孫婷婷 王文舉 婁文月 劉 峰 張 旭 王 玲 陳玉鳳 闕友雄,2 許莉萍,2 李大妹,2 蘇亞春,2,*

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甘蔗脂氧合酶基因的克隆與表達(dá)分析

孫婷婷1王文舉1婁文月1劉 峰1張 旭1王 玲1陳玉鳳1闕友雄1,2許莉萍1,2李大妹1,2蘇亞春1,2,*

1福建農(nóng)林大學(xué) / 農(nóng)業(yè)部福建甘蔗生物學(xué)與遺傳育種重點(diǎn)實(shí)驗(yàn)室, 福建福州 350002;2福建農(nóng)林大學(xué) / 教育部作物遺傳育種與綜合利用重點(diǎn)實(shí)驗(yàn)室, 福建福州 350002

LOX屬于脂氧合酶超家族(lipoxygenase superfamily), 是脂肪氧化途徑的重要因子, 廣泛參與植物生長(zhǎng)發(fā)育的調(diào)節(jié)和對(duì)外界刺激的抵御。本研究基于甘蔗(spp.)轉(zhuǎn)錄組數(shù)據(jù)庫(kù), 通過(guò)RT-PCR技術(shù), 首次從新臺(tái)糖22號(hào)(ROC22)蔗芽中克隆獲得基因(GenBank登錄號(hào)為MK106188)的cDNA全長(zhǎng)序列。生物信息學(xué)分析顯示,基因cDNA序列長(zhǎng)度為2813 bp, 開(kāi)放讀碼框全長(zhǎng)2664 bp, 編碼887個(gè)氨基酸, 其編碼蛋白的理論等電點(diǎn)為6.23, 不穩(wěn)定系數(shù)為39.77, 親水性平均值為-0.437, 無(wú)信號(hào)肽和跨膜結(jié)構(gòu), 但含有PLAT_LH2和Lipoxygenase活性位點(diǎn), 與高粱() LOX (XP_002466613.1)的氨基酸序列相似性高達(dá)95.96%。預(yù)測(cè)基因的編碼蛋白為酸性穩(wěn)定親水性非分泌蛋白, 屬于type I類(lèi)非傳統(tǒng)9-LOX。qPT-PCR分析結(jié)果顯示,基因在蔗芽組織中特異性表達(dá)。接種甘蔗黑穗病菌()后,基因的表達(dá)量在抗病品種崖城05-179中短暫上升, 但在感病品種ROC22中顯著下降。分別對(duì)瞬時(shí)表達(dá)基因的本氏煙()植株葉片接種煙草茄病鐮刀菌藍(lán)色變種(var.)和青枯菌(), 表型觀察、3,3’-二氨基聯(lián)苯胺(3,3’-diaminobenzidine, DAB)染色和煙草免疫相關(guān)基因的表達(dá)情況分析顯示,基因的過(guò)表達(dá)能夠增強(qiáng)本氏煙對(duì)煙草茄病鐮刀菌藍(lán)色變種的防御, 但對(duì)煙草青枯菌的作用與對(duì)照相比無(wú)明顯差異。研究還發(fā)現(xiàn),基因的表達(dá)受茉莉酸甲酯和水楊酸抑制下調(diào), 但受脫落酸、氯化鈉和聚乙二醇誘導(dǎo)上調(diào)。以上結(jié)果為深入研究甘蔗基因的功能提供參考資料。

甘蔗; 脂氧合酶; 生物信息學(xué); 實(shí)時(shí)熒光定量PCR; 生物和非生物脅迫

脂氧合酶(Lipoxygenase, EC1.13.11.12, LOX)是一種非血紅素、無(wú)硫鐵雙加氧酶, 每個(gè)多肽中含1個(gè)鐵原子, 是脂肪氧化途徑的重要因子, 廣泛存在于動(dòng)植物和真菌中[1-2]。LOX催化亞麻酸(linolenic acid, LNA)和亞油酸(leinoleic acid, LA)形成氫過(guò)氧化物(hydroperoxides, HPOs), 隨后經(jīng)過(guò)氫過(guò)氧化物裂解酶(hydroperoxide lyase, HPL)途徑、二乙烯醚合酶(divinyl ether synthase, DES)途徑、氫過(guò)氧化物異構(gòu)酶(peroxygenase, POX)途徑和丙二烯氧合酶(allene oxide synthase, AOS)途徑等至少7條支路形成一系列活性物質(zhì)參與防御外界刺激或者調(diào)控植物生長(zhǎng)發(fā)育[3-4]。根據(jù)加氧位置的不同, LOX在植物中分為傳統(tǒng)9-LOX、13-LOX和非傳統(tǒng)9/13-LOX三種類(lèi)型, 根據(jù)氨基酸氨基末端質(zhì)體轉(zhuǎn)運(yùn)肽的有無(wú)分為type I (包括傳統(tǒng)9-LOX、13-LOX和非傳統(tǒng)9/13-LOX)和type II (幾乎全部為13-LOX) 2種[5-7]。亞細(xì)胞定位研究表明, LOX蛋白主要存在于細(xì)胞質(zhì)、葉綠體、油體膜、微體膜和液泡等部位[8-9]。目前, 有關(guān)基因的分離和鑒定在大豆()[10]、擬南芥()[11]、水稻()[12]、番茄()[13]、葡萄()[14]和黃瓜()[15]等植物中被廣泛開(kāi)展。

大量研究表明,基因不僅參與調(diào)控植物生長(zhǎng)發(fā)育, 也參與植物對(duì)各種生物和非生物脅迫的防御[16-25]。Grechkin[17]發(fā)現(xiàn)大麥()、黃瓜和大豆在種子萌發(fā)早期會(huì)誘導(dǎo)特異的基因表達(dá)。劉南南等[19]將的反義植物表達(dá)載體轉(zhuǎn)入水稻植株中, 發(fā)現(xiàn)轉(zhuǎn)基因植株對(duì)水分脅迫、白葉枯病和稻瘟病均比非轉(zhuǎn)基因?qū)φ毡憩F(xiàn)敏感, 表明可能參與水稻對(duì)上述逆境脅迫的防御作用。Burow等[20]從花生()種子中分離出1個(gè)基因(1), 其在不成熟子葉中為組成型表達(dá), 在成熟子葉中能被茉莉酸甲酯(methyl jasmonate, MeJA)、傷害和曲霉屬()真菌高度誘導(dǎo)。邵琪等[21]對(duì)甜瓜()接種枯萎病菌(f. sp.)后, 甜瓜葉片和根部分別有13個(gè)和5個(gè)基因上調(diào)表達(dá), 表明基因可能參與甜瓜對(duì)該病原菌的防御反應(yīng)。此外, 研究表明基因與果實(shí)成熟過(guò)程中的乙烯(ethylene, ET)合成相關(guān)[22-23]。干旱脅迫后, 在5個(gè)人參()s基因中只有有響應(yīng)[24]。在辣椒()中,的表達(dá)能被MeJA、水楊酸(salicylic acid, SA)、過(guò)氧化氫(hydrogen peroxide, H2O2)、機(jī)械傷害和高鹽脅迫誘導(dǎo)上調(diào), 但在低溫條件下被抑制[25]。

