桃生長(zhǎng)勢(shì)相關(guān)基因PpSAUR73功能鑒定

楊麗，曹洪波，張學(xué)英，翟含含，李辛淼，彭佳偉，田義，陳海江

桃生長(zhǎng)勢(shì)相關(guān)基因功能鑒定

1河北農(nóng)業(yè)大學(xué)園藝學(xué)院，河北保定 071000；2喀什職業(yè)技術(shù)學(xué)院，新疆喀什 844000；3河北農(nóng)業(yè)大學(xué)山區(qū)研究所/河北省山區(qū)農(nóng)業(yè)技術(shù)創(chuàng)新中心/國(guó)家北方山區(qū)農(nóng)業(yè)工程技術(shù)研究中心，河北保定 071001

【目的】克隆桃樹(shù)勢(shì)相關(guān)基因，分析其對(duì)多種激素的響應(yīng)，鑒定其在調(diào)控?cái)M南芥生長(zhǎng)勢(shì)中的作用，為科學(xué)合理調(diào)控樹(shù)勢(shì)提供分子依據(jù)?！痉椒ā恳浴杏腕?號(hào)’為材料進(jìn)行激素處理，利用實(shí)時(shí)熒光定量分析在24 h內(nèi)的動(dòng)態(tài)響應(yīng)；以桃品種‘久艷’為材料克??；構(gòu)建過(guò)量表達(dá)載體，將其轉(zhuǎn)化擬南芥；對(duì)轉(zhuǎn)基因擬南芥進(jìn)行表型觀察，對(duì)同時(shí)播種的轉(zhuǎn)基因與野生型擬南芥進(jìn)行萌芽率統(tǒng)計(jì)，對(duì)萌發(fā)一致的生長(zhǎng)7 d擬南芥進(jìn)行根長(zhǎng)及下胚軸的測(cè)量，對(duì)萌發(fā)一致的擬南芥進(jìn)行不同濃度的激素處理；取7 d大小的兩個(gè)轉(zhuǎn)基因株系及野生型擬南芥材料進(jìn)行轉(zhuǎn)錄組測(cè)序，對(duì)差異表達(dá)基因進(jìn)行功能分析、KEGG通路富集分析，并分析調(diào)控基因?！窘Y(jié)果】能夠?qū)に靥幚碜龀隹焖夙憫?yīng)。過(guò)表達(dá)能夠影響擬南芥種子萌芽，轉(zhuǎn)基因擬南芥幼苗下胚軸及根長(zhǎng)較野生型長(zhǎng)，蓮座大，整體長(zhǎng)勢(shì)好于野生型，且降低了對(duì)生長(zhǎng)素的敏感性。過(guò)表達(dá)的轉(zhuǎn)錄組分析結(jié)果顯示，在兩個(gè)對(duì)比組中均表達(dá)的差異基因有128個(gè)，其中有84個(gè)上調(diào)基因，44個(gè)下調(diào)基因，并對(duì)20個(gè)表達(dá)量較高的差異基因進(jìn)行了描述。對(duì)過(guò)表達(dá)產(chǎn)生的差異表達(dá)基因進(jìn)行GO功能顯著性富集分析，結(jié)果表明差異表達(dá)基因在細(xì)胞組分方面富集的基因最多，定位在細(xì)胞質(zhì)、細(xì)胞膜、細(xì)胞器和細(xì)胞外區(qū)域。對(duì)差異表達(dá)基因進(jìn)行KEGG通路富集分析，結(jié)果表明差異表達(dá)基因主要富集到苯丙氨酸生物合成通路、植物激素信號(hào)轉(zhuǎn)導(dǎo)通路、淀粉蔗糖代謝通路等代謝通路。在苯丙氨酸生物合成通路中，能夠調(diào)控編碼過(guò)氧化物酶基因AT1G05260、AT3G01190、AT3G32980、AT5G15180表達(dá)上調(diào)，過(guò)氧化物酶與木質(zhì)素合成相關(guān)，木質(zhì)素含量與植株長(zhǎng)勢(shì)呈顯著相關(guān)，暗示過(guò)表達(dá)可能參與調(diào)控?cái)M南芥木質(zhì)素合成從而調(diào)控長(zhǎng)勢(shì)。在植物激素信號(hào)轉(zhuǎn)導(dǎo)通路中，生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)中的一些生長(zhǎng)素響應(yīng)基因等表達(dá)下調(diào)，脫落酸信號(hào)轉(zhuǎn)導(dǎo)途徑中磷酸酶蛋白表達(dá)上調(diào)，而脫落酸信號(hào)通路基因表達(dá)下調(diào)。能夠調(diào)控?cái)M南芥長(zhǎng)勢(shì)，參與多種激素信號(hào)轉(zhuǎn)導(dǎo)。【結(jié)論】能夠快速對(duì)激素做出響應(yīng)，且能夠調(diào)控轉(zhuǎn)基因擬南芥長(zhǎng)勢(shì)；過(guò)量表達(dá)導(dǎo)致的差異基因主要富集到苯丙氨酸生物合成通路、植物激素信號(hào)轉(zhuǎn)導(dǎo)通路、淀粉蔗糖代謝通路等代謝通路；調(diào)控IAA、ABA信號(hào)轉(zhuǎn)導(dǎo)，推測(cè)其在桃樹(shù)的生長(zhǎng)發(fā)育過(guò)程中起到了重要作用。

