王 珍 張曉莉 劉 淼 姚夢(mèng)楠 孟曉靜 曲存民,2 盧 坤,2 李加納,2,* 梁 穎,2,*

甘藍(lán)型油菜超量表達(dá)及中油821的轉(zhuǎn)錄差異表達(dá)分析

王 珍1,2,**張曉莉1,**劉 淼3姚夢(mèng)楠4孟曉靜1曲存民1,2盧 坤1,2李加納1,2,*梁 穎1,2,*

1西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院 / 油菜工程研究中心, 重慶 400715;2西南大學(xué)現(xiàn)代農(nóng)業(yè)科學(xué)研究院, 重慶 400715;3貴州大學(xué)農(nóng)業(yè)生物工程研究院 / 山地植物資源保護(hù)與種質(zhì)創(chuàng)新教育部重點(diǎn)實(shí)驗(yàn)室, 貴州貴陽(yáng) 550025;4江蘇沿江地區(qū)農(nóng)業(yè)科學(xué)研究所 / 南通市農(nóng)業(yè)科學(xué)研究院, 江蘇南通 210014

甘藍(lán)型油菜(L.)是我國(guó)重要的油料作物之一, 具有較高的產(chǎn)量及營(yíng)養(yǎng)價(jià)值。挖掘油菜廣譜抗逆基因并深入探究其分子機(jī)制, 對(duì)提高油菜抗逆性且保證產(chǎn)量及品質(zhì)具有重要意義。MAPK級(jí)聯(lián)(Mitogen-activated protein kinase cascades)是多種信號(hào)跨膜傳遞的交匯點(diǎn), 參與生長(zhǎng)發(fā)育、生物及非生物脅迫應(yīng)答等生物學(xué)過(guò)程。為了解甘藍(lán)型油菜基因的生物學(xué)功能, 本研究以超量表達(dá)(OE)和中油821 (CK)油菜為材料進(jìn)行數(shù)字基因表達(dá)譜分析。結(jié)果顯示, 與CK相比, 650個(gè)基因在OE油菜中差異表達(dá); 其中上調(diào)表達(dá)基因243個(gè), 下調(diào)表達(dá)基因407個(gè)。GO注釋共富集到生物學(xué)過(guò)程118條途徑, 其中18條與光合作用相關(guān)(包含77個(gè)差異表達(dá)基因); 分子功能7條途徑, 包括草酸氧化酶活性、錳離子結(jié)合、氧化還原酶活性等; 細(xì)胞組分38條途徑, 包括葉綠體、質(zhì)體、類囊體等。KEGG分析結(jié)果表明,參與調(diào)控油菜光合作用、碳代謝、次生代謝物生物合成等14條過(guò)程。此外, 利用實(shí)時(shí)熒光定量PCR驗(yàn)證8個(gè)候選基因在OE和CK油菜中的表達(dá), 其表達(dá)變化與測(cè)序結(jié)果基本一致。本研究結(jié)果為甘藍(lán)型油菜在生長(zhǎng)發(fā)育及光響應(yīng)等生物學(xué)過(guò)程中的功能分析奠定了基礎(chǔ), 也為農(nóng)作物廣譜抗逆遺傳育種提供了理論依據(jù)。

甘藍(lán)型油菜;; 差異表達(dá)基因(DEGs); 光合作用; 數(shù)字基因表達(dá)譜測(cè)序(DGE-seq)

油菜作為可食用葉類蔬菜及油用作物, 也用于花卉觀賞、生物燃料和動(dòng)物飼料等, 分布在全球不同氣候地區(qū)[1]。全球油菜四大主產(chǎn)區(qū)為中國(guó)、加拿大、印度次大陸及德國(guó)、法國(guó)、英國(guó)等歐洲產(chǎn)區(qū), 我國(guó)是世界第一的油菜種植大國(guó), 種植面積和總產(chǎn)量均已接近世界三分之一(FAOSTAT 2020; http://faostat.fao.org/), 在農(nóng)業(yè)生產(chǎn)中占據(jù)舉足輕重的地位。與白菜型油菜()和芥菜型油菜()相比, 甘藍(lán)型油菜()在植株的抗性和產(chǎn)量等方面都具有明顯的優(yōu)勢(shì), 是我國(guó)廣泛種植的栽培種[2]。由于全球氣候的變化以及栽培方式的改變, 連續(xù)陰雨天氣導(dǎo)致的播栽推遲、越冬季節(jié)遭遇低溫和干旱疊加災(zāi)害、光照不足及病蟲草害頻發(fā)等問(wèn)題, 正在嚴(yán)重制約著國(guó)民經(jīng)濟(jì)和油料產(chǎn)業(yè)的發(fā)展[3-5]。隨著綠色革命的發(fā)生, 育種目標(biāo)從單一高產(chǎn)轉(zhuǎn)向優(yōu)質(zhì)高抗高產(chǎn)等復(fù)合性狀目標(biāo), 使得培育減投增效和減損促穩(wěn)的作物新品種成為保障我國(guó)糧油供給的重大需求[6]。然而, 作物產(chǎn)量、品質(zhì)和生物與非生物抗逆性等重要農(nóng)藝性狀是由多個(gè)數(shù)量位點(diǎn)控制, 并且不同性狀之間存在連鎖性與模塊化調(diào)控[7-9]。因此, 挖掘油菜農(nóng)藝性狀形成的樞紐基因并解析其調(diào)控機(jī)理對(duì)油菜廣譜抗性新品種的培育具有重要的理論意義。

MAPK級(jí)聯(lián)(Mitogen-activated protein kinase cascades)是存在于所有真核生物中的保守信號(hào)轉(zhuǎn)導(dǎo)途徑, 參與植物的生長(zhǎng)發(fā)育、生物和非生物脅迫應(yīng)答過(guò)程, 包括病蟲害、低溫、高溫、干旱、紫外線等[10-14]。經(jīng)典的MAPK級(jí)聯(lián)由3種激酶組成, 包括上游MEKKs (MAPK kinase kinases)與MKKs (MAPK kinases)模塊和下游MAPKs模塊[15]。植物在感知到刺激信號(hào)后, MAPK級(jí)聯(lián)將信號(hào)逐級(jí)傳遞并放大, 進(jìn)而通過(guò)下游的轉(zhuǎn)錄因子、生物酶以及結(jié)構(gòu)蛋白等行使功能[13,16-17]。下游MAPKs模塊根據(jù)其保守TXY基序(磷酸化位點(diǎn))被分為A-group～D-group, A-group～C-group為TEY基序, D-group為TDY基序; 其中, MAPK3/6/10屬于A-group, MAPK4/5/11/12/13屬于B-group, MAPK1/2/7/14屬于C-group, MAPK8/9/15/16/17/ 18/19/20屬于D-group[14]。

