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      基于轉(zhuǎn)錄組測序篩選新疆野蘋果種子休眠解除過程中植物激素相關基因

      2024-12-31 00:00:00房震李靜馬娟張凱葉春秀
      果樹學報 2024年10期
      關鍵詞:差異表達基因內(nèi)源激素轉(zhuǎn)錄組

      摘" " 要: 【目的】研究新疆野蘋果(Malus sieversii)種子不同貯藏階段轉(zhuǎn)錄組差異和激素含量變化,篩選新疆野蘋果種子休眠解除過程中植物激素相關基因,為后續(xù)新疆野蘋果種子休眠解除激素調(diào)控機制研究提供依據(jù)?!痉椒ā恳孕陆疤O果種子為材料,對照未層積種子和經(jīng)4 ℃低溫沙藏層積處理(30、60、90、120 d)后種子進行轉(zhuǎn)錄組測序,采用酶聯(lián)免疫吸附法測定種子中的脫落酸(abscisic acid,ABA)、赤霉素(gibberellin,GA)、生長素(auxin,IAA)、細胞分裂素(cytokinin,CTK)含量以及乙烯氨基環(huán)丙烷羧酸氧化酶(1-aminocyclopropane-1-carboxylic acid oxidase,ACO)和氨基環(huán)丙烷羧酸合成酶(1-aminocyclopropane-1- carboxylic acid oxidase synthase,ACS)的活性?!窘Y(jié)果】新疆野蘋果種子GA、IAA、CTK含量隨貯藏時間的增加呈上升趨勢,而ABA含量呈下降趨勢。乙烯(ethylene,ETH)合成途徑ACO和ACS活性隨貯藏時間的增加呈增強趨勢。GO富集分析篩選出85個差異基因調(diào)控種子萌發(fā)(GO:0010029),114個GA相關差異基因,313個ABA相關差異基因和156個ETH相關差異基因。KEGG通路富集分析顯示,主要富集通路有植物激素信號轉(zhuǎn)導、MAPK信號通路-植物、內(nèi)質(zhì)網(wǎng)中的蛋白加工、淀粉和蔗糖代謝、糖酵解/糖原生成等。ABA信號傳導通路中有3個PYR/PYL基因下調(diào)表達,2個蛋白磷酸酶2C(PP2C-type protein phosphatases,PP2C)、3個蔗糖非發(fā)酵相關的蛋白激酶(sucrose non-fermenting-1-related protein kinase 2,SnRK2)上調(diào)表達和2個ABA分解代謝8'-羥化酶(8'-hydroxylases)基因表達顯著上調(diào)。GA信號轉(zhuǎn)導途徑中2個受體GID1(gibberellin insensitive dwarf 1)和6個負調(diào)控因子DELLA蛋白上調(diào)表達;ETH受體ETR(Ethylene receptor)的直接前體1-氨基環(huán)丙烷-1-羧酸(1-aminocyclopropane-1-carboxylic acid,ACC)和ACO基因上調(diào)表達?!窘Y(jié)論】新疆野蘋果種子經(jīng)低溫沙藏層積處理,ERF2-like表達量與ACO、ACS活性變化呈相反趨勢,PYR1-like、WRKY33基因表達量與ABA含量變化均下降,說明以上基因可能參與ABA信號通路調(diào)控新疆野蘋果種子休眠解除過程。

      關鍵詞:新疆野蘋果;轉(zhuǎn)錄組;內(nèi)源激素;差異表達基因;種子休眠

      中圖分類號:S661.1 文獻標志碼:A 文章編號:1009-9980(2024)10-1961-18

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

      FANG Zhen, LI Jing, MA Juan, ZHANG Kai, YE Chunxiu*

      (College of Forestry and Landscape Architecture, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China)

      Abstract: 【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-1 on 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-1 on 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-1 on 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 of 41.36 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-1 on 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 was 157.38 ng·g-1. The activity of ACS reached the maximum value of 418.92 ng·g-1 on 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 pathway-plant, 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 down-regulated. 【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.