桃；；基因表達(dá)；激素處理；信號(hào)轉(zhuǎn)導(dǎo)

0 引言

【研究意義】桃（L.）屬于薔薇科（Rosaceae）植物，落葉小喬木。桃樹(shù)營(yíng)養(yǎng)生長(zhǎng)旺盛，新梢生長(zhǎng)量大，一年可形成多次副梢，生產(chǎn)中經(jīng)常通過(guò)修剪以及噴施生長(zhǎng)調(diào)節(jié)劑對(duì)樹(shù)勢(shì)進(jìn)行控制，否則便會(huì)造成樹(shù)冠郁閉，影響樹(shù)體的通風(fēng)透光，從而影響果實(shí)品質(zhì)。因此，挖掘桃樹(shù)勢(shì)生長(zhǎng)調(diào)控基因，明確樹(shù)勢(shì)調(diào)控機(jī)制十分必要?！厩叭搜芯窟M(jìn)展】生長(zhǎng)素調(diào)節(jié)植物生長(zhǎng)發(fā)育，早期生長(zhǎng)素反應(yīng)基因介導(dǎo)其對(duì)植物生長(zhǎng)發(fā)育的基因組效應(yīng)。（）于1987年被發(fā)現(xiàn)，是最大的早期生長(zhǎng)素響應(yīng)基因家族[1]。在前人提出的生長(zhǎng)素介導(dǎo)細(xì)胞擴(kuò)增的酸生長(zhǎng)機(jī)制中發(fā)揮核心作用[2]。盡管早期發(fā)現(xiàn)了它們的生長(zhǎng)素反應(yīng)性，但其功能和作用方式長(zhǎng)期以來(lái)仍有待解析[3]。近年來(lái)，人們對(duì)在動(dòng)態(tài)調(diào)控生長(zhǎng)和適應(yīng)性調(diào)控生長(zhǎng)中的重要性以及SAUR蛋白作用的分子機(jī)制日益了解。目前，該基因家族已在擬南芥、棉花、茄子及葡萄等多個(gè)物種中得到鑒定[4-5]。一些主要在下胚軸或其他伸長(zhǎng)組織中表達(dá)，在調(diào)節(jié)細(xì)胞伸長(zhǎng)中發(fā)揮重要作用[6-7]。能夠通過(guò)負(fù)調(diào)節(jié)PP2C蛋白磷酸酶來(lái)調(diào)節(jié)質(zhì)膜H+-ATP酶活性，從而促進(jìn)細(xì)胞擴(kuò)增[7]。在擬南芥內(nèi)胚層中表達(dá)，參與側(cè)根發(fā)育，過(guò)表達(dá)的擬南芥會(huì)出現(xiàn)下胚軸伸長(zhǎng)，影響側(cè)根發(fā)育，使花瓣膨脹和花序莖發(fā)生扭曲[8]。此外，一些還可以對(duì)生長(zhǎng)素的合成運(yùn)輸產(chǎn)生負(fù)調(diào)控，過(guò)表達(dá)的擬南芥幼苗下胚軸變短且直，并且可以通過(guò)施加外源生長(zhǎng)素緩解這種癥狀[9]。在水稻中，過(guò)表達(dá)對(duì)生長(zhǎng)素的生物合成和運(yùn)輸產(chǎn)生負(fù)調(diào)控，降低生長(zhǎng)素的轉(zhuǎn)運(yùn)活性[10]，這表明不同的SAUR蛋白可能具有相反的功能。還可以受到大量其他上游因素的調(diào)控，從而根據(jù)內(nèi)部和環(huán)境信號(hào)動(dòng)態(tài)調(diào)節(jié)生長(zhǎng)[11-15]。此外，一些在葉片衰老、細(xì)胞分裂及響應(yīng)干旱、低溫、病蟲(chóng)害等過(guò)程中發(fā)揮作用[8-10]。【本研究切入點(diǎn)】桃是原產(chǎn)我國(guó)的一種極為重要的大宗水果，但其樹(shù)體偏大，普遍存在樹(shù)冠郁閉不便管理的問(wèn)題，大量使用人工導(dǎo)致生產(chǎn)成本增加[16-17]。筆者課題組前期在對(duì)不同樹(shù)勢(shì)的桃樹(shù)進(jìn)行轉(zhuǎn)錄組數(shù)據(jù)分析時(shí)獲得了與調(diào)控樹(shù)勢(shì)相關(guān)的18個(gè)關(guān)鍵基因，并對(duì)其中的進(jìn)行了功能鑒定，發(fā)現(xiàn)該基因過(guò)量表達(dá)促進(jìn)器官伸長(zhǎng)[18]。為進(jìn)一步探究其余的差異基因是否也存在相似或者相反的表型，對(duì)轉(zhuǎn)錄組中篩選得到的與樹(shù)勢(shì)相關(guān)的（XM_007201374.1）進(jìn)行功能鑒定，探究過(guò)表達(dá)擬南芥是否具有調(diào)控長(zhǎng)勢(shì)的功能，并觀察過(guò)表達(dá)擬南芥對(duì)激素的響應(yīng)情況。【擬解決的關(guān)鍵問(wèn)題】克隆桃并穩(wěn)定遺傳轉(zhuǎn)化擬南芥，進(jìn)行表型觀察及轉(zhuǎn)錄組測(cè)序分析，結(jié)合表達(dá)對(duì)激素的響應(yīng)，鑒定的基因功能，為調(diào)控桃樹(shù)體長(zhǎng)勢(shì)提供分子依據(jù)。

