Research Of Differential Gene Expression In The Developing Seed Of Brassica Napus Under High Temperature Stress

Brassica napus L.is one of the main oil crops in our country,even to the world.With the development of global warming trend,high temperature forced maturation which occurred during the ripening of B.napus greatly affects the yield and quality of rapeseed.This study focus on the high temperature forced maturation of B.napus using differential display to figure out the differentially expressed gene profile.Twenty one inbred lines were planted in two same automatic controlled climate chambers with two repeats,40 plants each.The light,humidity,water and other growth conditions of two climate chambers were controlled the same except the temperature.The temperature treatment was taken 20 days after flowering in one climate chamber,with 35～40℃from 9:00 am to 16:00 pm,while the another climate chamber as control with temperature kept at 25～30℃.Pods were collected 30 days after flowering to sample the young seeds for differential display. For each lines,kilo-grain weight,oil content,protein content,soluble sugar content were measured after ripen to screen the temperature sensitive and non-sensitive genotypes.Finally,we got high temperature sensitive lines R54,R64 and R65,high temperature non- sensitive lines R51,R60,R67 and R69.Young seeds from one temperature sensitive line(R54) and one non-sensitive line(R60) were sampled for differential display.After RNA extraction,reverse transcription into cDNA then digested with two types of restriction endonuclease- Ase I and Taq I.Totally,256 pairs of primer combination were screened in the selective amplification after adaptor ligation and preamplification, and 5 groups of differentially expressed fragments reserved steadily.Sequences were BLAST in NCBI,and 2 groups of the fragments resembled highly homologous to Arabidopsis thaliana protease-associated(PA) domain-containing protein(AT1G63690),and B.napus mitochondrial DNA Separately.These fragments may be a reflection of differentially expression of B.napus.under high-temperature-stress.Another fragment homologous to the sequence of B.rapa,but no function reported for this sequence.RT-PCR was carried out with the same materials in 2008,which confirmed that these fragments do have a relatively reduced expression under high temperature stress.The research of HSP70(heat shock protein 70) shows that it plays an important role in mitochondria.Mitochondrial is the most important energy metabolism in vivo biological cells.We deduced that the fragment obtained in this experiment which has the homologous sequence with B. napus,mitochondrial genome might be specially expressed under high temperature stress, cooperatively synthesis of heat shock proteins and regulates the response of high temperature stress. This may be related to the energy metabolism in plants under high temperature stress.Another group of fragments which have the homologous sequence with a certain protease of the A.thaliana was also involved in intracellular protein metabolism.Although it is difficult to explain the differential expression under high temperature stress only by the fragments which were obtained from this experiment,and further experiments were needed to explain the detailed reaction mechanism and process caused by high temperature stress.However,the sequences information obtained in this experiment have an important role in elucidating the mechanism of differential expression under high temperature stress in the future.It makes great sense to further study of the mechanism of differential display under high temperature stress of B.napus.as well as an important guiding role in high temperature resistant variety breeding program.