      Key words: Malus sieversii; Transcriptome; Endogenous hormone; Differentially expressed gene; Seed dormancy

      新疆野蘋果[Malus sieversii (Ledeb.) Rome.]主要分布在中亞天山山脈[1],是現(xiàn)代栽培蘋果的祖先和珍貴的種質(zhì)資源,其種群遺傳多樣性豐富。作為世界蘋果基因庫的組成和中國栽培蘋果的砧木之一[2],具有保護價值和研究意義[3]。新疆野蘋果種子休眠受到種皮結(jié)構及生理生化指標雙重影響[4],進而影響新疆野蘋果育種工作的推進和種質(zhì)資源的維護。

      目前轉(zhuǎn)錄組學已應用在NaCl脅迫下篩選新疆野蘋果葉和根糖酵解途徑相關基因,發(fā)現(xiàn)磷酸丙糖異構酶(triosephosphate isomerase,TPI)、果糖-1,6二磷酸酶(fructose-1,6-bisphosphatase,F(xiàn)BPase)、磷酸烯醇式丙酮酸羧激酶[phosphoenolpyruvate carboxy kinase (ATP),pckA]、丙酮酸磷酸雙激酶(pyruvate,phosphate dikinase,PPDK)等基因的表達量顯著變化[5]。馬紅喜等[6]篩選獲得-3 ℃凍害新疆野蘋果組培苗光合調(diào)控相關基因PsbQ與PsbY。蘇永峰等[7]篩選新疆野蘋果組培苗應答凍害谷胱甘肽代謝相關基因,凍害脅迫響應與谷胱甘肽過氧化物酶(glutathione peroxidase,GPX)和谷胱甘肽轉(zhuǎn)硫酶(glutathione S-transferase,GST)表達上調(diào)有關。因此,研究新疆野蘋果種子休眠解除中的差異基因,對蘋果的遺傳育種及保護種質(zhì)資源提供基因資源。

      植物激素是植物產(chǎn)生的少量有機化合物,可以促進或抑制多種生理過程[8]。脫落酸(abscisic acid,ABA)和赤霉素(gibberellin,GA)在調(diào)控種子休眠和萌發(fā)中發(fā)揮主要作用,種子中的ABA濃度除了控制ABA生物合成外,還通過將ABA轉(zhuǎn)化為相酸(phasic acid)導致ABA失活來決定,負責轉(zhuǎn)化是ABA-8'-羥化酶(ABA-8'-hydroxylases),它是由CYP707A1和CYP707A2編碼的2個細胞色素P450(cytochrome P450,CYP450)[9]。Urbanova等[10]研究發(fā)現(xiàn)3種擬南芥(Arabidopsis)GA受體GID1(gibberellin insensitive dwarf 1)與GAI(GA-insensitive)、RGA(repressor-of-ga1-3)、RGL1(RGA-like1)、RGL2、RGL3等5種DELLA蛋白結(jié)合,作為GA信號轉(zhuǎn)導的阻遏物。其中RGL1在種子萌發(fā)中比GAI和RGA具有更重要的作用,但RGL2是擬南芥中響應GA種子萌發(fā)最重要的調(diào)節(jié)因子。乙烯(ethylene,ETH)是另一種發(fā)芽促進劑,減少休眠3(reduced dormancy 3)是ETH受體反應因子1(ethylene response factor 1,ETR1)的功能喪失突變體,ETR1通過延遲休眠1(delay of germination 1,DOG1)乙烯部分途徑控制種子休眠。ETR1抑制ERF12的表達,ERF12募集TOPLESS形成阻遏復合物并與DOG1啟動子結(jié)合,從而抑制DOG1的表達解除種子休眠[11]。馬娟等[12]發(fā)現(xiàn)新疆野蘋果種子低溫層積處理生長素(auxin,IAA)、GA3、細胞分裂素(cytokinin,CTK)含量增加,ABA含量降低,IAA、GA3和CTK促進種子解除休眠。轉(zhuǎn)錄組篩選分析花生(Arachis hypogaea)種子休眠過程中的ABA、GA、ETH、IAA相關差異表達基因,ABA合成和代謝基因調(diào)控花生種子休眠解除[13]。紫荊(Cercis chinensis Bunge)種子休眠解除主要與植物激素信號轉(zhuǎn)導和能量代謝通路差異基因的顯著變化相關[14]。對低溫處理豆梨(Pyrus calleryana Dence)[15]和強弱休眠花生種子[16]進行轉(zhuǎn)錄組測序,表明植物激素(ABA、GA、ETH、IAA)的信號轉(zhuǎn)導和生物合成在調(diào)控種子休眠維持和解除中起關鍵作用。前期的生理生化指標表明內(nèi)源激素對種子休眠解除具有顯著影響[12]。筆者在本研究中以新疆野蘋果種子為材料,選取未層積和低溫沙藏層積處理(30、60、90、120 d)的種子進行轉(zhuǎn)錄組測序,同時測定種子中植物激素ABA、GA、IAA、CTK含量、乙烯氨基環(huán)丙烷羧酸氧化酶(1-aminocyclopropane-1-carboxylic acid oxidase,ACO)和氨基環(huán)丙烷羧酸合成酶(1-aminocyclopropane-1- carboxylic acid oxidase synthase,ACS)的活性,分析在低溫層積過程中的激素變化,旨在揭示新疆野蘋果種子休眠解除植物激素相關的關鍵基因,為解析種子休眠解除的激素調(diào)控機制提供參考。