1 材料與方法

1.1 試驗(yàn)材料

試驗(yàn)于2022年在河北農(nóng)業(yè)大學(xué)園藝學(xué)院、保定市滿城區(qū)國(guó)家良種基地進(jìn)行。

本試驗(yàn)用于克隆基因的RNA提取自桃品種‘久艷’，砧木為‘毛桃’，采用常規(guī)田間管理。用于田間激素處理的RNA提取自桃品種‘中油蟠9號(hào)’，由保定市滿城區(qū)國(guó)家桃良種基地提供。

本研究使用的擬南芥為哥倫比亞型（Col-0）。

1.2 方法

1.2.1 構(gòu)建桃與擬南芥長(zhǎng)勢(shì)相關(guān)蛋白系統(tǒng)發(fā)育樹(shù) 通過(guò)文獻(xiàn)獲得擬南芥（）SAUR家族與長(zhǎng)勢(shì)相關(guān)的蛋白序列信息，將桃PpSAUR5、PpSAUR73和擬南芥SAUR蛋白序列置于MEGA7軟件中，使用ClustalW程序進(jìn)行多重序列比對(duì)，將得到的結(jié)構(gòu)采用鄰接法（Neighbor-Joining，NJ），參數(shù)默認(rèn)，Bootstrap鄰接值設(shè)置為1 000，構(gòu)建系統(tǒng)發(fā)育樹(shù)。

1.2.2 桃對(duì)激素的瞬時(shí)響應(yīng) 于2022年9月20日，選取長(zhǎng)至20 cm的‘中油蟠9號(hào)’嫩梢對(duì)其進(jìn)行生長(zhǎng)調(diào)節(jié)劑處理。試驗(yàn)以噴施0.7%無(wú)水乙醇溶液為對(duì)照，分別向葉面噴施100 mg?L-1吲哚-3-乙酸IAA、100 mg?L-1赤霉素（GA）、50 mg?L-1鄰氨甲酰苯甲酸（NPA）、50 mg?L-1的乙烯利（ETH）和100 mg?L-1的脫落酸（ABA）。取樣時(shí)間為處理后0 min、3 min、5 min、1 h、3 h和24 h。樣品采集部位為葉片，直接在液氮中冷凍后儲(chǔ)存于-80 ℃。

利用OminiPlant RNA Kit（康為世紀(jì)，北京，CW2598S）提取樣品RNA，采用HiFiScript gDNA Removal RT MasterMix（康為世紀(jì)，北京，CW2020M）試劑盒進(jìn)行反轉(zhuǎn)錄，合成cDNA第一鏈，用于quantitative real-time PCR（qRT-PCR）分析，儀器為L(zhǎng)ightCycler?96（Roche，德國(guó)）。選取桃樹(shù)（Prupe.6G163400）作為內(nèi)參基因[19]，每個(gè)樣品3次重復(fù)，用2-ΔΔCT方法計(jì)算基因相對(duì)表達(dá)量[20]。反應(yīng)條件設(shè)定：預(yù)變性95 ℃、300 s；95 ℃、10 s變性，58 ℃、10 s退火，72 ℃、10 s后延伸，循環(huán)45次。本研究所需引物見(jiàn)表1。

表1 qRT-PCR驗(yàn)證引物序列

1.2.3 桃轉(zhuǎn)基因擬南芥表型觀察 構(gòu)建PRI101-FLAG-過(guò)表達(dá)載體，利用花序浸染法轉(zhuǎn)化擬南芥[18]，獲得轉(zhuǎn)基因株系。在同一四分板MS培養(yǎng)基播種野生型與3個(gè)轉(zhuǎn)基因株系擬南芥種子，每個(gè)株系播種20粒種子，4 ℃春化2 d，之后放置在組培室培養(yǎng)，每12 h進(jìn)行一次萌發(fā)率統(tǒng)計(jì)（種子露出白色胚根即為成功萌發(fā)），試驗(yàn)重復(fù)3次。萌發(fā)率（%）=萌發(fā)種子數(shù)/培養(yǎng)皿中全部種子數(shù)×100%。游標(biāo)卡尺統(tǒng)計(jì)根長(zhǎng)、下胚軸長(zhǎng)度。