目前, MAPKs模塊的研究主要集中在A-group MAPK3/6和B-group MAPK4。研究報(bào)道, A-group MAPK3/6在擬南芥()、番茄()和水稻()等多種植物中均被發(fā)現(xiàn)與環(huán)境和激素應(yīng)答相關(guān), 包括響應(yīng)鹽脅迫、損傷、茉莉酸(jasmonic acid, JA)、水楊酸(Salicylic acid, SA)等[18-20]。擬南芥AtMAPK3/6通過(guò)MAPK級(jí)聯(lián)(AtMEKK1-AtMKK4/5- AtMAPK3/6)調(diào)控下游AtWRKY22/29轉(zhuǎn)錄因子, 進(jìn)而有效抵御細(xì)菌()和真菌()的侵害[21]。B-group AtMAPK4能夠與AtMKS1 (MAPK substrate 1)和AtWRKY33結(jié)合形成復(fù)合體, 調(diào)控植保素(Camalexin)的生物合成參與防御反應(yīng)[22]。此外, AtMAPK4還被報(bào)道通過(guò)MAPKs級(jí)聯(lián)參與滲透、干旱、冷害、鹽害脅迫應(yīng)答以及雄性特異性減數(shù)分裂胞質(zhì)分裂過(guò)程[23-26]。C-group MAPKs被報(bào)道可能主要在植物的非生物脅迫應(yīng)答過(guò)程中起調(diào)控作用。研究表明, 擬南芥AtMEKK17/18- AtMKK3-AtMAPK1級(jí)聯(lián)受到PYR/PYL/RCAR-SnRK2- PP2C (Pyrabactin resistance/Pyrabactin resistance-like/ Regulatory component of ABA receptor-SNF1-related protein kinases 2-Protein phosphatases 2C)復(fù)合體的激活而響應(yīng)ABA[27]。AtMKK3-AtMAPK1-AtRBK1 (Rho-like GTPases from plants-binding kinase 1)級(jí)聯(lián)通過(guò)生長(zhǎng)素信號(hào)途徑參與細(xì)胞擴(kuò)張過(guò)程[28]。此外, MAPK1還被報(bào)道在擬南芥及豌豆()中響應(yīng)H2O2, 正向調(diào)控種子萌發(fā)過(guò)程[29-31]。與A-group～C-group不同的是, D-group MAPKs為植物特有, 可不依賴于經(jīng)典的MAPKs級(jí)聯(lián)途徑行使功能[32]。研究發(fā)現(xiàn), 玉米參與ABA、SA、JA、低溫、滲透、病原體等多種外源信號(hào)分子和脅迫應(yīng)答[33]。番茄通過(guò)糖和生長(zhǎng)素代謝與信號(hào)傳遞而特異性調(diào)節(jié)減數(shù)分裂后的花粉發(fā)育過(guò)程[34]。這些研究表明, 植物是具有廣譜抗逆性的保守基因。因此, 甘藍(lán)型油菜基因的生物學(xué)功能研究對(duì)油菜廣譜抗性品種的選育具有重要意義。

本課題組前期分別對(duì)擬南芥、芥菜()、黑芥()、甘藍(lán)()、白菜()以及甘藍(lán)型油菜的MAPKs基因家族進(jìn)行比對(duì)分析, 在甘藍(lán)型油菜中獲得29個(gè)基因(C-group包含5個(gè)基因), 其中(同源基因)在不同生長(zhǎng)時(shí)期及不同組織器官中均有表達(dá), 并且能夠響應(yīng)JA、ABA、H2O2、損傷、核盤菌()、干旱及弱光脅迫誘導(dǎo)[35-36]。我們前期以BnMAPK1為誘餌, 通過(guò)酵母雙雜交文庫(kù)篩選發(fā)現(xiàn)BnMAPK1可能參與多種激素信號(hào)途徑、生物與非生物脅迫應(yīng)答、光合作用、次生代謝物的生物合成與代謝等生物學(xué)過(guò)程[37]。目前, 對(duì)下游MAPKs模塊的研究主要通過(guò)上游MEKKs和MKKs模塊與下游MAPKs模塊之間的相互作用進(jìn)行功能分析, 受限于該級(jí)聯(lián)的逐級(jí)激活, 對(duì)C-group的生物學(xué)功能還知之甚少。本試驗(yàn)以不依賴于MAPK級(jí)聯(lián)上游模塊的超量表達(dá)轉(zhuǎn)基因油菜及其受體為材料, 利用數(shù)字基因表達(dá)譜測(cè)序(digital gene expression profiling sequencing, DGE-seq)技術(shù)對(duì)調(diào)控的差異表達(dá)基因進(jìn)行篩選及分析, 以深入解析在甘藍(lán)型油菜中的基因功能、下游應(yīng)答因子及其調(diào)控途徑。本研究為逐步完善甘藍(lán)型油菜的分子功能奠定基礎(chǔ), 同時(shí)也為MAPK級(jí)聯(lián)在油菜優(yōu)質(zhì)高抗高產(chǎn)品種改良中的應(yīng)用提供理論支撐。

