heat shock,of which 934 genes were up-regulated and 2102 genes were down-regulated after heat shock.These DEGs were mainly enriched in glutathione metabolism,phenylpropanoid biosynthesis and α-linolenic acid metabolism.A total of 2734 heat shock response genes were screened as seed thermotolerant and seed thermosensitive strains shared DEGs,1039 of which were up-regulated and 1695down-regulated after heat shock in group T.In group S,1157 genes were up-regulated and 1577 were down-regulated after heat shock,and the DEGs were mainly enriched in amino acid metabolism,nitrogen metabolism and α-linolenic acid metabolism.Alanine,aspartate and glutamate metabolism,nitrogen metabolism,phenylpropanoid biosynthesis and other metabolic pathways were involved in heat shock response of Brassica napus.Glutathione metabolism,phenylpropanoid biosynthesis,α-linolenic acid and other pathways may be related to the formation of heat-tolerance trait in seed thermotolerant Brassica napus(T).