1.2.4轉(zhuǎn)基因擬南芥轉(zhuǎn)錄組測(cè)序分析 選取MS培養(yǎng)基中生長(zhǎng)7 d的野生型與轉(zhuǎn)基因擬南芥幼苗用液氮速凍，干冰運(yùn)輸至百邁客公司，文庫(kù)質(zhì)檢合格后，使用Illumina NovaSeq6000測(cè)序平臺(tái)進(jìn)行測(cè)序，利用百邁客云平臺(tái)進(jìn)行數(shù)據(jù)分析。

2 結(jié)果

2.1 桃PpSAUR73與擬南芥長(zhǎng)勢(shì)相關(guān)蛋白的系統(tǒng)發(fā)育樹(shù)

為進(jìn)一步研究PpSAUR73與擬南芥的系統(tǒng)進(jìn)化關(guān)系，采用MEGA7軟件構(gòu)建了桃、擬南芥長(zhǎng)勢(shì)相關(guān)的SAUR蛋白系統(tǒng)進(jìn)化樹(shù)。PpSAUR73與AtSAUR14、15、19蛋白的關(guān)系較近（圖1）。

2.2 桃PpSAUR73對(duì)激素的瞬時(shí)響應(yīng)

為了探究桃對(duì)各種激素的響應(yīng)情況，對(duì)‘中油蟠9號(hào)’嫩梢進(jìn)行激素噴施并取樣，定量分析的表達(dá)量。結(jié)果顯示，桃能在噴施IAA后3 min內(nèi)出現(xiàn)快速響應(yīng)，表達(dá)量是對(duì)照（0 min）的3.1倍，在噴施后3 h時(shí)表達(dá)量達(dá)到對(duì)照的14.9倍，在24 h時(shí)為對(duì)照的4.6倍；在噴施NPA后，的表達(dá)量呈先下降再上升趨勢(shì)，在1 h時(shí)的表達(dá)量?jī)H為對(duì)照的0.54倍，24 h時(shí)上升到與對(duì)照持平水平；在噴施ABA后，表達(dá)量整體呈上升趨勢(shì)，最高在3 h時(shí)，為對(duì)照的3.4倍；在噴施乙烯后，的表達(dá)量呈上升下降再上升下降的趨勢(shì)，1 h時(shí)表達(dá)量最高，為對(duì)照的2.4倍（圖2）。

圖1 PpSAUR73與擬南芥長(zhǎng)勢(shì)相關(guān)蛋白的系統(tǒng)發(fā)育樹(shù)

圖2 PpSAUR73表達(dá)對(duì)激素的響應(yīng)分析

2.3 PpSAUR73轉(zhuǎn)基因擬南芥表型觀察

為探究的基因功能，構(gòu)建了過(guò)表達(dá)載體，遺傳轉(zhuǎn)化擬南芥，獲得轉(zhuǎn)基因擬南芥。結(jié)果顯示，種子萌芽率在24 h時(shí)出現(xiàn)顯著性差異，野生型擬南芥的萌芽率僅45%，但轉(zhuǎn)基因擬南芥能夠達(dá)到70%，甚至96%（圖3）。表明過(guò)量表達(dá)能夠影響種子萌芽。

對(duì)萌發(fā)7 d的轉(zhuǎn)基因擬南芥的根長(zhǎng)、下胚軸進(jìn)行觀察統(tǒng)計(jì)。結(jié)果顯示，相較于野生型，轉(zhuǎn)基因株系的根長(zhǎng)顯著增加，分別增加了32%、43%和43%；下胚軸長(zhǎng)度分別顯著增加60%、59%和74%（圖4），表明也具有促進(jìn)器官伸長(zhǎng)的功能。此外，過(guò)表達(dá)的擬南芥較野生型的蓮座大，一直到結(jié)果期，過(guò)表達(dá)的擬南芥都表現(xiàn)出長(zhǎng)勢(shì)顯著好于野生型。

Col-0：野生型；SAUR73-1、SAUR73-14、SAUR73-2：轉(zhuǎn)基因擬南芥株系。不同小寫(xiě)字母表示差異顯著（P＜0.05）。下同