      1 材料和方法

      1.1 材料

      試驗材料為新疆塔城額敏縣野果林新疆野蘋果[M. sieversii(Ledeb.)Rome.]種子,對照組為室溫保存5個時期(0、30、60、90、120 d)的種子樣品,處理組M0d是CK0d清水浸泡24 h,4 ℃低溫沙藏層積4個時期(30、60、90、120 d)的種子樣品,標記為M30d、M60d、M90d和M120d。樣品迅速于液氮中冷凍,之后在-80 ℃超低溫冰箱中保存?zhèn)溆?。各時期種子3次生物學重復。種子萌發(fā)率測定,將層積不同時期的種子均勻擺放于培養(yǎng)皿脫脂棉表面,蓋上皿蓋置于自然光下,定期換水并觀察種子發(fā)芽情況,各時期處理30粒種子,3次重復。

      1.2 植物激素和乙烯合成途徑酶濃度檢測

      采用雙抗體夾心酶聯(lián)免疫吸附法(ELISA)測定植物激素ABA、GA、IAA、CTK含量,ETH合成途徑ACC氧化酶和ACC合成酶活性,試劑盒購自睿信生物科技有限公司。

      1.3 RNA提取與轉(zhuǎn)錄組測序分析

      所有新疆野蘋果種子樣本轉(zhuǎn)錄組測序分析委托新疆康普森有限公司。使用Trizol法提取5個時期種胚的總RNA,檢測RNA樣品的濃度和完整性。完成轉(zhuǎn)錄組測序文庫構建,使用Illumina平臺進行PE150測序,得到150 bp的雙端測序reads。利用Trimmomatic[17]進行數(shù)據(jù)質(zhì)量過濾,去除read中包含的接頭序列和質(zhì)量低于20的堿基,過濾后的數(shù)據(jù)按照長度進行篩選,去掉長度小于50 bp或者只有一端的reads。過濾之后得到的高質(zhì)量reads稱為Clean reads。金冠蘋果參考基因組和基因模型注釋文件直接從基因組網(wǎng)站下載,使用Hisat2[18] v2.0.5建立參考基因組索引,將成對的Clean reads與蘋果參考基因組進行比對,獲得Mapped Data。根據(jù)基因長度和Htseq-count統(tǒng)計映射到該基因上的讀數(shù),計算出每個基因的每百萬堿基對測序的轉(zhuǎn)錄本序列片段數(shù)(fragments per kilobase of transcript sequence per millions base pairs sequenced,F(xiàn)PKM)進行基因表達量的量化。

      1.4 差異表達分析、DEGs功能注釋和富集分析

      差異表達分析使用DESeq2[19],采用皮爾遜(Pearson)相關系數(shù)作為生物重復之間的相關性指標[20]。通過DESeq2發(fā)現(xiàn)Padj<0.05的基因被歸為普通差異表達基因(differentially expressed genes,DEGs)。研究各組合的顯著差異表達基因,選取錯誤發(fā)現(xiàn)率(1 discovery rate,F(xiàn)DR)<0.05且差異倍數(shù)|log2(Fold Change)|≥2作為篩選標準。采用層次聚類分析DEGs的表達模式,并將DEGs與基因本體(gene ontology,GO)[21]和(kyoto encyclopedia of genes and genomes,KEGG)[22]數(shù)據(jù)庫比較,使用topGO R和clusterProfiler R[23]軟件包來測試GO和KEGG通路中DEGs的統(tǒng)計富集,通過KEGG通路顯著性富集來確定植物激素相關差異表達基因。

