Analysis On Transcriptome Of Genes Involved In Peanut Seed Dormancy Release (Arachis Hypogaea L.)

Peanut(Arachis hypogaea L.)is an important oil crop and economic crop in China,yield,area and exports of which come out top in the world.Because it rain during peanut harvested period,it was found that peanut seeds in plants happened germination.This can damage end-use quality such peant yield,quality and seed quality.Seed dormancy is an important agronomic trait as it relates to peanut germination in plants.Most of peanut varieties widely cultivated have weaker seed dormancy,therefore,breeding for an intermediate level of dormancy in peanut is highly desirable.In this study,we screened peanut variety with strong dormancy and analysed the main factors affecting seed dormancy.To understand the molecular mechanisms of switches from dormancy to germination in peanut seeds underlying the role of ethephon,we preformed transcriptome analyses among imbibed dormant seeds as control and dormancy-released seeds treated by exogenous ethephon,and compared the expression of unigenes related to hormone including ABA,GA,ETH and auxin.These results would provide valuable reference for the following research about analysing gene function and breeding new peanut varieties with moderate dormancy.The results are followed:1.The seed dormancy of different varieties was different and the germination rate of dry seed harvested after mature from 16 peanut varieties varied from 0%to 100%.Huayu 52 has a strong seed dormancy.Endogenous hormone analysis showed that the high ABA content,low GA content and low GA/ABA ratio played the positive role to maintain seed dormancy.In contrast to dormant seed,endogenous GA content in dormant Seed treated by ethephon rose rapidly,the content of ABA decreased,and the ratio of GA/ABA increased rapidly.2.To profile peanut seed transcriptome,mRNA from seeds of cultivar Huayu52 different samples(B02,AE1,AE2 and AE3)were used to construct four cDNA libraies.102845,104546,111535 and 110489 Unigenes were detected in B02,AE1,AE2,AE3 four libraries,respectively.Different expression Unigenes of different samples were selected by Fold Change>2 and Pvalue<0.05.Compared between B02 and AE1,we found that 2027 Unigenes were differentially expressed,with more up-regulated Unigenes(1555)than down-regulated ones(472)Unigenes.And 6921 Unigenes were differentially expressed between B02 and AE2 samples with more up-regulated Unigenes(4586)than down-regulated ones(2335).The expression of 4192 genes was up-regulated while 2967 genes were down-regulated in AE3 to compare with B02.We performed hierarchical clustering of the DEGs using the Euclidean distance method associated with complete-linkage,hoping to further illustrate the relationships between DEGs with various expression patterns.We defined 9 clusters according to the cluster results.Expecially,genes encoding gibberellin 20 oxidase,abscisic acid 8’hydroxylase,EREBP-like factor,heat shock protein,aminocyclopropanecarboxylate oxidase(ACO)were significantly changed during the process from dormancy to germination in peanut seeds.3.Gene full-length of auxin-reprssed protein gene was screened from seed of peanut cultivar Huayu52 by RNA-seq,and designated as AhARP in this article.The whole sequence of AhARP was 918 bp and its open reading frame was 363 bp,encoding a 121 amino acid residuces.The deduced protein molecular weight was 13.44 kD and its theoretical isoelectric point was 9.64.Sequenc alignment of the deduced amino acids of AhARP revealed high degree of similarity with auxin-reprssed protein gene(AAZ20292.1)of peanut.The results of Real-time RT-PCR showed that AhARP gene was at a higher level in dormant variety than in non-dormant variety;The expression of AhARP was induced distinctly in dormancy-released seed by ethephon.It indicated that AhARP may be correlated to the seed dormancy of peanut.4.Based on transcriptomic analyses,we compared the expression of unigenes related to ABA,GA,ETH and auxin.The results showed that there were 15,40,60,and 56 unigenes associated with GA,ABA,ETH,and auxin,respectively,which were significanted differentially expressed unigenes during the process from dormancy to germination.The results of Real-time RT-PCR showed that the expressions of AhNCED2 and AhCYP707A1 were induced distinctly by exogenous ethephon in seed dormancy released process.In dormant and non-dormant seed imbibition and germination processes,there were different roles between expresses of AhNCED2 and AhCYP707A1.AhNCED2 played a positive role in maintaining seed dormancy,while AhCYP707A1 played a positive role for seed dormancy breaking.Based on transcriptome analysis of peanut seed,1-amino 1-amiocyclopropane-l-carboxylic acid oxidase(ACO)gene was obtained.The accession number was KP298003 in GeneBank.The results of Real-time RT-PCR showed that the expression of AhACO1 was induced distinctly by exogenous ethephon.During the process of seed imbibitions,AhACO1 expression was down-regulation in dormant seed,while was up-regulation in non-dormant seed,suggested that AhACO1 may have close relationship with peanut seed germination.5.The full-length cDNA sequence of a gene related to seed germination(AhSGR)was obtained,which encoded unknown protein.The protein had DOG1 conserved domain in 44-122AA,a molecular weight of 33.39 kD,and pI of 5.04,which was located in nucleus.It had no signal peptide and splice site.According to allignment similarity by Blastp,We speculated that AhSGR may be a transcription factor.The results of real-time quantitative PCR showed that the expression of AhSGR gene was closely correlated to the seed germination.