圖4 野生型及轉(zhuǎn)基因擬南芥表型觀察

2.4 PpSAUR73過(guò)表達(dá)影響擬南芥根系對(duì)IAA和GA的敏感性

為了探究對(duì)激素的響應(yīng)，對(duì)野生型與過(guò)表達(dá)擬南芥進(jìn)行了不同濃度的IAA、GA及NPA處理（圖5）。結(jié)果顯示，與野生型相比，轉(zhuǎn)基因株系對(duì)IAA的敏感性大幅度降低。與Col-0相比，50 nmol?L-1IAA對(duì)3個(gè)轉(zhuǎn)基因擬南芥根長(zhǎng)生長(zhǎng)的抑制率分別降低了11%、4%和6%；75 nmol?L-1IAA對(duì)轉(zhuǎn)基因擬南芥根長(zhǎng)生長(zhǎng)的抑制率分別降低約8%、6%和4%；100 nmol?L-1IAA對(duì)轉(zhuǎn)基因擬南芥根長(zhǎng)生長(zhǎng)的抑制率分別降低約7%、7%和6%，隨著處理濃度增大，IAA對(duì)各擬南芥根系的抑制作用逐漸增大。與野生型相比，轉(zhuǎn)基因株系對(duì)GA與NPA的敏感性明顯增加。50 μmol?L-1的GA對(duì)各擬南芥根長(zhǎng)生長(zhǎng)的抑制差異并不顯著，均約為23%；100 μmol?L-1GA對(duì)不同擬南芥根長(zhǎng)生長(zhǎng)的抑制作用存在顯著性差異，分別約為28%、36%、38%和47%；150 μmol?L-1GA對(duì)擬南芥根長(zhǎng)生長(zhǎng)的抑制作用更加明顯，抑制率分別約為46%、53%、55%和57%。10和20 μmol?L-1NPA對(duì)轉(zhuǎn)基因擬南芥根長(zhǎng)生長(zhǎng)的抑制率均顯著高于野生型。由此可知，過(guò)量表達(dá)降低了植株對(duì)IAA的敏感性，提高了對(duì)GA與NPA敏感性。

圖5 IAA、GA及NPA對(duì)擬南芥根長(zhǎng)生長(zhǎng)的影響

2.5 轉(zhuǎn)錄組數(shù)據(jù)的實(shí)時(shí)熒光定量PCR驗(yàn)證

對(duì)野生型（CK）及SAUR73-1、SAUR73-14兩個(gè)轉(zhuǎn)基因擬南芥株系進(jìn)行轉(zhuǎn)錄組測(cè)定，從轉(zhuǎn)錄組中篩選出9個(gè)基因進(jìn)行qRT-PCR驗(yàn)證（圖6）。轉(zhuǎn)錄組測(cè)得的FPKM值與這9個(gè)基因相對(duì)表達(dá)量的變化趨勢(shì)一致，從而驗(yàn)證了RNA-seq數(shù)據(jù)的可靠性。

圖6 差異表達(dá)基因的qRT-PCR驗(yàn)證

2.6 差異基因表達(dá)分析

將SAUR73-1、SAUR73-14分別與CK進(jìn)行差異比較分析（圖7），顯示在兩個(gè)對(duì)比組中均表達(dá)的差異基因有128個(gè)，其中有84個(gè)上調(diào)基因，44個(gè)下調(diào)基因，對(duì)這些差異基因的表達(dá)繪制熱圖，篩選出20個(gè)表達(dá)量相對(duì)高的差異基因（表2）。

2.7 差異基因功能分析

將CK與SAUR73-1、SAUR73-14的共同差異基因進(jìn)行GO功能顯著性富集分析。結(jié)果表明，差異表達(dá)基因在細(xì)胞組分方面富集的基因最多，定位在細(xì)胞質(zhì)、細(xì)胞膜、細(xì)胞器和細(xì)胞外區(qū)域等；其次是生物學(xué)過(guò)程方面富集的基因，與細(xì)胞過(guò)程、代謝過(guò)程、生物調(diào)節(jié)和對(duì)刺激的反應(yīng)等有關(guān)；在分子功能方面富集的基因較少，與其他物質(zhì)結(jié)合、催化活性、核酸結(jié)合轉(zhuǎn)錄因子活性、結(jié)構(gòu)分子活性、抗氧化活性以及分子功能調(diào)節(jié)劑等有關(guān)（圖8-A）。對(duì)差異表達(dá)基因進(jìn)行KEGG通路富集分析（圖8-B），結(jié)果顯示，差異基因主要富集到苯丙氨酸生物合成通路、植物激素信號(hào)轉(zhuǎn)導(dǎo)通路、淀粉蔗糖代謝通路、MAPK信號(hào)通路。其中，苯丙氨酸生物合成通路與植物激素信號(hào)轉(zhuǎn)導(dǎo)通路可能與生長(zhǎng)勢(shì)密切相關(guān)。

2.8 苯丙氨酸代謝合成途徑

對(duì)苯丙氨酸代謝合成通路進(jìn)行分析（圖9），挖掘差異表達(dá)基因（表3），發(fā)現(xiàn)差異基因中AT1G05260、AT3G01190、AT3G32980、AT5G15180 4個(gè)編碼過(guò)氧化物酶基因表達(dá)上調(diào)，而過(guò)氧化物酶在木質(zhì)素的合成過(guò)程中發(fā)揮關(guān)鍵作用[21]，推測(cè)過(guò)表達(dá)可能參與調(diào)控木質(zhì)素合成。