      1.5 差異表達轉(zhuǎn)錄因子分析

      通過植物轉(zhuǎn)錄因子數(shù)據(jù)庫(plant transcription factor database,PTFDB)與DEGs進行比對,篩選差異表達轉(zhuǎn)錄因子(transcription factor,TFs)。采用皮爾遜相關系數(shù)作為轉(zhuǎn)錄因子與植物激素有關DEGs的相關性指標,利用TBtools[24]繪制熱圖分析差異轉(zhuǎn)錄因子的表達模式。

      1.6 qRT-PCR驗證

      隨機選擇與種子休眠和激素相關的9個DEGs進行實時熒光定量(qRT-PCR),使用Primer 5設計引物(表1)。以不同時期的新疆野蘋果種子RNA為模板,使用SYBR Green I染料法qPCR預混液(Enzy Artisan,Q204)進行qRT-PCR:反應條件為預變性95 ℃ 30 s;三步擴增95 ℃ 10 s;60 ℃ 15 s;72 ℃ 30 s;40個循環(huán);溶解95 ℃ 10 s;60 ℃ 60 s;95 ℃ 15 s;冷卻37 ℃ 30 s。以β-actin為內(nèi)參基因,根據(jù)2-ΔΔCt算法[25]計算相對表達量,每個處理3次重復。

      1.7 數(shù)據(jù)分析

      使用Excel進行數(shù)據(jù)統(tǒng)計,使用SPSS 25 Duncan新復極差法進行差異顯著性檢驗,使用Origin 2022 軟件繪圖。

      2 結(jié)果與分析

      2.1 新疆野蘋果種子不同貯藏時期植物激素含量、酶活性及萌發(fā)率比較

      新疆野蘋果種子ABA含量隨貯藏時間的增加呈下降趨勢(圖1-A),貯藏0 d時ABA含量(w,后同)最高為80.22 ng·g-1,在貯藏60 d時ABA含量顯著低于0、30、90 d三個時期(p<0.05),120 d時ABA含量達到最低為43.67 ng·g-1;GA含量隨貯藏時間的增加呈上升趨勢(圖1-B),貯藏0 d時GA含量(b,后同)最低為78.42 pmol·g-1,120 d時GA含量顯著高于其他時期(p<0.05),達到最高為170.67 pmol·g-1;IAA含量隨貯藏時間的增加呈上升趨勢(圖1-C),貯藏60 d時IAA含量顯著高于其他時期,達到高41.36 nmol·g-1;CTK含量在貯藏時間0~30 d時呈下降趨勢(圖1-D),貯藏30 d時CTK含量顯著低于其他時期(p<0.05),達到最低值為31.34 ng·g-1,在貯藏30~60 d時CTK含量呈顯著上升趨勢;表明該時期CTK能促進種子同化物的積累。ETH生物合成途徑中的ACC氧化酶(ACO)和ACC合成酶(ACS)兩個關鍵酶在貯藏過程中的活性變化趨勢不一致(圖1-E、F)。在貯藏90 d時,ACO活性為262.52 ng·g-1,顯著高于其他時期(p<0.05),貯藏30 d時,ACO活性最低為157.38 ng·g-1。ACS的活性在貯藏120 d時達到最大值418.92 ng·g-1,顯著高于0、30、60 d三個時期(p<0.05)。說明低溫沙藏層積促進GA、IAA、CTK、ACC氧化酶和ACC合成酶的合成,且ETH合成途徑的兩個酶可能對低溫更加敏感。種子萌發(fā)率在低溫沙藏層積30、60、90、120 d時萌發(fā)率分別為0%、7%、95%、100%。種子萌發(fā)率與GA、IAA含量及ACO活性變化趨勢一致,與ABA含量變化趨勢相反。

      2.2 層積期間種子的轉(zhuǎn)錄組測序數(shù)據(jù)

