| Wedelia trilobata was listed as one of the world’s most harmful 100 species of alien invasive by the World Conservation Union(IUCN).Our previous study showed that W.trilobata has a stronger heat resistance to high temperature than Wedelia chinensis,but the molecular mechanism was not clear.The leaf transcriptome of W.trilobata and W.chinensis at 40 ℃ and 28 ℃ for 20 h was sequenced using an Illumina HiSeqTM 2500 platform.Compared with the differences of the two species in gene expression under the high temperature stress,seaked related high temperature resistant function genes of W.trilobata and predicted the genes’ function and metabolic pathway.The 4 kinds of genes’ expression,which encoding different functional protein in high temperature stress was verified using real-time PCR.The main conclusions are as follows:1.A total of 28.58 Gb clean data was received and all samples’ clean data was reached 6.52 Gb.A total of 492,983 transcripts were obtained by using Trinity.A total of 88,504 unigenes with a mean length of 1,488 bp was obtained by further sequence splicing.57,851 unignes(65.4%)were annotated using BLASTX searches against the CGO,GO,KEGG,KOG,Pfam,Swisspriot and nr databases.2.RPKM standardization processing method was used to identify the results before and after high temperature stress of the two species,W.trilobata and W.chinensis had 5,566 and 4,429genes’ expression changed significantly.1,837 genes of W.chinensis and 2,413 genes of W.trilobata were broadly divided into 3 categories(biological processes,cellular components and molecular function)using GO.The classification of the two species was similar.In the biological process,the enrichment function involved the metabolic process(1,675 genes of W.trilobata and 1,311 genes of W.chinensis),the cell process(1,321 and 1,006)and the single cell organism process(1,164 and 868).In the classification of cell groups,the 2 branches of abundant genes are cell fraction(1,029 and 863),membrane structure(806 and 682)and so on.In the molecular function category,the enrichment function mostly is the catalytic activity(1,231 and 925),binding function(1,121 and 840)and so on.The result showed that the amount genes of the two species under high temperature stress involved in the regulation of metabolism and apoptosis,proliferation and differentiation,oxidoreductase reaction,enzymatic hydrolysis,molecule binding,transporters,the structure of cell membrane and the process of membrane binding organelle.The results also showed that there are a large number of genes co-expression of the two species,including 268 co-up-regulated genes and 240 co-down-regulated genes.3.Using KEGG channel noted the two kinds of Wedelia founded that the process about endoplasmic reticulum pathway(103 and 76),ribosome synthesis(38 and 96),carbon metabolism(49 and 51),photosynthesis(30 and 36),amino acid biosynthesis(40 and 52),plant hormone signal transduction(39 and 34)all have different degrees of up and down,indicating that these genes play an important role in the regulation of Wedelia under high temperature stress.4.Using real-time PCR to analyzed the 4 genes’ expression which related heat resistance,including CL9177Contig1(HSP20-like chaperones superfamily protein),Group2_Unigene_BMK.52427(Hsp20/alpha crystal protein family),Group1_Unigene_BMK.20387(Rubisco)and CL19212Contig1(S-Adenosylmethionine dependent Methyltransferases)high temperature stress for three time periods(3 h,12 h,20 h)to validate the gene expression level.The results showed that the CL9177Contig1 superfamily protein of W.trilobata and W.chinensis reached the highest at 3 h(15.56)and 12 h(4.9),Group2_Unigene_BMK.52427 r-eached the highest at 20 h(7.64)and 12 h(5.96).Group1_Unigene_BMK.20387 r-eached the highest at 20 h(5.52)and 12 h(3.43).CL19212Contig1 reached the highest at 3 h(6.35)and 12 h(4.28).These 4 genes’ expression of W.trilobata were higher than W.chinensis.It shows that W.trilobata has better heat resistance. |
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