A：樣品間差異表達(dá)基因維恩圖；B：共同差異表達(dá)基因熱圖

2.9 植物激素信號(hào)轉(zhuǎn)導(dǎo)通路

KEGG通路富集顯示（圖10），過(guò)表達(dá)擬南芥的差異基因在生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)、脫落酸信號(hào)轉(zhuǎn)導(dǎo)通路富集。對(duì)上述通路中的差異表達(dá)基因進(jìn)行分析，發(fā)現(xiàn)過(guò)量表達(dá)下調(diào)生長(zhǎng)素的響應(yīng)基因AT1G16510（）、AT1G56150（）、AT1G75580（）、AT3G12830（）、AT4G34770（）表達(dá)，其中基因AT1G16510（）與AT1G75580（）還可對(duì)植物的衰老進(jìn)行調(diào)控，AT1G56150（）表達(dá)對(duì)ABA信號(hào)傳導(dǎo)和葉綠體的功能狀態(tài)進(jìn)行調(diào)控[22]。在脫落酸信號(hào)轉(zhuǎn)導(dǎo)通路，過(guò)量表達(dá)上調(diào)磷酸酶蛋白AT3G11410（）表達(dá)，下調(diào)脫落酸信號(hào)通路基因AT5G05440（），編碼ABA信號(hào)傳導(dǎo)負(fù)調(diào)節(jié)器，而能夠調(diào)節(jié)絲氨酸、蘇氨酸磷酸酶活性。盡管是生長(zhǎng)素響應(yīng)基因，但對(duì)脫落酸通路有調(diào)控作用，并且田間激素噴施試驗(yàn)也發(fā)現(xiàn)對(duì)脫落酸有響應(yīng)。

表2 SAUR73部分共同差異基因的信息描述

3 討論

3.1 PpSAUR73對(duì)激素的響應(yīng)

廣泛參與細(xì)胞生理發(fā)育過(guò)程，涉及植物生長(zhǎng)發(fā)育的激素和環(huán)境調(diào)控過(guò)程，而植物激素之間也存在相互作用，互相影響[23]。作為生長(zhǎng)素響應(yīng)基因，擬南芥能夠?qū)AA作出快速響應(yīng)[8]，在IAA處理30 min后，—都發(fā)生兩到三倍的上調(diào)[24]，過(guò)表達(dá)—能夠?qū)TH作出響應(yīng)，突變體對(duì)ETH的敏感性降低[25]。本研究通過(guò)對(duì)田間桃樹(shù)進(jìn)行激素噴施，發(fā)現(xiàn)對(duì)IAA能夠做出快速響應(yīng)，并且發(fā)現(xiàn)其也能對(duì)ABA、ETH作出響應(yīng)。轉(zhuǎn)擬南芥影響種子萌芽，而種子萌芽受到赤霉素的調(diào)控[26]，但轉(zhuǎn)錄組數(shù)據(jù)中并沒(méi)有富集到赤霉素信號(hào)轉(zhuǎn)導(dǎo)，推測(cè)是在種子萌芽期間影響赤霉素水平，但在幼苗及成苗以后不影響。以上均說(shuō)明可能參與多種激素信號(hào)轉(zhuǎn)導(dǎo)過(guò)程。擬南芥種子的萌發(fā)、根長(zhǎng)、下胚軸長(zhǎng)度等與激素水平有關(guān)，且激素在調(diào)控樹(shù)勢(shì)生長(zhǎng)中也具有重要的作用，有研究表明蘋(píng)果倒貼皮和矮化中間砧可以降低韌皮部IAA水平從而使樹(shù)體矮化[27]；ABA水平與核桃實(shí)生苗生長(zhǎng)勢(shì)呈負(fù)相關(guān)[28]；GA處理24 h后，栽培種和野生種曼陀羅種子的發(fā)芽率和幼苗的鮮質(zhì)量、干質(zhì)量、根長(zhǎng)以及根表面積均顯著提高[29]。本研究發(fā)現(xiàn)擬南芥萌發(fā)、根長(zhǎng)、下胚軸長(zhǎng)度都發(fā)生了變化，能夠促進(jìn)器官伸長(zhǎng)，猜測(cè)該基因可能具有影響激素水平的功能，而激素是調(diào)控樹(shù)體生長(zhǎng)發(fā)育的關(guān)鍵因素之一，由此推測(cè)，該基因也可能通過(guò)影響激素水平而調(diào)控桃樹(shù)樹(shù)勢(shì)。