1 材料與方法

1.1 試驗(yàn)材料

供試材料超量表達(dá)轉(zhuǎn)基因油菜(OE)及對(duì)照受體材料中油821 (CK)由重慶市油菜工程技術(shù)研究中心提供[38]。()的開(kāi)放閱讀框(open reading frame, ORF)通過(guò)限制性內(nèi)切酶I和I連接至pCAMBIA2301M超量表達(dá)載體后, 遺傳轉(zhuǎn)化至CK中。取飽滿一致的T3世代OE及CK種子, 播種于直徑12 cm的花盆中。待真葉長(zhǎng)出后, 對(duì)轉(zhuǎn)基因植株進(jìn)行陽(yáng)性鑒定。首先, 每株植株選取一片真葉涂刷300 mg L-1Basta, 3 d后觀察葉片是否黃化。隨后, 選取無(wú)黃化的植株提取葉片DNA, 分別使用35S-F+OCS-R和Bar-F+Bar-R引物檢測(cè)和(Basta抗性基因)的插入。對(duì)PCR條帶正確的植株進(jìn)一步提取RNA, 分別使用BnMAPK1- qF+BnMAPK1-qR和BnACT7-F+BnACT7-R引物進(jìn)行實(shí)時(shí)熒光定量PCR (qRT-PCR)檢測(cè)和內(nèi)參基因()的表達(dá)水平(引物序列見(jiàn)表1)。檢測(cè)后的陽(yáng)性植株每盆定苗3株, 種植于人工氣候光照培養(yǎng)箱(Conviron, 加拿大)。光照及溫度條件設(shè)置為16 h光照(25℃)/8 h黑暗(16℃), 光照強(qiáng)度800 μmol m?2s?1, 濕度50%～60%。分別選取1月齡(4～5葉)苗期健康且長(zhǎng)勢(shì)一致的OE和CK組植株各6株, 每組材料中每3棵植株的第2～3片真葉取樣作為混合樣本, 迅速放入液氮中冷凍, 貯存于?80℃超低溫冰箱備用, 用于總RNA的提取。

1.2 甘藍(lán)型油菜葉片總RNA的提取、DGE-seq文庫(kù)構(gòu)建與測(cè)序

采用TRIzol Reagent (Thermo Fisher Scientific, 美國(guó)), 參照說(shuō)明書提取甘藍(lán)型油菜葉片總RNA, 并檢測(cè)RNA的完整性、濃度及純度。無(wú)明顯降解和污染的RNA樣品委托上海美吉生物醫(yī)藥科技有限公司進(jìn)一步評(píng)估RNA質(zhì)量。檢測(cè)合格的RNA樣品用于DGE-seq文庫(kù)構(gòu)建及測(cè)序。使用Oligo(dT)磁珠(Thermo Fisher Scientific)富集分離mRNA。隨后采用Truseq RNA sample prep Kit (Illumina, 美國(guó))將mRNA打斷, 以短片段mRNA為模板合成雙鏈cDNA并純化, 末端補(bǔ)平后, 3￠端加A并連接接頭。然后進(jìn)行PCR擴(kuò)增及純化, 使用TBS380和Agilent 2100對(duì)富集的文庫(kù)進(jìn)行定量、稀釋及質(zhì)量檢測(cè)。最后采用Truseq PE Cluster Kit v3-cBot-HS (Illumina)對(duì)合格的文庫(kù)進(jìn)行橋式擴(kuò)增生成clusters, 利用Illumina HiSeq平臺(tái)進(jìn)行測(cè)序。原始數(shù)據(jù)(Raw data)已上傳至NCBI (National Center for Biotechnology Information) SRA (Sequence Read Archive)數(shù)據(jù)庫(kù)(BioProject ID: PRJNA595447; Accession ID: SRR10681566, SRR10762584, SRR10681565, SRR10681567)。

表1 轉(zhuǎn)基因植株鑒定和差異表達(dá)基因驗(yàn)證所用引物

1.3 DGE-seq數(shù)據(jù)處理及差異表達(dá)基因分析

DGE-seq的Raw data使用FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/)和BBTools- BBDuk (https://jgi.doe.gov/data-and-tools/bbtools/)進(jìn)行質(zhì)控, 獲得Clean data。以Genoscope數(shù)據(jù)庫(kù)甘藍(lán)型油菜為參考基因組(http://www.genoscope.cns.fr/brassicanapus/), 分別采用StringTie[39]和HISAT2[40]對(duì)Clean data進(jìn)行序列組裝拼接和mapping比對(duì)。通過(guò)DESeq2 RLE (Relative Log Expression)算法[41]對(duì)比對(duì)結(jié)果進(jìn)行歸一化、統(tǒng)計(jì)分析及差異表達(dá)(OE vs. CK)鑒定。為保證差異表達(dá)基因(differentially expressed genes, DEGs)的分析質(zhì)量, 剔除在一組材料中的FPKM (Fragments Per Kilobase of exon model per Million mapped fragments)值均為0的基因, 篩選閾值設(shè)置為adj<0.05和|log2Fold Change |>1。使用TBtools[42]對(duì)DEGs在OE及CK各植株中的表達(dá)進(jìn)行可視化展示, 根據(jù)DEGs進(jìn)行均一化分析及聚類分析; 熱圖顏色為各DEGs的表達(dá)情況, 以log2(FPKM+1)均一化數(shù)值表示。

1.4 差異表達(dá)基因的GO和KEGG pathway富集分析

利用GOstats平臺(tái)[43]對(duì)DEGs進(jìn)行GO (Gene Ontology)和KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway顯著性富集分析。按照生物學(xué)過(guò)程、分子功能和細(xì)胞組分三大類別對(duì)DEGs進(jìn)行GO注釋和富集分析, 閾值設(shè)置為adj<0.05。GO富集以柱狀圖進(jìn)行可視化展示, X軸和Y軸分別表示GO Term名稱和富集顯著性。根據(jù)代謝通路對(duì)KEGG pathway進(jìn)行分類和富集分析, 閾值設(shè)置為value<0.05。KEGG pathway使用R軟件包對(duì)各信號(hào)通路的DEGs富集進(jìn)行可視化展示, X軸和Y軸分別表示富集顯著性和通路名稱, 氣泡大小和顏色分別表示富集基因數(shù)量和富集因子(該代謝通路中富集的DEGs數(shù)量占該通路所有注釋基因總數(shù)的百分比)。

1.5 候選DEGs的表達(dá)模式分析及實(shí)時(shí)熒光定量PCR (qRT-PCR)驗(yàn)證

結(jié)合GO和KEGG pathway富集分析, 篩選相關(guān)生物學(xué)途徑及其對(duì)應(yīng)DEGs。根據(jù)本研究中心前期甘藍(lán)型油菜中油821苗期逆境RNA-Seq數(shù)據(jù)(NCBI Database, BioProject ID: PRJNA680826), 對(duì)候選基因在苗期正常生長(zhǎng)(Mock)及弱光(Shading)處理?xiàng)l件下的表達(dá)模式進(jìn)行比較分析。根據(jù)前期甘藍(lán)型油菜中雙11品種各生長(zhǎng)時(shí)期與組織器官的RNA-Seq數(shù)據(jù)(National Genomics Data Center, BioProject ID: PRJCA001246)[44], 對(duì)候選DEGs進(jìn)行時(shí)空表達(dá)模式分析, 包括種子萌發(fā)期、苗期、蕾薹期、初花期、盛花期以及下胚軸、子葉、根系、莖、葉片、主序頂端、種子。使用TBtools對(duì)DEGs的log2(FPKM+1)值均一化并進(jìn)行可視化分析。