      五個休眠解除時期總共得到209.45 G的高質(zhì)量數(shù)據(jù)(表2),各樣本Q30堿基百分比均高于92.9%,說明樣品測序數(shù)據(jù)以及從頭組裝后的質(zhì)量較高。將優(yōu)化后的高質(zhì)量數(shù)據(jù)序列與金冠蘋果(M. domestica)的參考基因組進行比對(表3),比對效率為85.09%~94.06%,比對效率經(jīng)分析后如果高于70%,說明所選參考基因組可滿足信息分析需求。reads在參考基因組不同區(qū)域的分布情況,每個樣品沒有比對到基因間區(qū)的reads在5.94%~26.81%,可能來源于ncRNA或少許DNA片段污染;reads在基因組外顯子區(qū)域在66.44%~91.47%,說明物種的參考基因注釋較為完善。

      在差異表達分析時使用Pearson相關系數(shù)進行計算,樣品間基因表達水平相關性是檢驗試驗可靠性和樣本選擇是否合理的重要指標。根據(jù)各樣本所有基因的FPKM值計算組內(nèi)及組間樣本的相關性系數(shù),繪制成熱圖(圖2),可直觀顯示組間樣本差異及組內(nèi)樣本重復情況。不同顏色代表皮爾遜相關系數(shù)的大小,顏色越偏向紅色代表樣品間相關系數(shù)的絕對值越大。生物學重復樣品間R2均大于0.7,相關系數(shù)越接近1,表明樣品之間表達模式的相似度越高。相關性分析確保后續(xù)的差異基因分析得到更可靠的結(jié)果。

      2.3 顯著差異基因分析

      顯著差異基因分析表明DEGs在低溫層積過程中顯著富集(圖3-A)。M120d_CK120d相對于M0d_CK0d和M30d_CK30d、M60d_CK60d和M90d_CK90d中有更多的顯著DEGs,這表明M120d中具有更多的DEGs來調(diào)控種子萌發(fā)和生理變化。使用R軟件包pheatmap進行基因和樣品的雙向?qū)哟尉垲惙治觯▓D3-B),同處理不同重復間的基因表達模式相似,可能功能相似或參與生物學過程相同,而同時期不同處理間差異基因表達模式存在較大差異。韋恩圖展示了層積和對照比較組合共有或獨有的差異基因數(shù)(圖4)。M30d_CK30d與M60d_CK60d時期顯著差異表達基因、顯著差異上調(diào)基因和顯著差異下調(diào)基因分別共有7384、4875和3236個,表明在低溫層積兩個時期(M30d_CK30d與M60d_CK60d)之間有更多的顯著差異基因參與種子休眠解除。

      2.4 DEGs功能注釋和富集分析

      對差異基因進行顯著富集GO條目(表4)分析,進一步了解DEGs的生物學功能。在生物過程中,有85個DEGs富集在調(diào)控種子萌發(fā)(GO:0010029),調(diào)控種子萌發(fā)中有45個DEGs下調(diào)表達,40個DEGs上調(diào)表達;在赤霉素響應(GO:0009739)和脫落酸響應(GO:0009737)中分別有114個和313個DEGs,其中赤霉素響應中有60個DEGs上調(diào)表達,54個DEGs下調(diào)表達;其中脫落酸響應中有169個DEGs上調(diào)表達,144個DEGs下調(diào)表達;富集在乙烯響應有156個DEGs,99個DEGs上調(diào)表達,57個下調(diào)表達。分子功能中,富集DEGs最高的是DNA結(jié)合轉(zhuǎn)錄因子活性(GO:0003700),449個上調(diào)表達,294個下調(diào)表達。細胞組成中,葉綠體薄膜(GO:0009941)中有271個上調(diào)表達,89個下調(diào)表達。說明植物激素相關調(diào)控基因?qū)ΨN子休眠與萌發(fā)的影響較大。

      對DEGs進行GO富集(圖5),生物過程主要參與滲透壓和缺水的反應、脫落酸響應、鹽脅迫的反應、轉(zhuǎn)錄調(diào)控、調(diào)控種子萌發(fā)和赤霉素響應等。細胞組分主要有葉綠體基質(zhì)、葉綠體包膜和類囊體。分子功能主要與DNA結(jié)合轉(zhuǎn)錄因子活性、磷酸酶活性和蛋白質(zhì)同源二聚體活化有關。新疆野蘋果種子低溫層積休眠解除過程中,多個植物激素生物學過程保持活躍變化,表明植物激素在種子休眠解除活動中發(fā)揮作用。