A：SAUR73共同差異表達(dá)基因的GO功能類別；B：SAUR73共同差異表達(dá)基因的KEGG代謝通路富集

表3 苯丙氨酸合成代謝通路中涉及的差異基因

圖 9 苯丙氨酸合成代謝通路圖

圖10 植物激素信號(hào)轉(zhuǎn)導(dǎo)中差異表達(dá)基因聚類熱圖

3.2 過(guò)表達(dá)PpSAUR73使器官伸長(zhǎng)

擬南芥中發(fā)現(xiàn)多個(gè)基因能夠調(diào)控細(xì)胞伸長(zhǎng)，定位在細(xì)胞質(zhì)膜上，在下胚軸和根中高表達(dá)，正向調(diào)控?cái)M南芥的生長(zhǎng)和生長(zhǎng)素轉(zhuǎn)運(yùn)，過(guò)表達(dá)使細(xì)胞擴(kuò)張導(dǎo)致下胚軸長(zhǎng)度增加[6,24-25,30]；過(guò)表達(dá)會(huì)導(dǎo)致細(xì)胞與器官的伸長(zhǎng)并且影響頂鉤的發(fā)育[11]；在擬南芥中發(fā)現(xiàn)過(guò)表達(dá)融合蛋白能夠使下胚軸、雄蕊絲變長(zhǎng)，并且發(fā)現(xiàn)下胚軸中積累的IAA較多[6]；—通過(guò)影響擬南芥的乙烯受體信號(hào)傳導(dǎo)并促進(jìn)植物生長(zhǎng)，使子葉、蓮座葉變大，葉片表皮細(xì)胞面積變大[25]；過(guò)表達(dá)—也均被發(fā)現(xiàn)能夠促進(jìn)細(xì)胞伸長(zhǎng)[6,24,30-35]。筆者實(shí)驗(yàn)室前期研究也發(fā)現(xiàn)，過(guò)表達(dá)能夠使擬南芥器官伸長(zhǎng)[18]。與前人研究一致，本研究中的表型試驗(yàn)表明過(guò)表達(dá)能夠使轉(zhuǎn)基因擬南芥的根及下胚軸伸長(zhǎng)、蓮座變大。

3.3 轉(zhuǎn)錄組分析

過(guò)表達(dá)影響生長(zhǎng)素水平、生長(zhǎng)素極性轉(zhuǎn)運(yùn)和生長(zhǎng)素通路基因的表達(dá)[6,8,24,36]。同樣在本研究轉(zhuǎn)錄組測(cè)序中發(fā)現(xiàn)，過(guò)表達(dá)對(duì)IAA信號(hào)轉(zhuǎn)導(dǎo)發(fā)生調(diào)控，下調(diào)生長(zhǎng)素的響應(yīng)基因AT1G16510（）、AT1G56150（）、AT1G75580（）、AT3G12830（）、AT4G34770（）表達(dá)，其中AT1G16510（）與AT1G75580（）基因還可對(duì)植物的衰老進(jìn)行調(diào)控。過(guò)表達(dá)上調(diào)擬南芥表達(dá)，下調(diào)的表達(dá)。由此猜測(cè)過(guò)表達(dá)也對(duì)擬南芥的IAA水平產(chǎn)生影響。前人研究表明，已有許多對(duì)脫落酸有反應(yīng)[11]，如與脫落酸信號(hào)轉(zhuǎn)導(dǎo)相關(guān)[10,37]。同樣，本研究發(fā)現(xiàn)能夠調(diào)控IAA信號(hào)轉(zhuǎn)導(dǎo)，且能夠調(diào)控ABA信號(hào)轉(zhuǎn)導(dǎo)，這與相似。轉(zhuǎn)錄組數(shù)據(jù)顯示在野生型與SAUR73-1、SAUR73-14株系中均表達(dá)的差異基因有128個(gè)，在差異基因的KEGG代謝途徑分析中顯示，有4個(gè)編碼過(guò)氧化物酶基因表達(dá)上調(diào)，而過(guò)氧化物酶在木質(zhì)素的合成過(guò)程中發(fā)揮關(guān)鍵作用，而已有研究表明木質(zhì)素水平與長(zhǎng)勢(shì)呈顯著相關(guān)[38-43]，這暗示很可能是通過(guò)促進(jìn)木質(zhì)素的合成來(lái)調(diào)控植株的生長(zhǎng)勢(shì)。前人研究發(fā)現(xiàn)可通過(guò)與PP2C.D磷酸酶相互作用并抑制其磷酸酶活性，使質(zhì)外體酸化，細(xì)胞壁松弛，促進(jìn)細(xì)胞的伸長(zhǎng)生長(zhǎng)[2,7,24,44-45]。本研究轉(zhuǎn)錄組測(cè)序結(jié)果顯示，對(duì)PP2CA表達(dá)量產(chǎn)生了上調(diào)，但是否存在與相似的功能還需要進(jìn)一步鑒定。