甘蔗(spp.)是主要的糖料作物和生物能源作物之一, 具有重要的生物學(xué)與經(jīng)濟(jì)價(jià)值。由于甘蔗遺傳背景復(fù)雜、生育周期長(zhǎng)等特點(diǎn), 使得在培育高產(chǎn)和抗逆優(yōu)良新品種時(shí), 基因工程育種比傳統(tǒng)育種方法凸顯優(yōu)勢(shì)[26-27]。因此, 挖掘優(yōu)良抗性基因, 為甘蔗抗逆基因工程育種提供可靠的基因資源, 有助于促進(jìn)甘蔗產(chǎn)業(yè)發(fā)展。前人研究表明植物基因在多種逆境脅迫中扮演重要的角色, 屬于較為重要的抗性基因[16-25], 但至今尚未從甘蔗中獲得, 鑒于此, 本研究從甘蔗中分離獲得了同源基因, 并對(duì)其開(kāi)展了生物信息學(xué)、組織特異性以及不同外源脅迫下的表達(dá)模式分析。此外, 本研究還在本氏煙()葉片中瞬時(shí)表達(dá)該基因, 并進(jìn)行煙草()不同病原菌接種后的表型觀察、3,3’-二氨基聯(lián)苯胺(3,3’- diaminobenzidine, DAB)染色和煙草免疫相關(guān)標(biāo)記基因的檢測(cè)。該研究為進(jìn)一步了解基因的功能和發(fā)掘甘蔗抗逆基因資源奠定了基礎(chǔ)。

1 材料與方法

1.1 材料處理

甘蔗抗黑穗病品種崖城05-179、感黑穗病品種ROC22和甘蔗黑穗病菌()混合冬孢子均來(lái)源于農(nóng)業(yè)部福建甘蔗生物學(xué)與遺傳育種重點(diǎn)實(shí)驗(yàn)室基地。在37℃條件下將采集自田間的甘蔗黑穗病鞭子烘干, 置4℃冰箱保存?zhèn)溆谩?/p>

田間隨機(jī)選取6株長(zhǎng)勢(shì)一致的ROC22成熟甘蔗植株, 連根挖起, 清水洗凈后, 分別取乳白色幼嫩蔗根、+1葉(最高可見(jiàn)肥厚帶下第1片葉)、蔗芽、蔗皮和蔗肉后, 立即液氮速凍和–80℃保存, 用于甘蔗組織特異性表達(dá)分析。

選取4個(gè)月大且長(zhǎng)勢(shì)一致的ROC22組培苗, 置清水中, 在28℃下光照16 h/黑暗8 h復(fù)性培養(yǎng)1周后進(jìn)行處理[28]。組培苗葉面分別噴施100 μmol L-1MeJA (含0.1%乙醇和0.05%吐溫-20, v/v)、5 mmol L-1水楊酸(含0.01%吐溫–20, v/v)和100 μmol L-1脫落酸(abscisic acid, ABA)。MeJA和SA處理0、3、12和24 h后及ABA處理0、0.5、3、6和24 h后立即將整株組培苗用液氮速凍, 用于分析目的基因在不同植物激素脅迫下的表達(dá)模式。

挑選生長(zhǎng)健壯且長(zhǎng)勢(shì)一致的崖城05-179和ROC22成熟蔗莖, 切成雙芽莖段, 用稀釋1200倍的80%多菌靈可濕性粉劑浸泡3 h, 再于流動(dòng)的清水中浸泡24 h, 然后擺盤(pán)并用濕紗布覆蓋置培養(yǎng)箱(32℃, 光照16 h/黑暗8 h)催芽培養(yǎng)。待蔗芽長(zhǎng)至2 cm左右, 一部分材料用5×106個(gè) mL-1(含0.01%吐溫-20, v/v)黑穗病菌孢子懸浮液針刺接種蔗芽, 以無(wú)菌水(含0.01%吐溫-20, v/v)接種蔗芽作為對(duì)照組, 所有處理材料在28℃下光照16 h/黑暗8 h培養(yǎng)[29], 分別于接種后0、24、48和72 h立即取5個(gè)單芽于液氮中速凍和–80℃保存。另一部分未接種的ROC22蔗芽材料繼續(xù)水培至4~5葉齡, 然后挑選長(zhǎng)勢(shì)一致的種莖苗分別置250 mmol L-1氯化鈉(sodium chloride, NaCl)和25.0%聚乙二醇8000 (polyethylene glycol 8000, PEG-8000)水溶液中, 脅迫處理0、0.5、3、6和24 h后立即取+1葉于液氮中速凍, 取3株為1個(gè)重復(fù), 共3次生物學(xué)重復(fù), 于–80℃保存?zhèn)溆谩?/p>

1.2 總RNA的提取及cDNA第1鏈合成

所有植物樣品總RNA的提取及cDNA第1鏈的合成參考Liu等[30]的方法, 其中, 基因克隆和表達(dá)分析用的模板分別按照RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania)和PrimerScript RT-PCR Kit (Takara, 中國(guó)大連)試劑盒說(shuō)明書(shū)合成。

1.3 ScLOX1基因克隆

依據(jù)課題組已獲得的轉(zhuǎn)錄組注釋文庫(kù)[31], 篩選到1條與同源的unigene序列(unigene ID:c151059.graph_c1), 并根據(jù)其序列設(shè)計(jì)基因特異引物-cDNAF/-cDNAR (表1), 以ROC22蔗芽cDNA為模板進(jìn)行PCR擴(kuò)增。PCR程序?yàn)?4℃預(yù)變性4 min; 94℃變性30 s, 55℃退火30 s, 72℃延伸3 min, 共35個(gè)循環(huán); 72℃延伸10 min。隨后對(duì)PCR產(chǎn)物進(jìn)行回收純化、克隆與測(cè)序鑒定[32]。