為驗(yàn)證DGE-seq數(shù)據(jù)及候選差異表達(dá)基因的可靠性, 采用Primer 3 (https://primer3.ut.ee/)在線工具[45]設(shè)計(jì)qRT-PCR特異引物(表1), 檢測(cè)8個(gè)DEGs在OE及CK材料中的表達(dá)。根據(jù)操作手冊(cè)使用TaKaRa SYBR Premix ExII Kit (Takara, 美國(guó)), 采用qTOWER2.2 quantitative real-time PCR thermal cycler system (Analytik Jena, 德國(guó))進(jìn)行qRT-PCR反應(yīng)。每個(gè)樣品3次重復(fù), 內(nèi)參基因?yàn)?。反?yīng)程序?yàn)? 95℃ 30 s; 95℃ 5 s, 60℃ 45 s, 40個(gè)循環(huán); 60℃ 45 s。反應(yīng)結(jié)束后, 采用qPCR soft 3.1 system (Analytik Jena)分析Ct值, 并使用2?ΔΔCt算法[46]分析各DEGs的相對(duì)表達(dá)量。

2 結(jié)果與分析

2.1 BnMAPK1超量表達(dá)與對(duì)照甘藍(lán)型油菜的DGE-seq篩選到650個(gè)DEGs

分別采用35S-F+OCS-R和Bar-F+Bar-R引物對(duì)OE植株進(jìn)行PCR鑒定, 前者產(chǎn)物長(zhǎng)度約為1800 bp, 后者約為530 bp (附圖1-A, B), 表明目的基因與抗性基因已整合至植物基因組中。利用qRT-PCR對(duì)CK及OE植株的相對(duì)表達(dá)量進(jìn)行檢測(cè)(附圖1-C), 選取長(zhǎng)勢(shì)一致且表達(dá)水平接近的植株進(jìn)行DGE-seq分析。將DGE-seq質(zhì)控后的Clean data與甘藍(lán)型油菜基因組進(jìn)行比對(duì), 并對(duì)所有基因在各轉(zhuǎn)錄本中的表達(dá)進(jìn)行差異顯著性分析。根據(jù)基因在各樣本間的|log2Fold Change|>1及adj<0.05篩選標(biāo)準(zhǔn), 并且同組材料中至少一個(gè)轉(zhuǎn)錄本的FPKM≠0, 與CK相比, 在OE轉(zhuǎn)基因甘藍(lán)型油菜中共獲得650個(gè)DEGs, 其中上調(diào)與下調(diào)的DEGs分別為243、407個(gè), 分別占總DEGs的37.38%、62.62% (圖1和附表1)。表達(dá)上調(diào)與下調(diào)2～4倍的DEGs分別為91、101個(gè), 分別占總上調(diào)與總下調(diào)DEGs的37.45%、24.82%; 表達(dá)上調(diào)與下調(diào)4～8倍的DEGs分別為73個(gè)和117個(gè), 分別占總上調(diào)與總下調(diào)DEGs的30.04%、28.75% (附圖1-D和附表1)。表明, DEGs的表達(dá)水平變化主要集中在8倍以內(nèi), 表現(xiàn)出高度的不均一性。

圖1 BnMAPK1超量表達(dá)(OE)及對(duì)照(CK)甘藍(lán)型油菜間的650個(gè)差異表達(dá)基因(DEGs)熱圖

Fig. 1 Heatmap of 650 differentially expressed genes (DEGs)between-overexpression (OE) and the control (CK) in rapeseed

熱圖顏色代表DEGs均一化后的log2(FPKM+1)數(shù)值, |log2Fold Change|>1且adj.< 0.05。

The color in the heatmap represents the log2(FPKM+1) value of DEGs after normalization, with |log2Fold Change|>1 andadj.< 0.05.

2.2 BnMAPK1調(diào)控甘藍(lán)型油菜光合作用基因的表達(dá)

對(duì)650個(gè)DEGs進(jìn)行GO功能注釋, 分別富集到生物學(xué)過(guò)程、分子功能和細(xì)胞組分118、7和38條pathways (附表2)。其中, 生物學(xué)過(guò)程主要包括光合作用(GO:0015979)、響應(yīng)蔗糖(GO:0009744)和響應(yīng)細(xì)菌(GO:0009617)等; 分子功能主要包括草酸氧化酶活性(GO:0050162)、錳離子結(jié)合(GO:0030145)和氧化還原酶活性(GO:0016491)等; 細(xì)胞組分主要包括葉綠體(GO:0009507)、質(zhì)體(GO:0009536)和類囊體(GO:0009579)等(圖2-A和附表2)。值得注意的是, GO生物學(xué)過(guò)程共富集到18條與光合作用相關(guān)的pathways, 包括光合作用光反應(yīng)(GO:0019684)、光系統(tǒng)II的組裝(GO:0010207)和光合電子傳遞鏈(GO:0009767)等(圖2-B和附表3)。其中, 與光合作用相關(guān)的DEGs共77個(gè), 上調(diào)DEGs僅有3個(gè)(3.90%), 下調(diào)基因74個(gè)(96.10%); 這些基因主要參與光反應(yīng)、光系統(tǒng)II、氣孔復(fù)合體形態(tài)建成、光合色素、四吡咯合成及代謝、電子傳遞以及對(duì)藍(lán)光、紅光與遠(yuǎn)紅光的響應(yīng)過(guò)程(附圖2和附表4)。

圖2 BnMAPK1超量表達(dá)(OE)及對(duì)照(CK)甘藍(lán)型油菜DGE-seq中DEGs的Top 10 GO注釋分析(A)及光合作用相關(guān)的GO-BP途徑富集分析(B)

GO分類分別為生物學(xué)過(guò)程、分子功能和細(xì)胞組分。GO:0006091表示前體代謝物和能量的產(chǎn)生; GO:0016623表示氧化還原酶活性, 作用于供體的醛基或氧代基團(tuán), 氧為受體; GO:0016903表示氧化還原酶活性, 作用于供體的醛基或氧代基團(tuán); GO:0003868表示4-羥基苯基丙酮酸雙加氧酶活性。DEGs: 差異表達(dá)基因。

GO category including biological process, molecular function, and cellular component. GO:0006091 represents the generation of precursor metabolites and energy; GO:0016623 represents the oxidoreductase activity, acting on the aldehyde or oxo group of donors, oxygen as acceptor;GO:0016903 represents the oxidoreductase activity, acting on the aldehyde or oxo group of donors; GO:0003868 represents 4-hydroxyphenylpyruvate dioxygenase activity. DEGs: differentially expressed genes.