4 結(jié)論

本研究發(fā)現(xiàn)能夠快速對(duì)激素做出響應(yīng)，且能夠調(diào)控轉(zhuǎn)基因擬南芥長(zhǎng)勢(shì)；過(guò)量表達(dá)導(dǎo)致的差異基因主要富集到苯丙氨酸生物合成通路、植物激素信號(hào)轉(zhuǎn)導(dǎo)通路、淀粉蔗糖代謝通路等代謝通路；調(diào)控IAA、ABA信號(hào)轉(zhuǎn)導(dǎo),推測(cè)其在桃樹(shù)的生長(zhǎng)發(fā)育過(guò)程中發(fā)揮重要作用，研究結(jié)果為采用分子手段控制樹(shù)勢(shì)提供了參考。

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Functional Identification of Peach Gene

YANG Li1, CAO HongBo1, ZHANG XueYing1, ZHAI HanHan2, LI XinMiao1, PENG JiaWei1, TIAN Yi3, CHEN HaiJiang1

1Horticultural Department, Agricultural University of Hebei, Baoding 071000, Hebei;2Kashgar Vocational and Technical College, Kashgar 844000, Xinjiang;3Mountainous Areas Research Institute, Hebei Agricultural University/Technology Innovation Center for Agriculture in Mountainous Areas of Hebei Province/National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071001, Hebei

【Objective】 The object of this study was to isolate a peach potential-related gene, to analyze its expression response to hormones, and to identify its role in regulating seedling growth in transgenic Arabidopsis, so as to provide the molecular basis for the regulation of tree potential. 【Method】 Using Zhongyou Pan 9 as the material for hormone treatment, the real-time fluorescence quantitative analysis was used to analyze the dynamic response ofwithin 24 hours.was cloned from the peach variety Jiuyan. PpSAUR73 overexpression vector was constructed and transformed into. Phenotypic observation of genetical modified Arabidopsis was carried out, and the germination rate statistics of both genetically modified and wild-type Arabidopsis sown simultaneously were performed too. The root length and hypocotyl of 7-day growing Arabidopsis with consistent germination were measured, and Arabidopsis with consistent germination was treated with different concentration hormone. Transcriptome sequencing was performed using 7-day-old seedlings, and the differentially expressed genes were analyzed by functional analysis, KEGG pathway enrichment analysis, and regulatory genes analysis, respectively.【Result】could respond quickly to hormone treatments. The overexpression ofcould affect the germination of Arabidopsis seeds. The hypocotyl and root length of seedlings were longer than those of wild type. In addition, the rosette of transgenic Arabidopsis was larger, and the overall growth potential was larger than wild type. The transgenic Arabidopsis showed decreased sensitivity to auxin. The transcriptome analysis of overexpressingshowed that there were 128 differentially expressed genes in both control groups, including 84 up-regulated genes and 44 down-regulated genes, and 20 differentially expressed genes were described. The GO function significant enrichment analysis of the differentially expressed genes generated by overexpression ofshowed that the differentially expressed genes were the most abundant in cell components, located in cytoplasm, cell membrane, organelle and extracellular regions. KEGG pathway enrichment analysis on differentially expressed genes were conducted, and the results showed that the differentially expressed genes in pairwise comparisons CK vsand CK vswere mainly enriched in phenylalanine biosynthesis pathway, plant hormone signal transduction pathway, starch sucrose metabolic pathway and other metabolic pathways. In the phenylalanine biosynthesis pathway,could regulate the upregulation of peroxidase encoding genes,,and. Peroxidases were associated with lignin synthesis, and lignin content was significantly correlated with plant growth, suggesting that overexpression ofmight be involved in regulating lignin synthesis inand thus growth. In plant hormone signal transduction pathway, the expression of some auxin responsive genes of,,,andin abscisic acid signal transduction pathway was up-regulated, and the expression of abscisic acid signal pathway genewas down-regulated.could regulate the growth ofand participate in multiple hormone signal transduction pathways. 【Conclusion】This study found thatcould quickly respond to hormones and regulate the growth in transgenic. The differentially expressed genes caused by overexpressed genes caused by overexpression ofwere mainly enriched in metabolic pathways, such as phenylalanine biosynthesis pathway, plant hormone signaling pathway, and starch sucrose metabolism pathway.also played an important role in IAA and ABA signal transduction pathways, it was speculated that it played an important role in the growth and development of peach trees.

peach;; gene expression; hormone treatment; signal transduction

10.3864/j.issn.0578-1752.2023.20.012

2023-03-24；

2023-06-30

財(cái)政部和農(nóng)業(yè)農(nóng)村部：國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系資助項(xiàng)目（CARS-30-2-03）、河北省重點(diǎn)研發(fā)計(jì)劃（20326804D）、熱雜果現(xiàn)代種業(yè)科技創(chuàng)新團(tuán)隊(duì)（21326310D）

楊麗，E-mail：1097125451@qq.com。通信作者陳海江，chenhaijiang2001@163.com。通信作者田義，tianyi@hebau.edu.cn

（責(zé)任編輯 趙伶俐）