表1 本研究所用引物序列及用途

(續(xù)表1)

引物名稱(chēng)Primer name引物序列Primer sequence (5'-3')用途Purpose NtHSR201-RGCTCAGTTTAGCCGCAGTTGTGqRT-PCR after transient overexpression NtHSR203-FTGGCTCAACGATTACGCAqRT-PCR after transient overexpression NtHSR203-RGCACGAAACCTGGATGGqRT-PCR after transient overexpression NtHSR515-FTTGGGCAGAATAGATGGGTAqRT-PCR after transient overexpression NtHSR515-RTTTGGTGAAAGTCTTGGCTCqRT-PCR after transient overexpression NtPR-1a/c-FAACCTTTGACCTGGGACGACqRT-PCR after transient overexpression NtPR-1a/c-RGCACATCCAACACGAACCGAqRT-PCR after transient overexpression NtPR2-FTGATGCCCTTTTGGATTCTATGqRT-PCR after transient overexpression NtPR2-RAGTTCCTGCCCCGCTTTqRT-PCR after transient overexpression NtPR3-FCAGGAGGGTATTGCTTTGTTAGGqRT-PCR after transient overexpression NtPR3-RCGTGGGAAGATGGCTTGTTGTCqRT-PCR after transient overexpression NtEFE26-FCGGACGCTGGTGGCATAATqRT-PCR after transient overexpression NtEFE26-RCAACAAGAGCTGGTGCTGGATAqRT-PCR after transient overexpression NtAccdeaminase-FTCTGAGGTTACTGATTTGGATTGGqRT-PCR after transient overexpression NtAccdeaminase-RTGGACATGGTGGATAGTTGCTqRT-PCR after transient overexpression NtEF-1α-FTGCTGCTGTAACAAGATGGATGCqRT-PCR after transient overexpression NtEF-1α-RGAGATGGGGACAAAGGGGATTqRT-PCR after transient overexpression

1.4 ScLOX1基因生物信息學(xué)分析

分別利用ORF (open reading frame) Finder、NCBI conserved domains、ProtParam、SWISSMODEL、Rastop、SignalP 4.0 Server、TMHMM Server v. 2.0和ProtScale軟件分析基因序列的理化特性、保守結(jié)構(gòu)域、一級(jí)結(jié)構(gòu)、三級(jí)結(jié)構(gòu)、信號(hào)肽、跨膜結(jié)構(gòu)和親疏水性。參考Wang等[5]的方法, 利用DNAMAN 6.0軟件將基因編碼的氨基酸序列與NCBI下載的其他同源植物L(fēng)OX氨基酸序列進(jìn)行多序列比對(duì)。參考曹嵩曉等[4]的方法, 利用MEGA 6.06軟件, 選取包括5個(gè)玉米()、2個(gè)水稻、6個(gè)番茄、4個(gè)擬南芥、3個(gè)煙草等在內(nèi)的48個(gè)LOX蛋白與基因編碼的氨基酸構(gòu)建系統(tǒng)進(jìn)化樹(shù)。

1.5 實(shí)時(shí)熒光定量PCR (quantitative real-time PCR, qRT-PCR)分析

根據(jù)基因序列設(shè)計(jì)特異性定量PCR引物, 命名為-QF和-QR (表1), 選用甘油醛-3-磷酸脫氫酶基因(glyceraldehyde-3- phosphate dehydrogenase,)作為內(nèi)參基因, 利用SYBR Green染料法[30], 定量檢測(cè)基因在不同甘蔗組織(根、芽、葉、蔗肉和蔗皮)以及在甘蔗黑穗病菌、植物激素(SA、MeJA、ABA)、模擬鹽(NaCl)和干旱(PEG)等脅迫下的表達(dá)特性。每個(gè)樣品設(shè)3次生物學(xué)重復(fù)和3次技術(shù)重復(fù), 參照2–DDCT法[33]計(jì)算基因相對(duì)表達(dá)量。采用DPS 7.05對(duì)數(shù)據(jù)進(jìn)行顯著性分析, Origin 8.0軟件作柱狀圖。

1.6 瞬時(shí)表達(dá)分析

根據(jù)基因序列設(shè)計(jì)gateway入門(mén)載體引物-Gate-F/-Gate-R (表1), 以質(zhì)粒pMD 19-T-為模板進(jìn)行RT-PCR擴(kuò)增和膠回收純化。利用gateway克隆方法構(gòu)建入門(mén)載體(pDONR-221-)。以獲得的pDONR-221-質(zhì)粒為模板, 利用M13-F/M13-R引物(表1), 進(jìn)行PCR擴(kuò)增、膠回收純化、重組過(guò)表達(dá)載體(pEarleyGate 203-)構(gòu)建, 具體參考Liu等[30]的方法。將pEarleyGate 203-轉(zhuǎn)入農(nóng)桿菌Gv3101, 參考Choi等[34]方法, 收集和重懸菌體后, 注射5~8葉齡且長(zhǎng)勢(shì)一致的本氏煙葉片, 每3株一組作為生物學(xué)重復(fù)。將本氏煙植株置24℃培養(yǎng), 1 d后取注射葉進(jìn)行目的基因檢測(cè)和8個(gè)煙草免疫相關(guān)標(biāo)記基因[包括過(guò)敏反應(yīng)(Hypersensitive reaction, HR)標(biāo)記基因、和; 水楊酸途徑相關(guān)基因; 茉莉酸(jasmonate, JA)途徑相關(guān)基因和; 乙烯合成依賴(lài)基因和] (表1)表達(dá)模式分析。培養(yǎng)2 d時(shí), 取注射葉片進(jìn)行DAB染色[35]。另外, 將瞬時(shí)過(guò)表達(dá)目的基因的本氏煙培養(yǎng)1 d后, 分別接種煙草病原真菌茄病鐮刀菌藍(lán)色變種(var.)和煙草病原細(xì)菌青枯菌(), 培養(yǎng)1 d和7 d后分別進(jìn)行表型觀察、DAB染色和煙草免疫相關(guān)標(biāo)記基因表達(dá)模式分析[30,36]。