為進(jìn)一步了解可能參與的代謝途徑與信號(hào)轉(zhuǎn)導(dǎo)過(guò)程, 對(duì)650個(gè)DEGs進(jìn)行KEGG pathway富集分析。結(jié)果顯示, 650個(gè)DEGs主要分布在14條KEGG pathways中, 主要包括光合作用(ko00195)、碳代謝(ko01200)和丙酮酸代謝(ko00620)等(附表5和圖3)。其中, 光合作用pathway最顯著, 共有9個(gè)DEGs, 它們?cè)贕O pathway中也被富集到, 這些結(jié)果表明可能在甘藍(lán)型油菜光合作用中扮演重要角色。

2.3 BnMAPK1可能負(fù)調(diào)控甘藍(lán)型油菜光系統(tǒng)與電子傳遞相關(guān)基因的表達(dá)

對(duì)GO和KEGG pathway均富集到的9個(gè)DEGs進(jìn)一步分析發(fā)現(xiàn), 它們?cè)诔勘磉_(dá)后均發(fā)生顯著下調(diào)(表2)。9個(gè)基因分別參與光合作用pathway中光系統(tǒng)I、光系統(tǒng)II和光合電子傳遞過(guò)程(圖4)。其中,(,)與(,)基因參與光系統(tǒng)I, 分別下調(diào)9.73倍和6.87倍;(,)、(,)與(,)基因參與光系統(tǒng)II, 分別下調(diào)8.48、5.32和3.06倍;(,,,,)基因參與光合電子傳遞過(guò)程, 分別下調(diào)35.79、24.12、5.72和4.01倍(表2、圖4和圖5-A)。表明可能參與光合作用中光系統(tǒng)I、光系統(tǒng)II與電子傳遞受體的表達(dá)調(diào)控。

圖3 BnMAPK1超量表達(dá)(OE)及對(duì)照(CK)甘藍(lán)型油菜DGE-seq中DEGs的Top 10 KEGG pathway富集分析

DEGs: 差異表達(dá)基因。DEGs: differentially expressed genes.

表2 BnMAPK1超量表達(dá)(OE)與對(duì)照(CK)甘藍(lán)型油菜的候選光合作用差異表達(dá)基因列表

(續(xù)表2)

圖4 BnMAPK1超量表達(dá)(OE)及對(duì)照(CK)甘藍(lán)型油菜DEGs光合作用(ko00195)通路圖

DEGs: 差異表達(dá)基因。DEGs: differentially expressed genes.

2.4 光合作用光系統(tǒng)I、光系統(tǒng)II與電子傳遞相關(guān)DEGs的表達(dá)模式分析

利用本課題組前期的甘藍(lán)型油菜中油821品種正常生長(zhǎng)與弱光處理的RNA-seq數(shù)據(jù), 分析9個(gè)DEGs對(duì)弱光脅迫的應(yīng)答模式。結(jié)果顯示, 與正常生長(zhǎng)條件相比,()成員在弱光條件下的基因表達(dá)上調(diào), 是正常生長(zhǎng)環(huán)境下該基因表達(dá)量的1.24倍; 其余8個(gè)DEGs的表達(dá)均下調(diào), 是正常生長(zhǎng)條件下基因表達(dá)量的0.30～0.91倍(圖5-B)。根據(jù)本研究室甘藍(lán)型油菜中雙11品種不同組織器官及不同生長(zhǎng)時(shí)期的RNA-seq數(shù)據(jù), 對(duì)9個(gè)DEGs的時(shí)空表達(dá)模式進(jìn)行分析。結(jié)果顯示, 光系統(tǒng)I和電子傳遞受體()基因在各生長(zhǎng)時(shí)期及各組織器官中幾乎不表達(dá); 其余7個(gè)DEGs在根系中的表達(dá)均較低, 在葉片(子葉、幼葉與成熟葉)中的表達(dá)最高, 在莖、主序頂端及幼嫩種子(Se_10d)中也有表達(dá)(圖5-C)。葉片作為甘藍(lán)型油菜光合作用的主要場(chǎng)所, 光合作用直接影響其生長(zhǎng)發(fā)育、產(chǎn)量及抗性等過(guò)程。因此, 我們推測(cè)基因可能在光合作用途徑, 尤其是光系統(tǒng)及電子傳遞過(guò)程中扮演著重要角色, 進(jìn)而可能參與調(diào)控甘藍(lán)型油菜的生長(zhǎng)發(fā)育和光響應(yīng)等過(guò)程。

圖5 BnMAPK1參與甘藍(lán)型油菜光合作用途徑的候選基因的表達(dá)模式分析

A: 候選基因在DGE-seq中的差異表達(dá); B: 候選基因在甘藍(lán)型油菜中油821品種中的弱光應(yīng)答模式; C: 候選基因在甘藍(lán)型油菜中雙11品種不同時(shí)期不同組織中的時(shí)空表達(dá)模式。Ro_48h: 種子萌發(fā)48 h根系; Ro_s: 苗期根系; Ro_b: 蕾薹期根系; Ro_i: 初花期根系; Ro_f: 盛花期根系。Hy_48h: 種子萌發(fā)48 h下胚軸; St_b: 蕾薹期莖; St_i: 初花期莖; St_f: 盛花期莖。Co_48h: 種子萌發(fā)48 h子葉; LeY_s: 苗期幼葉; LeY_b: 蕾薹期幼葉; LeY_i: 初花期幼葉; LeY_f: 盛花期幼葉。LeM_b: 蕾薹期成熟葉; LeM_i: 初花期成熟葉; LeM_f: 盛花期成熟葉。IT_i: 初花期主序頂端; IT_f: 盛花期主序頂端。Se_10d: 花后10 d種子; Se_21d: 花后21 d種子; Se_30d: 花后30 d種子; Se_40d: 花后40 d種子。熱圖顏色代表DEGs均一化后的log2(FPKM+1)數(shù)值, |log2Fold Change|>1且adj<0.05。

