基于转录组测序筛选新疆野苹果种子休眠解除过程中植物激素相关基因

Screening of plant hormone-associated genes during seed dormancy release in Malus sieversii based on transcriptome sequencing

【目的】研究新疆野苹果(Malus sieversii)种子不同贮藏阶段转录组差异和激素含量变化,筛选新疆野苹果种子休眠解除过程中植物激素相关基因,为后续新疆野苹果种子休眠解除激素调控机制研究提供依据。【方法】以新疆野苹果种子为材料,对照未层积种子和经4℃低温沙藏层积处理(30、60、90、120 d)后种子进行转录组测序,采用酶联免疫吸附法测定种子中的脱落酸(abscisic acid,ABA)、赤霉素(gibberellin,GA)、生长素(auxin,IAA)、细胞分裂素(cytokinin,CTK)含量以及乙烯氨基环丙烷羧酸氧化酶(1-aminocyclopropane-1-carboxylic acid oxidase,ACO)和氨基环丙烷羧酸合成酶(1-aminocyclopropane-1-carboxylic acid oxidase synthase,ACS)的活性。【结果】新疆野苹果种子GA、IAA、CTK含量随贮藏时间的增加呈上升趋势,而ABA含量呈下降趋势。乙烯(ethylene,ETH)合成途径ACO和ACS活性随贮藏时间的增加呈增强趋势。GO富集分析筛选出85个差异基因调控种子萌发(GO:0010029),114个GA相关差异基因,313个ABA相关差异基因和156个ETH相关差异基因。KEGG通路富集分析显示,主要富集通路有植物激素信号转导、MAPK信号通路-植物、内质网中的蛋白加工、淀粉和蔗糖代谢、糖酵解/糖原生成等。ABA信号传导通路中有3个PYR/PYL基因下调表达,2个蛋白磷酸酶2C(PP2C-type protein phosphatases,PP2C)、3个蔗糖非发酵相关的蛋白激酶(sucrose non-fermenting-1-related protein kinase 2,SnRK2)上调表达和2个ABA分解代谢8'-羟化酶(8'-hydroxylases)基因表达显著上调。GA信号转导途径中2个受体GID1(gibberellin insensitive dwarf 1)和6个负调控因子DELLA蛋白上调表达;ETH受体ETR(Ethylene receptor)的直接前体1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylic acid,ACC)和ACO基因上调表达。【结论】新疆野苹果种子经低温沙藏层积处理,ERF2-like表达量与ACO、ACS活性变化呈相反趋势,PYR1-like、WRKY33基因表达量与ABA含量变化均下降,说明以上基因可能参与ABA信号通路调控新疆野苹果种子休眠解除过程。