2 結(jié)果與分析

2.1 ScLOX1基因克隆及理化性質(zhì)分析

從甘蔗轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中挖掘并克隆獲得了基因序列(GenBank登錄號(hào)為MK106188), 該基因cDNA全長(zhǎng)2813 bp, ORF長(zhǎng)度為2664 bp, 編碼887個(gè)氨基酸(圖1)?；蚓幋a蛋白的分子式為C4532H7006N1238O1314S16, 理論相對(duì)分子量為100.37 kDa, 理論等電點(diǎn)為6.23, 不穩(wěn)定系數(shù)為39.77, 親水性平均值為-0.437, 無(wú)信號(hào)肽和跨膜結(jié)構(gòu)。由圖2可知, ScLOX1的三級(jí)結(jié)構(gòu)主要由α-螺旋、β-折疊和無(wú)規(guī)則卷曲組成, 且含有1個(gè)鐵原子。保守結(jié)構(gòu)域預(yù)測(cè)顯示, ScLOX1蛋白含有PLN02337型結(jié)構(gòu)域, 具有LOX相關(guān)蛋白的活性位點(diǎn)PLAT_LH2和Lipoxygenase (圖3)。由此推測(cè), ScLOX1為酸性穩(wěn)定親水性非分泌LOX蛋白。

圖1 ScLOX1基因核苷酸序列及其推導(dǎo)的氨基酸序列

下畫(huà)線部分為克隆引物; *表示終止密碼子。

The sequences underlined are cloning primers; * indicates stop codon.

圖2 ScLOX1蛋白三級(jí)結(jié)構(gòu)預(yù)測(cè)

2.2 系統(tǒng)進(jìn)化樹(shù)分析

參考曹嵩曉等[4]的方法, 將ScLOX1與48個(gè)其他物種LOX蛋白的氨基酸序列構(gòu)建系統(tǒng)進(jìn)化樹(shù)(圖4)。結(jié)果顯示, ScLOX1與OsLOX1 (DQ389164)、r9- LOX1 (AB099850)、ZmLOX1 (AF271894)和ZmLOX3 (AF329371)聚合在type I單子葉植物分支上, 此分支為非傳統(tǒng)的LOX。利用DNAMAN軟件將ScLOX1氨基酸序列與NCBI查找的5個(gè)單子葉植物L(fēng)OX氨基酸序列進(jìn)行同源序列比對(duì)(圖5)顯示, ScLOX1與水稻(, DQ389164)、玉米(, AF271894)、大麥(, L37358)、高粱(, XP_002466613)和谷子(, XP_ 004982082) LOX氨基酸序列相似性分別為77.25%、63.00%、61.05%、95.96%和90.65%。上述6個(gè)LOX序列均含有LOX蛋白保守區(qū)域, 包括底物結(jié)合位點(diǎn)(Domain I)、氧結(jié)合位點(diǎn)(Domain II)和鐵結(jié)合有關(guān)的保守氨基酸殘基。此外, ScLOX1蛋白含有蘇氨酸/纈氨酸(Thr/Val, 594/595)和精氨酸(Arg, 745), 具有9-LOX特性。分析表明, ScLOX1屬于type I類(lèi)非傳統(tǒng)9-LOX。

2.3 ScLOX1基因在甘蔗各組織中的表達(dá)

利用qRT-PCR技術(shù)分析基因在成熟甘蔗植株ROC22不同組織中的相對(duì)表達(dá)情況(圖6), 結(jié)果顯示,基因只在蔗芽組織中特異性表達(dá)。

2.4 ScLOX1基因在黑穗病菌脅迫下的表達(dá)分析

由圖7可知,基因的表達(dá)量在甘蔗抗病品種崖城05-179受黑穗病菌侵染24 h時(shí)有所上升, 在48 h和72 h時(shí)下降。在感病品種ROC22中, 隨著黑穗病菌侵染時(shí)間的延長(zhǎng),基因的表達(dá)量在24 h時(shí)顯著下降, 且在24~72 h基本保持不變。相比而言,基因的表達(dá)量在抗病品種接種黑穗病孢子第1天略有上升, 而在感病品種中已顯著下降, 同時(shí)兩者的表達(dá)量相差約456.29%。

2.5 本氏煙葉片瞬時(shí)過(guò)表達(dá)ScLOX1基因?qū)Σ煌≡{迫的響應(yīng)情況

分別將對(duì)照空載pEarleyGate 203 ()和重組載體pEarleyGate 203-()轉(zhuǎn)入農(nóng)桿菌Gv3101, 然后注射本氏煙葉片。在注射1 d時(shí), RT-PCR分析顯示,基因在葉片中有表達(dá)(圖8-A)。DAB染色結(jié)果顯示, 瞬時(shí)過(guò)表達(dá)基因的葉片顏色與對(duì)照相比無(wú)明顯差異(圖8-B)。通過(guò)qRT-PCR檢測(cè)瞬時(shí)過(guò)表達(dá)基因1 d后葉片中8個(gè)煙草免疫相關(guān)基因的表達(dá)情況(圖8-C)顯示, HR標(biāo)記基因、JA途徑相關(guān)基因和以及ET合成依賴(lài)基因的表達(dá)量顯著上調(diào), 分別為對(duì)照的2.62、2.31、3.70和5.46倍, 其余基因表達(dá)量與對(duì)照相比無(wú)顯著差異, 推測(cè)基因在本氏煙葉片中的瞬時(shí)過(guò)表達(dá)可能誘導(dǎo)植物免疫反應(yīng)。

對(duì)已注射攜帶空載和重組載體農(nóng)桿菌1 d的本氏煙葉片分別接種了煙草青枯菌和茄病鐮刀菌藍(lán)色變種。在接種青枯菌1 d時(shí),本氏煙葉片和本氏煙葉片均未觀察到明顯的病癥(圖8-D)。葉片中的、、和的表達(dá)量與對(duì)照相比無(wú)顯著差異,和的表達(dá)量顯著低于對(duì)照,和的表達(dá)量顯著高于對(duì)照(圖8-E)。接種青枯菌7 d時(shí),和的本氏煙葉片均出現(xiàn)病斑, 但兩者的病癥差異不大(圖8-D)。葉片中的、和的表達(dá)量與對(duì)照相比無(wú)顯著差異,、和的表達(dá)量顯著低于對(duì)照,和的表達(dá)量顯著高于對(duì)照(圖8-E)。此外, 處理組與對(duì)照組接種1 d和7 d的本氏煙葉片DAB染色結(jié)果無(wú)明顯差異(圖8-D)。