      KEGG通路富集分析顯示(圖6),種子在不同時期低溫沙藏層積過程中,主要富集的通路有植物激素信號轉(zhuǎn)導、MAPK信號通路-植物、內(nèi)質(zhì)網(wǎng)中的蛋白加工、淀粉和蔗糖代謝、糖酵解/糖原生成等。ABA、GA、ETH等激素信號轉(zhuǎn)導通路相關基因參與種子休眠解除等過程,淀粉和蔗糖代謝通路參與種胚的碳代謝過程,為種胚提供營養(yǎng)物質(zhì)。糖原生成代謝通路參與細胞蛋白質(zhì)的合成和代謝過程,為種胚萌發(fā)提供氮源和能量。

      2.5 植物激素信號轉(zhuǎn)導和代謝差異表達基因的篩選

      根據(jù)KEGG通路分析,篩選在植物激素信號轉(zhuǎn)導和代謝通路上富集的差異表達基因,分析新疆野蘋果種子不同低溫沙藏層積階段的激素和代謝相關差異基因表達模式(圖7),基于已報道的參與植物激素信號轉(zhuǎn)導相關基因,參考蘋果全基因組序列,篩選ABA、GA、ETH、IAA等激素差異表達基因。在貯藏處理下新疆野蘋果植物激素相關差異基因均表現(xiàn)為上調(diào)基因數(shù)多于下調(diào)基因數(shù)。在新疆野蘋果種子低溫沙藏層積過程中,ABA受體PYR/PYL有12個基因上調(diào)和3個基因下調(diào)表達,ABA信號轉(zhuǎn)導因子PP2C和正調(diào)控因子GA受體GID1基因呈上調(diào)表達趨勢,SnRK2有3個基因上調(diào)和1個基因下調(diào)表達,ABA分解代謝8'-羥化酶2個基因呈上調(diào)表達。GA信號轉(zhuǎn)導起負調(diào)控作用的生長抑制因子DELLA蛋白有6個基因上調(diào)和1個基因下調(diào)表達,表明低溫沙藏層積處理增強GA信號轉(zhuǎn)導。ETH、IAA在新疆野蘋果種子不同層積階段中呈現(xiàn)出顯著差異表達,ETH受體ETR、正調(diào)控信號因子EIL(ETHYLENE INSENSITIVE 3/ EIN3-LIKE)、ETH的直接前體ACC和ACC氧化酶(ACO)呈上調(diào)表達;IAA相關基因5個上調(diào)和1個下調(diào)表達,生長素上調(diào)小RNA(small auxin up-regulated,SAUR)上調(diào)表達,表明低溫沙藏層積促進IAA的合成。

      細胞發(fā)育生理變化在種子休眠到休眠解除過程中發(fā)揮作用,篩選發(fā)現(xiàn)許多DEGs參與細胞分裂和生長發(fā)育。細胞周期蛋白A(cyclin-A)和細胞周期蛋白D(cyclin-D)調(diào)控細胞有絲分裂活動,細胞周期蛋白基因上調(diào)表達,滿足種子休眠解除的營養(yǎng)生長需求。另外,篩選到與蔗糖代謝有關的基因,蔗糖分解基因(sucrose synthase,SUS)和內(nèi)切葡聚糖酶(endoglucanase,EG)在低溫沙藏層積過程中上調(diào)表達;蔗糖轉(zhuǎn)化酶13、14和1(sugar transport protein,STP)上調(diào)表達,而STP5下調(diào)表達,這說明蔗糖代謝提供種子發(fā)育所需的能量。

      2.6 轉(zhuǎn)錄因子分析

      M0d_CK0d、M30d_CK30d、M60d_CK60d、M90d_CK90d、M120d_CK120d五個比較組中差異顯著TFs分別為1237、5080、5141、2978和6797個(圖8-A)。在M0d_CK0d中,832個TFs上調(diào)和405個TFs下調(diào);在M30d_CK30d中,3341個TFs上調(diào)和1739個TFs下調(diào);在M60d_CK60d中,3364個TFs上調(diào)和1777個TFs下調(diào);在M90d_CK90d中,2028個TFs上調(diào)和950個TFs下調(diào);在M120d_CK120d中,4527個TFs上調(diào)和2270個TFs下調(diào)。