【Objective】The study aimed to study the transcriptome differences and hormone content changes of the seeds of Malus sieversii at different stages of stratification in order to screen the plant hormones genes related to dormancy release and provide a basis for subsequent studies on the hormonal regulation mechanism of seed dormancy release in M.sieversii.【Methods】The seeds of M.sieversii were used as materials,transcriptome sequencing was performed on the seeds at different stages of the stratification at 4℃(0,30,60,90 and 120 d).The content of abscisic acid(ABA),gibberellins(GA),auxin(IAA),cytokinin(CTK),the activities of ethylene 1-aminocyclopropane-1-carboxylate oxidase(ACO)and 1-aminocyclopropane-1-carboxylate oxidase synthase(ACS)were measured using an enzyme-linked immunosorbent assay.【Results】The ABA content of the seeds showed a decreasing trend with the increase of stratification time,the maximum content was 80.22 ng·g-1on 0 d of the storage,the content on 60 d of the storage was significantly lower than that at the three periods of 0,30 and 90 d(p<0.05),the content reached a minimum of 43.67 ng·g-1on 120 d.The GA content showed an increasing trend with the increase of the storage time,the content was the lowest at 78.42 pmol·g-1on 0 d,and the content on 120 d significantly higher than those of the other periods(p<0.05),and reached a maximum of 170.67 pmol·g-1.The IAA content showed an increasing trend with the storage time,and the content on 90 d was significantly higher than those of the other periods,reaching a maximum value of41.36 nmol·g-1.The CTK content on 90 d was significantly higher than those of the other periods,reaching a minimum value of 43.67 ng·g-1,it showed a decreasing trend in the storage time of 0-30 d,the content on 30 d was significantly lower than those of the other periods(p<0.05),reaching a minimum value of 31.34 ng·g-1,and showed an increasing trend in the storage time of 30-60 d,suggesting that CTK would promote the accumulation of seed assimilates in this period.The activities of ACC oxidase and ACC synthase in the ethylene biosynthesis pathway showed inconsistent trends during the storage.The activity of ACO was 262.52 ng·g-1on 90 d of the storage,which was significantly higher than that of other periods(p<0.05),and the activity of ACO was the lowest on 30 d of the storage,which was157.38 ng·g-1.The activity of ACS reached the maximum value of 418.92 ng·g-1on 120 d of the storage,which was significantly higher than that of the three periods of 0,30 and 60 d(p<0.05).It indicated that the stratification promoted the synthesis of GA,IAA,CTK,ACC oxidase and ACC synthase,and two enzymes of the ethylene synthesis pathway might be more sensitive to low temperature.There were more significant DEGs on M120d_CK120d compared with M0d_CK0d,M30d_CK30d,M60d_CK60d and M90d_CK90d,suggesting that there were more DEGs on M120d for the regulation of seed germination and physiological changes There were a total of 7384,4875 and 3236 significantly and differentially expressed genes,significantly and differentially up-regulated genes and significantly and differentially down-regulated genes on the M30d_CK30d and M60d_CK60d periods,respectively.The gene ontology enrichment of DEGs was performed,and the biological processes were mainly involved in the response to osmotic stress and water deprivation,abscisic acid response,response to salt stress,transcriptional regulation,regulation of seed germination,and gibberellin response.The cellular components were mainly chloroplast stroma,chloroplast envelope and thylakoid.The molecular functions were mainly related to DNA-binding transcription factor activity,phosphatase activity and protein homodimer activation.The multiple plant hormone biological processes remained active and changed during the course of stratification of the seeds,suggesting that they would play a role in seed dormancy release activities.The KEGG pathway enrichment analysis showed that the main pathways enriched in seeds at different periods of the stratification were plant hormone signaling,MAPK signaling pathwayplant,protein processing in endoplasmic reticulum,starch and sucrose metabolism,and glycolysis/glycogenesis.Among them,plant hormones would play a key role in regulating seed dormancy and germination in M.sieversii,and hormone signal transduction pathway-related genes such as ABA,GA,and ETH might be involved in the processes such as seed dormancy release.The starch and sucrose metabolic pathways were involved in the process of carbon metabolism in the seed embryo,providing carbon source for the seed embryo.The glycogen production metabolic pathway was involved in the synthesis and metabolism of cellular proteins,providing nitrogen source and energy for seed embryo germination.The ABA receptor PYR/PYL had 12 genes up-regulated and 3 genes down-regulated.The ABA signal transducer protein phosphatases 2C(PP2C)and positive regulator GA receptor gibberellin insensitive dwarf 1(GID1)genes were up-regulated Three genes were up-regulated and one gene was down-regulated for the sucrose non-fermenting-1-related protein kinase 2(SnRK2),and two genes were up-regulated for the ABA catabolic hydroxylases 8'-hydroxylases.The GA signaling the negatively regulated growth inhibitor DELLA protein was up-regulated by 6 genes and down-regulated by 1 gene,indicating that low-temperature stratification treatment enhanced GA signaling.The ETH and IAA were significantly and differentially expressed at different stratification stages of M.sieversii embryos.The ETH receptor ETR,ETHYLENE INSENSITIVE 3/EIN3-LIKE,1-aminocyclopropane-1-carboxylic acid,a direct precursor of ETH,and ACO were up-regulated expression.The 5 IAA-related genes were up-regulated and one was down-regulated,and small auxin up-regulated RNA was up-regulated,indicating that the stratification would promot IAA synthesis.The cell cycle protein genes were up-regulated to meet the nutritional growth of the seed dormancy release.In addition,the genes related to sucrose metabolism were screened,sucrose synthase and endoglucanase were up-regulated during the stratification.The sucrose transport protein STP13 and STP14 were up-regulated,while STP5 and STP10 were downregulated.【Conclusion】The expression of the ERF2-like showed an opposite trend to the changes of ACO and ACS activities,and the expression of the PYR1-like and WRKY33 genes decreased in relation to the changes of ABA content,suggesting that the above genes might be involved in the ABA signaling pathway to regulate the process of dormancy release process of M.sieversii seeds.