圖3 ScLOX1氨基酸序列的保守結(jié)構(gòu)域

圖4 ScLOX1與其他植物L(fēng)OX蛋白構(gòu)建的系統(tǒng)發(fā)育樹(shù)

ScLOX1用▲標(biāo)識(shí)。玉米LOXs: ZmLOX1 (AF271894)、ZmLOX3 (AF329371)、ZmLOX6 (DQ335764)、ZmLOX10 (DQ335768)、ZmLOX11 (DQ335769); 水稻LOXs: OsLOX1 (DQ389164)、r9-LOX1 (AB099850); 番茄LOXs: TomLOXA (U09026)、TomLOXB (U09025)、TomLOXC (U37839)、TomLOXD (U37840)、TomLOXE (AY008278)、TomLOXF (FJ617476); 擬南芥LOXs: AtLOX1 (NM_104376)、AtLOX2 (AY062611)、AtLOX3 (AJ249794)、AtLOX4 (NM_105911); 煙草LOXs: NaLOX1 (X84040)、NaLOX2 (AY254348)、NaLOX3 (AY254349); 馬鈴薯LOXs: StLOX1 (X95513)、StLOX2 (X96405)、StLOX3 (X96406); 大豆LOXs: GmLOX9 (EU003576)、GmLOX10 (EU003577); 花生LOX: PnLOX2 (DQ068249); 豌豆LOX: LOXN2 (AJ749702); 桃LOXs: PpLOX1 (EU883638)、PpLOX2 (FJ029110)、PpLOX3 (FJ032015)、PpLOX4 (EF568783); 葡萄LOXs: VvLOXA (FJ858255)、VvLOXC (FJ858256)、VvLOXO (FJ858257); 橄欖LOXs: OeLOX (EU678670)、Oep1LOX2 (EU513352)、Oep2LOX2 (EU513353); 辣椒LOXs: CaLOX1 (FJ377708)、CaLOX2 (JQ219046); 楊樹(shù)LOXs: PdLOX1 (DQ131178)、PdLOX2 (DQ131179); 杏LOXs: LOX1:Pd:1 (AJ404331)、LOX1:Pd:2 (AJ418043); 菜豆LOX: PvLOX6 (EF196866); 甘藍(lán)LOX: BoLOX (EF123056); 鬼箭錦雞兒LOX: CjLOX (EF530043); 茶樹(shù)LOX: CasLOX1 (EU195885); 榛子LOX: CaLOX (AJ417975)。

ScLOX1 is marked with a black triangle.LOXs: ZmLOX1 (AF271894), ZmLOX3 (AF329371), ZmLOX6 (DQ335764), ZmLOX10 (DQ335768), ZmLOX11 (DQ335769);LOXs: OsLOX1 (DQ389164), r9-LOX1 (AB099850);LOXs: TomLOXA (U09026), TomLOXB (U09025), TomLOXC (U37839), TomLOXD (U37840), TomLOXE (AY008278), TomLOXF (FJ617476);LOXs: AtLOX1 (NM_104376), AtLOX2 (AY062611), AtLOX3 (AJ249794), AtLOX4 (NM_105911);LOXs: NaLOX1 (X84040), NaLOX2 (AY254348), NaLOX3 (AY254349);LOXs: StLOX1 (X95513), StLOX2 (X96405), StLOX3 (X96406);LOXs: GmLOX9 (EU003576), GmLOX10 (EU003577);LOX: PnLOX2 (DQ068249);LOX: LOXN2 (AJ749702);LOXs: PpLOX1 (EU883638), PpLOX2 (FJ029110), PpLOX3 (FJ032015), PpLOX4 (EF568783);LOXs: VvLOXA (FJ858255), VvLOXC (FJ858256), VvLOXO (FJ858257);LOXs: OeLOX (EU678670), Oep1LOX2 (EU513352), Oep2LOX2 (EU513353);LOXs: CaLOX1 (FJ377708), CaLOX2 (JQ219046);LOXs: PdLOX1 (DQ131178), PdLOX2 (DQ131179);LOXs: LOX1:Pd:1 (AJ404331), LOX1:Pd:2 (AJ418043);LOX: PvLOX6 (EF196866);LOX: BoLOX (EF123056);LOX: CjLOX (EF530043);LOX: CasLOX1 (EU195885);LOX: CaLOX (AJ417975).

圖5 甘蔗ScLOX1與其他植物L(fēng)OX氨基酸序列比對(duì)

文本框表示底物結(jié)合結(jié)構(gòu)域(Domain I和II)和C末端保守區(qū)域。箭頭表示與鐵結(jié)合有關(guān)的保守氨基酸殘基。加號(hào)為T(mén)H(V)/R(K), 是一個(gè)對(duì)確定加氧位置特異性至關(guān)重要的基序。OsLOX1: 水稻(DQ389164); ZmLOX1: 玉米(AF271894); HvLOXC: 大麥(L37358); SbLOX4: 高粱(XP_002466613); SiLOX4: 谷子(XP_ 004982082)。

The substrate-binding domain (Domains I and II) and C-terminus conserved regions are shown by text boxes. An arrow indicates a conserved amino acid residue related to iron binding. The plus sign indicates TH(V)/R(K), a motif proposed to be essential for determining oxygen-adding positional specificity. OsLOX1:(DQ389164); ZmLOX1:(AF271894); HvLOXC:(L37358); SbLOX4:(XP_ 002466613); SiLOX4:(XP_004982082).

圖6 ScLOX1基因在甘蔗ROC22不同組織中的表達(dá)情況

以甘油醛-3-磷酸脫氫酶基因(glyceraldehyde-3-phosphate dehydrogenase,)為內(nèi)參基因。數(shù)據(jù)點(diǎn)為平均值±標(biāo)準(zhǔn)誤(= 3)。Leaf: 葉; Stem pith: 蔗肉; Bud: 蔗芽; Epidermis: 蔗皮; Root: 根。

Usingas an internal reference gene. Data points are means ± SE (= 3).

