【作者】 陈静；

【导师】 万建民； 翟虎渠；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2015， 博士

【摘要】 花生是我国重要的油料作物和经济作物,单产、面积、出口均居世界前列。近年来我国花生在收获时期多逢阴雨天气,花生在植株上发芽现象严重,严重影响花生的产量、品质和种用质量,其主要根源是目前选育推广的花生新品种休眠性较弱。种子休眠性愈来愈成为花生重要的农艺性状,本研究筛选到强休眠的花生品种,并探讨了影响花生休眠性的主要因素,通过转录组学方法挖掘花生种子休眠解除过程以及萌发相关的候选基因,以阐明花生种子休眠向萌发转换的分子机制,研究结果为后续开展休眠功能基因研究、分子育种实践、培育适度休眠性的花生品种提供有价值信息。主要研究结果如下:1.不同花生品种(系)种子休眠性有差异,干种子发芽率变幅为0%～100%;强休眠品种花育52号鲜种子发芽率和干种子发芽率均为0%。内源激素分析表明,高水平内源ABA含量、低水平GA含量以及低GA/ABA比值对维持花生吸胀种子的休眠状态有积极作用。休眠态种子与乙烯利解除休眠种子相比较,乙烯利解除休眠种子的内源GA含量迅速上升,ABA含量下降,GA/ABA比值迅速上升。2.利用RNA-seq技术对花生吸胀休眠态种子B02(CK)、乙烯利处理吸胀休眠态种子2.5h AE1、种子露白AE2、种子萌发AE3四个不同时期的样品进行转录组测序,得到374006个长度大于200 bp的转录本,大小471Mb;得到121412个Unigenes,大小94Mb。B02(CK),AE1,AE2,AE3四个文库中分别获得102845,104546,1111535和110489 Unigenes。通过与B02(CK)相比较(Fold Change≥2 and Pvalue≤0.05),AE1中有1555个unigenes上调表达,472unigenes下调表达;AE2中有4586个unigenes上调表达,2335unigenes下调表达;AE3中有4192个unigenes上调表达,2967 unigenes下调表达。筛选到四个样品中的共表达差异unigenes为1206个,通过聚类分析将差异表达unigenes聚类分成9组。其中赤霉素20氧化酶、脱落酸8’羟化酶、EREBP-like因子、ACC氧化酶、热激蛋白等综合调控花生种子休眠解除以及萌发。通过荧光定量进行了验证,证实转录组分析可靠。3.通过转录组测序获得生长素抑制蛋白基因(AhARP),该基因全长为918 bp,基因开放阅读框为363 bp,编码121个氨基酸,推导出蛋白质分子量为13.44 kD,理论等电点pI9.64。序列比对表明该序列与花生生长素抑制蛋白基因(AAZ20292.1)一致。荧光定量PCR结果表明,AhARP基因在种子休眠阶段表达量较高,种子休眠解除后表达量降低;乙烯利解除吸胀种子休眠过程中,AhARP基因受到明显诱导;说明AhARP基因可能与花生种子休眠有关。4.基于转录组分析结果,检测到15个与GA、40个与ABA、60个与ETH、56个与auxin相关的unigenes在花生种子休眠解除过程中表现显著差异表达。荧光定量PCR结果显示,ABA合成关键基因AhNCED2和代谢关键基因AhCYP707A1在种子休眠解除过程中均受外源乙烯利诱导,表达差异显著;在休眠和无休眠种子吸胀萌发过程中,AhNCED2和AhCYP707A1的表达趋势不同,AhNCED2对于种子休眠维持发挥积极作用,而AhCYP707A1对于种子休眠解除发挥积极作用。获得了乙烯合成途径中的关键基因AhACO1,GeneBank中登录号为KP298003。荧光定量PCR结果表明AhACO1基因受到乙烯利诱导表达;休眠种子吸胀过程中AhACO1下调表达,无休眠种子吸胀萌发过程中AhACO1上调表达,推测AhACO1可能与花生种子萌发密切相关。5.获得与花生种子萌发相关的基因AhSGR,该基因编码未知蛋白,在44-122AA处含有DOG1保守结构域,分子量33.39kD,等电点为5.04,定位于细胞核内,未发现信号肽及其剪切位点,推测AhSGR可能是转录因子。荧光定量检测表明该基因与花生种子萌发密切相关。更多还原

【Abstract】 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.更多还原