A: the differential expression level of candidate genes in DGE-seq; B: shading stress response patterns of candidate genes inZhongyou 821; C: the spatial and temporal expression patterns of candidate genes in different tissues at different stages inZhongshuang11. Ro_48h: root at 48 h after seed germination; Ro_s: root at seedling stage; Ro_b: root at bolting stage; Ro_i: root at initial bloom stage; Ro_f: root at full bloom stage. Hy_48h: hypocotyl at 48 h after seed germination; St_b: stem at bolting stage; St_i: stem at initial bloom stage; St_f: stem at full bloom stage. Co_48h: cotyledons at 48 h after seed germination; LeY_s: young leaf at seedling stage; LeY_b: young leaf at bolting stage; LeY_i: young leaf at initial bloom stage; LeY_f: young leaf at full bloom stage. LeM_b: mature leaf at bolting stage; LeM_i: mature leaf at initial bloom stage; LeM_f: mature leaf at full bloom stage. IT_i: tip of main inflorescence at initial bloom stage; IT_f: tip of main inflorescence at full bloom stage. Se_10d: seed at 10 d after flowering; Se_21d: seed at 21 d after flowering; Se_30d: seed at 30 d after flowering; Se_40d: seed at 40 d after flowering. The color in the heatmap represents the log2(FPKM+1) value of DEGs after normalization, with |log2Fold Change|>1 andadj<0.05.

2.5 差異表達(dá)基因的qRT-PCR驗(yàn)證

為驗(yàn)證DGE-seq結(jié)果的準(zhǔn)確性, 設(shè)計(jì)特異引物對(duì)超量表達(dá)及對(duì)照植株的8個(gè)DEGs進(jìn)行qRT-PCR驗(yàn)證。采用作為內(nèi)參基因, 計(jì)算8個(gè)DEGs的相對(duì)表達(dá)量。其中, 5個(gè)下調(diào)DEGs與光系統(tǒng)復(fù)合體及電子傳遞受體相關(guān), 包括、、、和(), 在OE植株中的表達(dá)量是CK的0.10～0.33倍(圖6-A～E)。3個(gè)上調(diào)DEGs與生長(zhǎng)發(fā)育相關(guān), 包括(,)、(,)和(,)。被報(bào)道通過(guò)生長(zhǎng)素與ABA信號(hào)途徑正向調(diào)控?cái)M南芥的根系和花藥的發(fā)育過(guò)程[47], 雜草地膚()突變后導(dǎo)致維管束系統(tǒng)發(fā)育受阻且光合作用效率顯著下降[48]。CDC5作為植物、動(dòng)物和真菌中保守的MYB相關(guān)蛋白, 正向調(diào)控初級(jí)mRNA的轉(zhuǎn)錄后加工[49]; 同時(shí), CDS作為細(xì)胞周期調(diào)節(jié)劑, 還參與擬南芥莖尖分生組織的細(xì)胞分裂過(guò)程[50]?；蚴强缍_(kāi)花植物所必需的基因,被報(bào)道可能作為負(fù)調(diào)控因子參與梅花()和番茄的開(kāi)花過(guò)程[51-52]。qRT-PCR結(jié)果顯示,、和基因在OE油菜中的表達(dá)量分別為CK植株的2.14、4.18和10.20倍(圖6-F～H)。將qRT-PCR結(jié)果與DGE-seq數(shù)據(jù)進(jìn)行比較分析, 結(jié)果如圖6-I～J所示, 8個(gè)DEGs的測(cè)序表達(dá)變化趨勢(shì)與qRT-PCR中的表達(dá)變化結(jié)果基本一致, 同時(shí)DGE-seq數(shù)據(jù)與qRT-PCR結(jié)果一致性高(2=0.9686), 表明DGE-seq數(shù)據(jù)的可靠性, 可以用來(lái)分析在甘藍(lán)型油菜中的基因功能。

圖6 BnMAPK1超量表達(dá)(OE)及對(duì)照(CK)甘藍(lán)型油菜DGE-seq中DEGs的qRT-PCR驗(yàn)證結(jié)果

A～H: 8個(gè)DEGs的相對(duì)表達(dá)量; I: 8個(gè)DEGs在DGE-seq中的差異表達(dá)倍數(shù); J: 8個(gè)DEGs qRT-PCR與DGE-seq的相關(guān)性分析。DEGs: 差異表達(dá)基因。

A–H: the relative expression levels of eight DGEs; I: the differential expression folds of eight DEGs in DGE-seq; J: the correlation between qRT-PCR and DGE-seq of eight DEGs. DEGs: differentially expressed genes.

3 討論

MAPK級(jí)聯(lián)是植物調(diào)控生長(zhǎng)發(fā)育過(guò)程、響應(yīng)生物與非生物脅迫的主要信號(hào)通路[32,53-54]。下游的MAPKs模塊已在擬南芥、水稻、玉米、小麥、棉花及番茄等多種植物中被鑒定, 其中C-group基因被報(bào)道參與生長(zhǎng)素誘導(dǎo)的細(xì)胞擴(kuò)張, 響應(yīng)損傷、JA、ABA、SA、ROS、H2O2及鹽脅迫等[27-29,32,55-58]。值得注意的是, 不同物種對(duì)相同的刺激會(huì)做出不同的應(yīng)答, 且對(duì)應(yīng)的信號(hào)轉(zhuǎn)導(dǎo)途徑也不相同[59]。結(jié)合前期對(duì)甘藍(lán)型油菜BnMAPKs家族的鑒定與逆境應(yīng)答模式分析以及BnMAPK1的酵母雙雜交文庫(kù)篩選發(fā)現(xiàn),基因在多種生物與非生物脅迫應(yīng)答中具有重要調(diào)控作用, 如病原菌、激素信號(hào)途徑、高溫、干旱、光照等[35-37]。隨著高通量組學(xué)技術(shù)的不斷發(fā)展及其應(yīng)用成本的降低, 采用轉(zhuǎn)錄水平的測(cè)序已經(jīng)成為快速解析基因分子功能及其下游調(diào)控基因的手段之一。本研究對(duì)超量表達(dá)轉(zhuǎn)基因油菜及其對(duì)照植株進(jìn)行DEG-Seq分析發(fā)現(xiàn),超量表達(dá)后共有650個(gè)基因差異表達(dá)(附表1), 這些基因?qū)樵诟仕{(lán)型油菜中的生物學(xué)功能、相關(guān)的信號(hào)途徑及其分子機(jī)制的研究提供基礎(chǔ)。