圖7 不同基因型甘蔗與黑穗病菌互作過(guò)程中ScLOX1基因的表達(dá)量

以甘油醛-3-磷酸脫氫酶基因(glyceraldehyde-3-phosphate dehydrogenase,)作為內(nèi)參。柱上不同的字母代表在< 0.05時(shí)顯著性的差異。數(shù)據(jù)點(diǎn)為平均值±標(biāo)準(zhǔn)誤(= 3)。Yacheng 05-179代表甘蔗抗黑穗病品種; ROC22代表甘蔗感黑穗病品種。

Usingas an internal reference gene. Bars superscripted by different lowercase letters are significantly different at< 0.05. Data points are means ± SE (= 3). Yacheng05-179 is a smut-resistant sugarcane variety. ROC22 is a smut-susceptible sugarcane variety.

注射和的本氏煙葉片在接種茄病鐮刀菌藍(lán)色變種1 d后均無(wú)明顯的病癥出現(xiàn)(圖8-F), 此時(shí), 瞬時(shí)表達(dá)的葉片中的表達(dá)量顯著高于對(duì)照,、、和的表達(dá)量顯著低于對(duì)照,和的表達(dá)量與對(duì)照相比無(wú)顯著差異(圖8-G)。接種茄病鐮刀菌藍(lán)色變種7 d時(shí),和葉片均出現(xiàn)大片病斑, 但對(duì)照發(fā)病相對(duì)較嚴(yán)重(圖8-F)。此外, 瞬時(shí)表達(dá)基因的本氏煙葉片中、和的表達(dá)量顯著低于對(duì)照, 但、、和的表達(dá)量均顯著高于對(duì)照(圖8-G)。接種1 d和7 d的葉片經(jīng)DAB染色后均有深棕色斑點(diǎn)出現(xiàn), 但處理組與對(duì)照組差異不明顯(圖8-F)。

2.6 ScLOX1基因在不同植物激素脅迫下的表達(dá)分析

利用qRT-PCR分析ROC22組培苗中基因在不同植物激素脅迫下的表達(dá)情況(圖9)顯示, MeJA處理后,的轉(zhuǎn)錄本積累量顯著下降, 且在3~24 h基本不變。SA處理后,基因表達(dá)量在3 h時(shí)維持在對(duì)照水平, 12 h時(shí)顯著下降, 為對(duì)照的0.15倍, 24 h時(shí)有所上升但仍顯著低于對(duì)照水平。受ABA脅迫后,基因表達(dá)量在0.5~3.0 h時(shí)基本保持不變, 在6 h顯著上升并呈現(xiàn)表達(dá)高峰, 為對(duì)照的10.88倍, 24 h時(shí)有所下降但仍顯著高于對(duì)照水平。

2.7 ScLOX1基因在干旱和鹽脅迫下的表達(dá)分析

由圖10可知,基因在正常培養(yǎng)條件下的ROC22種莖苗葉片(0 h)中不表達(dá), 但在NaCl和PEG脅迫下被誘導(dǎo)表達(dá)。隨著NaCl處理(模擬鹽脅迫)時(shí)間的延長(zhǎng),基因的表達(dá)量在0.5 h顯著上升, 在3 h時(shí)達(dá)到峰值, 其后顯著下降且與0.5 h的基因表達(dá)量無(wú)顯著差異。利用PEG模擬干旱脅迫后,基因在0.5~6.0 h被誘導(dǎo)上調(diào)表達(dá)且維持在一定的水平, 并在24 h時(shí)顯著上調(diào)表達(dá)且達(dá)到峰值。

3 討論

LOX屬于脂氧合酶超家族(lipoxygenase superfamily), 在植物中以多基因家族形式存在, 參與植物生長(zhǎng)發(fā)育的調(diào)節(jié)和對(duì)逆境脅迫的抵御[7]。本研究從甘蔗中首次分離鑒定出1個(gè)基因, 該基因推導(dǎo)的編碼蛋白屬于type I類(lèi)非傳統(tǒng)9-LOX。前人研究報(bào)道, 單子葉植物type I類(lèi)可能參與植物對(duì)害蟲(chóng)和病原菌的防御[4]。例如, 水稻參與植物對(duì)機(jī)械損傷和稻褐飛虱()攻擊的響應(yīng)[5]。在玉米中,基因在玉米籽苗中能被機(jī)械損傷和MeJA誘導(dǎo)[37],基因能夠響應(yīng)曲霉菌(spp.)對(duì)種子的入侵[38]。

水稻的轉(zhuǎn)錄本可以在未成熟的種子和新發(fā)芽的籽苗中低量表達(dá)[5]?；蛟诮痿~(yú)草()的根、莖、葉和花中差異表達(dá), 其在花中的表達(dá)量最高[39]。在馬鈴薯()中,在根和塊莖中表達(dá)并參與塊莖的生長(zhǎng)發(fā)育;在葉片中表達(dá)并積極響應(yīng)損傷脅迫;在葉片和根中表達(dá), 其在植株受到損傷時(shí)轉(zhuǎn)錄本增加[40-41]。在菜豆()胚軸中,在生長(zhǎng)區(qū)大量表達(dá), 而在成熟區(qū)則幾乎不存在[42]。在本研究中,只在成熟ROC22植株的蔗芽中表達(dá)。上述結(jié)果表明,基因在植物不同組織器官和不同發(fā)育時(shí)期的表達(dá)存在差異。

(圖8)

A: RT-PCR檢測(cè)基因在接種攜帶和載體的農(nóng)桿菌菌株GV3101的本氏煙葉片1 d的表達(dá)情況。B: 對(duì)接種攜帶和載體的農(nóng)桿菌2 d的本氏煙葉片進(jìn)行DAB染色。(1)和(2)分別代表索尼相機(jī)和顯微鏡拍攝的圖像。C: 煙草免疫相關(guān)標(biāo)記基因在接種攜帶和載體的農(nóng)桿菌1 d的本氏煙葉片中的相對(duì)表達(dá)水平。D和F: 瞬時(shí)過(guò)表達(dá)基因1 d后接種煙草青枯菌和茄病鐮刀菌藍(lán)色變種1 d和7 d時(shí)的本氏煙葉片發(fā)病情況和DAB染色結(jié)果。(1)和(2)分別代表索尼相機(jī)和顯微鏡拍攝的圖像。E和G: 煙草免疫相關(guān)標(biāo)記基因在瞬時(shí)過(guò)表達(dá)基因的本氏煙葉片接種青枯菌和茄病鐮刀菌藍(lán)色變種1 d和7 d時(shí)的表達(dá)情況分析。煙草免疫相關(guān)標(biāo)記基因包括HR標(biāo)記基因、和, SA途徑相關(guān)基因, JA途徑相關(guān)基因和, ET合成依賴(lài)基因和。內(nèi)參基因?yàn)榛颉Ｖ喜煌淖帜复碓? 0.05時(shí)顯著性的差異。所有的點(diǎn)均為平均值±標(biāo)準(zhǔn)誤 (= 3)。a和b分別表示載體和3。