      采用皮爾遜相關系數(shù)分析8個TFs和9個DEGs間的相關性(圖8-B),bHLH144與ETR、DELLA、PP2C和PYR1呈顯著正相關(p<0.01);WRKY33、MYB86與SAUR32、IAA1和SnRK2.2呈顯著正相關(p<0.01);bHLH66與IAA1、SnRK2.2呈顯著正相關(p<0.01),MYB家族NACK1與IAA1、SnRK2.2和DELLA呈顯著正相關(p<0.01);MYB1R1與ETR和GID1B呈顯著負相關(p<0.01),WRKY22與ETR、PP2C和PYR1呈負相關(p<0.05),AP2/ERF與PP2C呈顯著負相關(p<0.01),與ETR、DELLA和SnRK2.2呈負相關(p<0.05)。

      2.7 新疆野蘋果種子休眠和激素差異基因的qRT-PCR驗證

      為驗證轉(zhuǎn)錄組數(shù)據(jù)結(jié)果,挑選9個與種子休眠和激素相關的DEGs進行qRT-PCR實驗(圖9),測定其在種子低溫層積期間的表達模式,結(jié)果顯示所選的9個DEGs的qRT-PCR表達模式大多與轉(zhuǎn)錄組測序結(jié)果基本一致。結(jié)合生物學重復的要求,說明轉(zhuǎn)錄組數(shù)據(jù)可靠。

      3 討 論

      轉(zhuǎn)錄組技術是分析種子休眠和萌發(fā)相關問題的常用方法之一。筆者在本研究中采用Illumina平臺對新疆野蘋果種子未層積和低溫沙藏層積過程樣品進行轉(zhuǎn)錄組測序,總共獲得209.45 G的高質(zhì)量數(shù)據(jù),與金冠蘋果的參考基因組進行比對,比對效率為85.09%~94.06%。陳靜等[13]利用RNA-seq技術對花生種子休眠和萌發(fā)四個時期的樣本進行轉(zhuǎn)錄組測序,四個時期共表達1206個差異unigenes。發(fā)現(xiàn)ABA 8'-hydroxylases、GA20ox、ACO、EREBP-like和熱激蛋白等調(diào)控花生種子休眠解除及萌發(fā),植物激素(GA、ABA、ETH、IAA)相關unigenes在花生種子休眠解除過程中呈顯著差異。獨行菜(Lepidium apetalum)低溫萌發(fā)停滯前后的種子,經(jīng)Illumina HiseqTM 2000高通量測序平臺轉(zhuǎn)錄組測序,獲得2108個差異基因,主要富集在轉(zhuǎn)錄調(diào)控和植物激素信號轉(zhuǎn)導等相關過程中[26]。魯強[27]基于比較轉(zhuǎn)錄組學研究15 ℃暖溫層積處理三椏苦(Melicope pteleifoli)種子休眠解除的分子機制,主要與植物激素信號傳導通路中ABA受體、GA受體、PP2C和DELLA蛋白等差異基因的顯著變化相關。

      筆者在本研究中對未層積和低溫沙藏層積處理新疆野蘋果種子樣品進行對比,篩選出85個顯著DEGs調(diào)控種子萌發(fā),114個GA相關的顯著DEGs,313個ABA相關的顯著DEGs和156個ETH相關的顯著DEGs。生物過程主要參與轉(zhuǎn)錄調(diào)控,分子功能主要與DNA結(jié)合轉(zhuǎn)錄因子活性、磷酸酶活性和蛋白質(zhì)同源二聚體活化有關。龍佳麗等[28]研究低溫處理前后甜菜DEGs功能富集,發(fā)現(xiàn)激素信號IAA和ABA信號通路與低溫脅迫密切相關。何鑫鑫等[29]使用RNA-Seq測序,分析了Wus2和IPT轉(zhuǎn)基因AC(Ailsa Craig)番茄類愈傷組織與普通下胚軸之間的基因表達差異,在激素信號轉(zhuǎn)導通路中富集到60個差異表達基因,其中上調(diào)表達基因34個,下調(diào)表達基因26個。KEGG通路富集分析顯示,新疆野蘋果種子在不同時期低溫沙藏層積過程中,主要富集的通路有植物激素信號轉(zhuǎn)導、MAPK信號通路-植物、內(nèi)質(zhì)網(wǎng)中的蛋白加工、淀粉和蔗糖代謝、糖酵解/糖原生成等。表明以上途徑在新疆野蘋果種子低溫沙藏層積過程中起積極作用。筆者在本研究中發(fā)現(xiàn),細胞周期蛋白基因、蔗糖分解基因和內(nèi)切葡聚糖酶在低溫沙藏層積過程中上調(diào)表達,促使種胚代謝活動增加,將貯藏物質(zhì)轉(zhuǎn)化為種子萌發(fā)營養(yǎng)物質(zhì)。