在自然界中, 光合作用是植物的能量來(lái)源, 直接決定植物的生長(zhǎng)發(fā)育和形態(tài)建成, 是植物生物與非生物脅迫應(yīng)答的基礎(chǔ)[60]。本研究對(duì)超量表達(dá)油菜650個(gè)DEGs的GO富集分析結(jié)果顯示, 這些差異基因主要集中在光合作用、響應(yīng)蔗糖和細(xì)菌、前體代謝物及能量的產(chǎn)生、小分子代謝過(guò)程等; 77個(gè)DEGs的注釋結(jié)果顯示參與調(diào)控光合作用過(guò)程, 包括光合電子傳遞和光合結(jié)構(gòu)相關(guān)的氣孔復(fù)合體與色素系統(tǒng)等, 其中僅有3個(gè)光合相關(guān)DEGs表達(dá)上調(diào)(附表2～附表4)。在植物的光呼吸作用中, 纈氨酸通過(guò)氧化脫羧和酯化以及去飽和形成β-羥基異丁酰輔酶A (HIBYL-CoA, β-hydroxyisobutyryl-CoA), 隨后經(jīng)過(guò)硫酯水解與氧化等過(guò)程逐步形成丙酰輔酶A[61]。研究報(bào)道, 擬南芥()基因編碼HIBYL-CoA水解酶, 參與脂肪酸β氧化、過(guò)氧化物酶體纈氨酸的分解過(guò)程和植物生長(zhǎng)素信號(hào)途徑, 并正向調(diào)控植物的耐冷性[61-62]。我們的DGE-seq數(shù)據(jù)顯示, 與對(duì)照植株相比,超量表達(dá)植株中()基因的表達(dá)水平顯著上調(diào)5.27倍(附表3)。因此, 推測(cè)可能在光合作用、生長(zhǎng)發(fā)育及冷脅迫應(yīng)答過(guò)程中扮演著重要角色。高等植物中, Tic20 (Translocon at the inner envelope membrane of chloroplasts 20)是葉綠體內(nèi)包膜前體蛋白的易位中心, 主要負(fù)責(zé)將細(xì)胞質(zhì)前體蛋白識(shí)別并轉(zhuǎn)運(yùn)至葉綠體基質(zhì)中行使光合功能[63]。擬南芥AtTic20分為2個(gè)亞族Group1 (AtTic20-I、-IV)和Group2 (AtTic20-II、-V), Group1在擬南芥中的功能較Group2更重要; 其中AtTic20-II在光合作用及胚胎和種子發(fā)育過(guò)程具有調(diào)控作用, 但各異構(gòu)體之間存在嚴(yán)重的功能冗余[63]。在我們的研究中,()基因在超量表達(dá)后顯著上調(diào)4.38倍, 而其他3種異構(gòu)體在DGE-seq中的無(wú)差異表達(dá)(附表3)。這些結(jié)果表明, 甘藍(lán)型油菜中BnTic20不同異構(gòu)體之間的功能重要性可能與擬南芥各不相同。與MAPKs相似的另一種絲氨酸/蘇氨酸蛋白激酶雷帕霉素靶蛋白TOR (Target of Rapamycin), 也是真核生物中非常保守的信號(hào)通路。胞質(zhì)磷酸核糖基焦磷酸合成酶PRS4 (Phosphoribosyl diphosphate synthase 4)作為一種關(guān)鍵調(diào)節(jié)酶, 是促進(jìn)植物TOR活性的必需基因, 在協(xié)調(diào)植物的生長(zhǎng)發(fā)育、代謝穩(wěn)態(tài)與營(yíng)養(yǎng)供應(yīng)中發(fā)揮著重要作用[64]。本研究中()基因在OE植株中顯著上調(diào)表達(dá)3.81倍(附表3)。有意思的是, 光自養(yǎng)植物的SnRK1 (Sucrose non-fermenting-1 (SNF1)-related kinase 1)和TOR在糖信號(hào)和能量傳遞中是以拮抗方式調(diào)節(jié)生長(zhǎng)發(fā)育[65], 前期研究發(fā)現(xiàn)可以招募BnSnRK1的PV42a亞基(BnaA06g10010D), 正向調(diào)控的轉(zhuǎn)錄表達(dá), 這些數(shù)據(jù)表明甘藍(lán)型油菜SnRK1-TOR信號(hào)網(wǎng)絡(luò)可能與MAPKs級(jí)聯(lián)途徑密切相關(guān)。此外, 前期發(fā)現(xiàn)中包含了大量光合相關(guān)的順式作用元件, 且的表達(dá)受到弱光脅迫的誘導(dǎo)[36]。表明可能在甘藍(lán)型油菜的光合作用過(guò)程中具有重要的調(diào)控作用。

高等植物的光合作用是一個(gè)非常復(fù)雜的生物過(guò)程, 涉及光系統(tǒng)I、光系統(tǒng)II、Cyt b6/f復(fù)合體(Cytochrome b6/f complex)、ATP合成酶以及其他電子傳遞相關(guān)因子等[66]。DGE-seq研究發(fā)現(xiàn),超量表達(dá)植株中光合相關(guān)的其他DEGs均表現(xiàn)為顯著下調(diào); 其中, 編碼光合作用通路(ko00195)光系統(tǒng)I中PSAF與PSAG亞基、光系統(tǒng)II中PSBO、PSBY與PSBS亞基以及光合電子傳遞中PetF亞基的9個(gè)DEGs均下調(diào)表達(dá)(表2和圖4)。研究報(bào)道, 高等植物中PSAF亞基的N段具有螺旋-螺旋模體, 使得植物的光系統(tǒng)I能夠有效的與電子遞體質(zhì)體藍(lán)素相結(jié)合, 并通過(guò)Cyt b6/f復(fù)合體保證電子傳遞的進(jìn)程[67]。PSAG亞基是植物和綠藻所特有的, PSAG與自身的2個(gè)跨膜螺旋構(gòu)成捕光復(fù)合體(LHC, light harvesting complex) I結(jié)合的表面, 同時(shí)其C端伸出的20個(gè)氨基酸為Cyt b6/f復(fù)合體提供結(jié)合表面[68]。本研究中,和基因在OE植株中顯著下調(diào), 編碼光系統(tǒng)I電子遞體鐵氧還蛋白的4個(gè)基因也下調(diào)表達(dá); 但編碼光系統(tǒng)I其他亞基及其他電子遞體如質(zhì)體藍(lán)素的相關(guān)基因均無(wú)差異表達(dá)(附表3和圖4), 這表明對(duì)光系統(tǒng)I的調(diào)控可能主要集中在電子傳遞過(guò)程。