A: RT-PCR analysis ofgene in theleaves after infiltration bystrain GV3101 carrying vectororfor 1 d. B: DAB staining withleaves 2 d after:-containingstrain infiltration. (1) and (2) represent images taken by SONY camera and microscope, respectively. C: Relative expression level of the tobacco immunity-associated marker genes in-transiently expressing leaves after infiltration for 1 d. D and F: The results of disease symptoms and DAB staining ofleaves after inoculation with1 d and theninfection withandvar.for 1 d and 7 d, respectively. (1) and (2) represent images taken by SONY camera and microscope, respectively. E and G: Analysis of the immunity related marker genes in theleaves byandvar.for 1 d and 7 d post inoculation. The tobacco immunity-associated marker genes including the HR marker genes,and, the SA pathway related genes, the JA pathway-associated genesand, and the ET synthesis depended genesand. Usingas an internal reference gene. Bars superscripted by different lowercase letters are significantly different at< 0.05. Data points are means ± SE (= 3). The vectors ofandare indicated by a and b, respectively.

圖9 ScLOX1基因在不同植物激素脅迫下的相對(duì)表達(dá)量

內(nèi)參基因?yàn)楦视腿?3-磷酸脫氫酶基因(glyceraldehyde-3-phosphate dehydrogenase,)。柱上不同的字母代表在< 0.05時(shí)顯著性的差異。數(shù)據(jù)點(diǎn)為平均值±標(biāo)準(zhǔn)誤(= 3)。

Usingas an internal reference gene. Bars superscripted by different lowercase letters are significantly different at< 0.05. Data points are means ± SE (= 3).

圖10 ScLOX1基因在NaCl和PEG脅迫下的表達(dá)情況

內(nèi)參基因?yàn)楦视腿?3-磷酸脫氫酶基因(glyceraldehyde-3-phos-phate dehydrogenase,)。柱上不同小寫(xiě)字母代表在< 0.05時(shí)顯著性的差異。數(shù)據(jù)點(diǎn)為平均值±標(biāo)準(zhǔn)誤(= 3)。

Usingas an internal reference gene. Bars superscripted by different lowercase letters are significantly different at< 0.05. Data points are means ± SE (= 3).

基因能參與調(diào)控植物對(duì)生物脅迫的防御作用。棉花()基因()在黃單胞菌()誘導(dǎo)的HR反應(yīng)高度表達(dá)[43]。在根瘤菌()脅迫下,的mRNA在14~16 d時(shí)有最大表達(dá)量[42]。辛翠花等[44]對(duì)抗晚疫病馬鈴薯轉(zhuǎn)基因株系DR1、DR3和晚疫病敏感型野生株DG接種晚疫病原菌()后, 馬鈴薯JA合成途徑關(guān)鍵酶基因和在葉片中的表達(dá)量均有不同程度的提高, 但轉(zhuǎn)基因株系葉片內(nèi)目標(biāo)基因的表達(dá)量高于野生型, 表明基因可能參與了馬鈴薯抗晚疫病反應(yīng)。在轉(zhuǎn)基因番茄植株中,的過(guò)表達(dá)增加了內(nèi)源JA的產(chǎn)生, 并且增強(qiáng)了植株對(duì)番茄葉霉菌()和高溫脅迫的抗性[45]。也有研究指出, 擬南芥和小麥()的表達(dá)能夠促進(jìn)禾谷鐮孢菌()的感染[46]。在本研究中, 不同甘蔗基因型的蔗芽接種甘蔗黑穗病菌后,基因的表達(dá)量在抗病品種崖城05-179中短暫上升, 而在感病品種ROC22中顯著下降。由于蔗芽是甘蔗黑穗病菌侵染的唯一通道[47],基因在蔗芽中的特異性表達(dá)暗示其可能在該甘蔗組織中積極參與對(duì)黑穗病菌的防御作用。據(jù)前人報(bào)道, 植物在防御病原菌侵染的過(guò)程中會(huì)由于細(xì)胞凋亡產(chǎn)生包括免疫相關(guān)基因表達(dá)、防御相關(guān)植物激素的累積、ROS和離子通量的增多等在內(nèi)的一系列生物反應(yīng)[34,48]。在瞬時(shí)表達(dá)基因的本氏煙葉片上接種煙草病原菌顯示,基因瞬時(shí)過(guò)表達(dá)后對(duì)煙草病原細(xì)菌青枯菌的防御作用相較于對(duì)照無(wú)明顯差異, 但能夠增強(qiáng)本氏煙對(duì)煙草病原真菌茄病鐮刀菌藍(lán)色變種的防御, 且接種7 d的本氏煙葉片的HR標(biāo)記基因和、SA途徑相關(guān)基因、JA途徑相關(guān)基因的表達(dá)量顯著上調(diào), 推測(cè)可能通過(guò)參與多個(gè)植物免疫基因的轉(zhuǎn)錄調(diào)控和過(guò)敏性壞死反應(yīng)來(lái)調(diào)節(jié)植物對(duì)病原菌的防御, 但針對(duì)不同類(lèi)型的病原菌其發(fā)揮的作用可能不同, 該研究結(jié)果有待穩(wěn)定遺傳轉(zhuǎn)化后作進(jìn)一步抗病驗(yàn)證。