      植物激素在種子休眠和萌發(fā)過程中,調(diào)節(jié)激素含量和信號轉(zhuǎn)導的平衡。在靜息狀態(tài)下ABA受體PYR/PYLs/RCARs以二聚體形式存在,與ABA結(jié)合時以單體形式與PP2Cs結(jié)合,通過抑制PP2Cs,解除PP2Cs對蛋白激酶SnRK2s的抑制,從而產(chǎn)生轉(zhuǎn)錄因子ABI5和RAV1被磷酸化,以激活下游ABA響應基因[30]。ABA信號傳導通路中PYR/PYL有3個基因下調(diào)表達,2個PP2C、3個SnRK2和2個ABA分解代謝8'-hydroxylases基因表達顯著上調(diào)。相關研究表明,種子特異性磷酸酶PP2C控制在種子發(fā)育過程中起作用的高活性ABA信號通路[31]。在水稻低溫處理后,受體PYR/PYL蛋白有1個基因顯著下調(diào),PP2C有5個差異基因顯著上調(diào),SnRK2有2個差異表達基因顯著上調(diào)[32]。以上說明新疆野蘋果ABA信號通路基因表達情況與水稻的結(jié)果相似。GA信號轉(zhuǎn)導中GA受體GID與負調(diào)控因子DELLA蛋白上調(diào)表達,在GA含量低時,DELLA蛋白會結(jié)合下游調(diào)控因子來抑制GA信號轉(zhuǎn)導;而GA含量高時,DELLA蛋白與受體GID1感知結(jié)合,可促進GID1/GA/DELLA復合體的形成,解除DELLA對下游調(diào)控因子的抑制,調(diào)控植物內(nèi)激素變化[33],說明低溫沙藏層積能促進GA合成。IAA相關基因5個上調(diào)表達,SAUR上調(diào)表達。ETH受體ETR、ETHYLENE INSENSITIVE 3、ACC氧化酶上調(diào)表達。這與新疆野蘋果種子低溫沙藏層積過程中ABA含量下降、IAA和GA含量增加結(jié)果一致[12],低溫沙藏層積促進了ACC氧化酶和ACC合成酶的合成。筆者在本研究中進一步證實植物激素信號轉(zhuǎn)導途徑在新疆野蘋果種子低溫沙藏層積中起到重要的調(diào)控作用,探究植物激素相關基因的表達,還需進一步研究新疆野蘋果種子休眠解除的分子機制。

      轉(zhuǎn)錄因子的主要作用是激活或阻遏基因的轉(zhuǎn)錄調(diào)控,其在植物發(fā)育、植物激素調(diào)節(jié)和逆境信號轉(zhuǎn)導等過程中發(fā)揮作用[34]。筆者在本研究中發(fā)現(xiàn)bHLH144與ETR、DELLA、PP2C和PYR1呈顯著正相關(p<0.01);WRKY33、MYB86與SAUR32、IAA1和SnRK2.2呈顯著正相關(p<0.01);bHLH66與IAA1、SnRK2.2呈顯著正相關(p<0.01),表明bHLH、WRKY和MYB等轉(zhuǎn)錄因子可能作為正調(diào)節(jié)因子參與激素相關基因的轉(zhuǎn)錄調(diào)控,在植物生長發(fā)育等過程中發(fā)揮作用。

      4 結(jié) 論

      綜合分析了未層積和低溫沙藏層積處理新疆野蘋果種子的轉(zhuǎn)錄組測序結(jié)果,挖掘新疆野蘋果種子休眠解除的相關基因,篩選出85個DEGs調(diào)控種子萌發(fā)、114個GA相關DEGs、313個ABA相關DEGs和156個ETH相關DEGs。ERF2-like表達量與ACO、ACS活性變化呈相反趨勢、PYR1-like、WRKY33基因表達量與ABA含量變化均下降,說明以上基因可能參與ABA信號通路調(diào)控新疆野蘋果種子休眠解除過程。

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