在光系統(tǒng)II中, 核基因編碼的PSBO亞基是高等植物和藍(lán)藻中均存在的非常保守的膜外在蛋白, 結(jié)合在類囊體腔一側(cè), 是光合放氧活性所必需的[69]。低分子質(zhì)量膜蛋白的PSBY亞基被報(bào)道也與光系統(tǒng)II相聯(lián)系, 可能是維持光系統(tǒng)II的結(jié)構(gòu)完整和雙體構(gòu)象以及葉綠素、類胡蘿卜素、脂分子的結(jié)合環(huán)境[70]。PSBS亞基也是真核光合生物所獨(dú)有的蛋白, 擬南芥突變體被報(bào)道不能進(jìn)行快速可逆的高能淬滅[71]。DGE-seq顯示, 編碼上述亞基的、與基因在OE植株中均顯著下調(diào); 并且編碼外周天線蛋白LHC II中LHCB1 (Chlorophyllbinding protein 1)和LHCB4 (Light-harvesting complex II chlorophyllbinding protein CP29)亞基的()和()基因也顯著下調(diào)表達(dá)(附表3)。在高等植物和一些藻類中, LHCB1與LHCB2、LHCB3組合形成三聚體, LHCB4與LHCB5、LHCB6組合形成LHC II外周天線, 在增強(qiáng)蛋白質(zhì)穩(wěn)定性、有效捕獲光能和控制能量耗散上發(fā)揮重要作用[72]。因此, 我們推測(cè)對(duì)光系統(tǒng)II的調(diào)控可能是通過(guò)控制光系統(tǒng)II-LHC II超級(jí)復(fù)合體的穩(wěn)定性而影響甘藍(lán)型油菜的光合作用過(guò)程。

盡管在甘藍(lán)型油菜光合作用中的功能還有待進(jìn)一步的研究, 但這些基于DGE-seq的數(shù)據(jù)為其基因功能及其調(diào)控網(wǎng)絡(luò)的深入研究奠定了基礎(chǔ), 同時(shí)也為甘藍(lán)型油菜新種質(zhì)資源的開(kāi)發(fā)和應(yīng)用提供了理論依據(jù)。

附圖和附表 請(qǐng)見(jiàn)網(wǎng)絡(luò)版: 1) 本刊網(wǎng)站http://zwxb. chinacrops.org/; 2) 中國(guó)知網(wǎng)http://www.cnki.net/; 3) 萬(wàn)方數(shù)據(jù)http://c.wanfangdata.com.cn/Periodicalzuowxb.aspx。

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Transcriptional differential expression analysis between-overexpression and Zhongyou 821 rapeseed (L.)

WANG Zhen1,2,**, ZHANG Xiao-Li1,**, LIU Miao3, YAO Meng-Nan4, MENG Xiao-Jing1, QU Cun-Min1,2, LU Kun1,2, LI Jia-Na1,2,*, and LIANG Ying1,2,*

1College of Agronomy and Biotechnology, Southwest University / Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China;2Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China;3Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education) / Institute of Agro-bioengineering College, Guizhou University, Guiyang 550025, Guizhou, China;4Jiangsu Yanjiang Institute of Agricultural Sciences / Nantong Academy of Agricultural Sciences, Nantong 210014, Jiangsu, China

Rapeseed (L.) is an important oilseed crop in China, with high yield and nutritional value. Exploring the mechanism of the broad-spectrum stress regulatory genes plays key roles for improving stress resistance, yield, and quality in rapeseed. Mitogen-activated protein kinase (MAPK) cascades are the junctions of several transmembrane signaling, and are involved in a wide variety of biological processes, including growth, development, biotic, and abiotic stress responses. In order to survey the biological function ofgene in rapeseed,-overexpression (OE) and Zhongyou 821 (CK) plants were used as the experimental materials to perform the digital gene expression profiling sequencing at one-month-old seedling stage. Compared with CK, a total of 650 differentially expressed genes were obtained in OE plants, including 243 up-regulated genes and 407 down-regulated genes. GO annotation showed that differentially expressed genes were mainly enriched in 118 pathways in biological process, among which 18 pathways related to photosynthesis (with 77 differentially expressed genes). There were seven pathways in molecular function such as oxalate oxidase activity, manganese ion binding, and oxidoreductase activity; and 38 pathways in cellular component such as chloroplast, plastid, and thylakoid. KEGG indicated thatwas involved in regulating 14 pathways in rapeseed, including photosynthesis, carbon metabolism, and biosynthesis of secondary metabolites. In addition, the relative expression levels of eight candidate genes were validated by qRT-PCR in OE and CK in rapeseed, demonstrating that the expression changes of these eight genes were generally consistent with the sequencing data. Our findings lay a foundation for the function ofin growth and development and light response in rapeseed and provide a theoretical basis for genetic breeding of crop resistance.

;; differentially expressed genes (DEGs); photosynthesis; digital gene expression profiling sequencing (DGE-seq)

10.3724/SP.J.1006.2023.24073

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(32101663, 31872876), 中國(guó)博士后科學(xué)基金項(xiàng)目(2021M692683), 重慶市自然科學(xué)基金項(xiàng)目(cstc2021jcyj-bshX0234), 重慶市博士后科研特別資助項(xiàng)目(2010010006157688)和高等學(xué)校學(xué)科創(chuàng)新引智基地項(xiàng)目111 (B12006)資助。

This study was supported by the National Natural Science Foundation of China (32101663, 31872876), the China Postdoctoral Science Foundation (2021M692683), the Chongqing Natural Science Foundation (cstc2021jcyj-bshX0234), the Chongqing Special Funding in Postdoctoral Scientific Research (2010010006157688), and the Project of Intellectual Base for Discipline Innovation in Colleges and Universities (the 111 Project of China) (B12006).

通信作者(Corresponding authors):梁穎, E-mail: yliang@swu.edu.cn; 李加納, E-mail: ljn1950@swu.edu.cn

同等貢獻(xiàn)(Contributed equally to this work)

王珍, E-mail: wangzhencq@swu.edu.cn; 張曉莉, E-mail: zhangxl806@163.com

2022-03-30;

2022-07-22;

2022-08-29.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20220829.1508.002.html

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).