近年來(lái), 植物基因在各種誘導(dǎo)因子(JA、SA和ABA)和非生物脅迫(鹽和干旱)下的表達(dá)模式被廣泛報(bào)道[11,15,43,49-50]。Lim等[49]在擬南芥中過(guò)表達(dá)了基因(-overexpressing,-OX), 發(fā)現(xiàn)與野生型相比,-OX植株在種子萌發(fā)和幼苗發(fā)育過(guò)程中對(duì)ABA、甘露醇和高鹽脅迫表現(xiàn)出更強(qiáng)的耐受性; 用高鹽和干旱條件脅迫處理生長(zhǎng)過(guò)程中的擬南芥,-OX植株同樣比野生型表現(xiàn)出更強(qiáng)的耐受性。在楊樹(shù)()中,和在MeJA和機(jī)械損傷誘導(dǎo)下上調(diào), 而受SA誘導(dǎo)均下調(diào)[50]。Porta等[43]用損傷、MeJA和ABA處理菜豆成熟葉片能誘導(dǎo)的累積; 在水分脅迫和ABA脅迫下,的表達(dá)量在菜豆胚軸生長(zhǎng)區(qū)減少, 而在成熟區(qū)增加。此外, 對(duì)黃瓜中的12個(gè)基因研究發(fā)現(xiàn), 10個(gè)基因(、、、、、、、、和)在MeJA誘導(dǎo)下上調(diào); 在ABA、冷害和鹽脅迫下, 8個(gè)基因(、、、、、、和)表達(dá)上調(diào)[15]。上述研究結(jié)果表明, 不同物種或同一物種的基因家族的不同成員之間存在功能分化。在本研究中,的表達(dá)量被JA和SA抑制下調(diào), 但在ABA、NaCl和PEG脅迫條件下上調(diào)表達(dá)。有研究報(bào)道, ABA是植物響應(yīng)非生物脅迫(如鹽脅迫)的關(guān)鍵調(diào)控因子[51]。SA、JA和乙烯則被認(rèn)為是植物系統(tǒng)獲得性抗性(systemic acquired resistance, SAR)和誘導(dǎo)系統(tǒng)性抗性(induced systemic resistance, ISR)的重要信號(hào)分子[52-53]。由此推測(cè),基因可能通過(guò)ABA介導(dǎo)的信號(hào)通路來(lái)積極響應(yīng)NaCl和PEG的脅迫。

4 結(jié)論

從甘蔗中挖掘與鑒定了1個(gè)基因。該基因cDNA序列全長(zhǎng)2813 bp, ORF全長(zhǎng)2664 bp, 編碼887個(gè)氨基酸, 預(yù)測(cè)其編碼蛋白為酸性穩(wěn)定親水性非分泌蛋白, 屬于type I類(lèi)非傳統(tǒng)9-LOX。在蔗芽中特異性表達(dá)。受黑穗病菌侵染后,基因的表達(dá)量在甘蔗抗病品種中短時(shí)間略微上調(diào), 但在感病品種中顯著下調(diào)。在本氏煙葉片中瞬時(shí)表達(dá), 發(fā)現(xiàn)基因能夠增強(qiáng)本氏煙對(duì)煙草病原真菌茄病鐮刀菌藍(lán)色變種的抵抗, 但對(duì)煙草病原細(xì)菌青枯菌的作用相較于對(duì)照無(wú)明顯差異。此外, 在外源脅迫下,基因的表達(dá)量被JA和SA抑制下調(diào), 但在ABA、NaCl和PEG脅迫條件下上調(diào)表達(dá), 推測(cè)基因可能通過(guò)ABA介導(dǎo)的信號(hào)通路來(lái)提高甘蔗對(duì)鹽和干旱脅迫的耐受性。

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Cloning and expression analysis of sugarcane lipoxygenase gene

SUN Ting-Ting1, WANG Wen-Ju1, LOU Wen-Yue1, LIU Feng1, ZHANG Xu1, WANG Ling1, CHEN Yu-Feng1, QUE You-Xiong1,2, XU Li-Ping1,2, LI Da-Mei1,2, and SU Ya-Chun1,2,*

1Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China;2Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

LOX, which belongs to the lipoxygenase superfamily, is an important factor for fat oxidation and widely involved in the regulation of plant growth and development and the resistance to external stimuli. In this study, based on sugarcane (spp.) transcriptome database, we first cloned a full-length cDNA sequence ofgene (GenBank accession number: MK106188) from ROC22 bud by RT-PCR. Bioinformatics analysis showed that the cDNA length ofgene was 2813 bp which had a 2664 bp length of open reading frame, encoding 887 amino acids. The theoretical isoelectric point, instability coefficient, and average hydrophilicity of the ScLOX1 protein were 6.23, 39.77, and-0.437, respectively. There were no signal peptide and transmembrane structure, but the PLAT_LH2 and lipoxygenase active sites in ScLOX1 protein. The similarity of amino acid sequences between ScLOX1 andLOX (XP_002466613.1) was 95.96%. The protein encoded bygene was predicted to be an acid-stable, hydrophilic, and non-secreted protein which belongs to the type I non-traditional 9-LOX. qRT-PCR results showed thatwas specifically expressed in sugarcane bud tissue. The expression level ofgene was transiently increased in the smut-resistant sugarcane variety Yacheng05-179 but significantly decreased in the smut-susceptible sugarcane variety ROC22 after inoculated with. When leaves ofwere transiently overexpressedgene and inoculated with tobacco pathogensvar.and, respectively, the results of phenotypic observation, 3,3’-diaminobenzidine (DAB) staining and expression analysis of tobacco immune-related genes revealed that the overexpression ofgene could enhance the defense ofto thevar., but had no significant difference with the control on the defense effect against. In addition, the expression level ofwas down-regulated by methyl jasmine and salicylic acid, but up-regulated by abscisic acid, sodium chloride and polyethylene glycol. The above results provide references for further study on the function of sugarcanegene.

sugarcane; lipoxygenase; bioinformatics; real-time flourescent quantitative PCR; biotic and abiotic stresses

2018-11-05;

2019-01-29;

2019-03-22.

10.3724/SP.J.1006.2019.84143

蘇亞春, E-mail: syc2009mail@163.com

E-mail: sunting3221@163.com

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(31501363), 福建省高校杰出青年科研人才計(jì)劃項(xiàng)目(蘇亞春-2017), 福建農(nóng)林大學(xué)杰出青年基金(xjq201630)和國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專(zhuān)項(xiàng)(CARS-17)資助。

This study was supported by the National Natural Science Foundation of China (31501363), the Research Funds for Distinguished Young Scientists in Fujian Provincial Department of Education (SYC-2017), the Research Funds for Distinguished Young Scientists in Fujian Agriculture and Forestry University (xjq201630) and the China Agriculture Research System (CARS-17).

URL: http://kns.cnki.net/kcms/detail/11.1809.s.20190321.1131.002.html