水稻黃綠葉突變體ygl3的鑒定與基因功能分析

許子怡，程行，沈奇，趙亞男，湯佳玉，劉喜

水稻黃綠葉突變體的鑒定與基因功能分析

許子怡，程行，沈奇，趙亞男，湯佳玉，劉喜

淮陰師范學(xué)院/江蘇省環(huán)洪澤湖生態(tài)農(nóng)業(yè)生物技術(shù)重點(diǎn)實(shí)驗(yàn)室/江蘇省區(qū)域現(xiàn)代農(nóng)業(yè)與環(huán)境保護(hù)協(xié)同創(chuàng)新中心，江蘇淮安 223300

【】為了豐富和加深人們對(duì)植物葉色形成的分子機(jī)理認(rèn)識(shí)，對(duì)水稻黃綠葉突變體（）進(jìn)行表型鑒定和基因克隆，闡明的分子功能，為解析調(diào)控水稻葉色形成的分子機(jī)理奠定基礎(chǔ)。從水稻中花11 CRISPR-Cas9敲除突變體庫(kù)中鑒定出2份穩(wěn)定遺傳的等位黃綠葉突變體，命名為與，對(duì)突變體的表型進(jìn)行鑒定，測(cè)定野生型和突變體苗期的葉綠素含量，運(yùn)用透射電鏡觀察野生型和突變體的葉綠體結(jié)構(gòu)。利用實(shí)時(shí)熒光定量PCR分析的組織表達(dá)模式，并使用BioXM 2.6軟件對(duì)YGL3及其同源蛋白序列進(jìn)行比對(duì)，采用酵母雙雜交方法篩選YGL3的互作蛋白。在苗期，與野生型相比，突變體葉片黃化，葉綠素、類(lèi)胡蘿卜素和總光合色素含量顯著降低。透射電鏡結(jié)果表明，突變體葉綠體形態(tài)異常，類(lèi)囊體片層結(jié)構(gòu)較少，而野生型葉綠體形態(tài)正常，類(lèi)囊體片層結(jié)構(gòu)排列有序。CRISPR-Cas9敲除位點(diǎn)鑒定結(jié)果表明，發(fā)生單堿基插入，導(dǎo)致蛋白翻譯提前終止，該基因編碼351個(gè)氨基酸的蛋白突變?yōu)?5個(gè)氨基酸的截短蛋白。與野生型相比，的表達(dá)水平在突變體中顯著下調(diào)。qRT-PCR結(jié)果表明在水稻根、穗、種子、葉鞘以及葉片中均有表達(dá)，且葉片中表達(dá)水平最高。編碼一個(gè)質(zhì)體定位的尿嘧啶核苷酸激酶。蛋白氨基酸序列比對(duì)表明YGL3蛋白在玉米、高粱、擬南芥等物種中均較為保守，與擬南芥同源蛋白氨基酸的同源性為59.4%。qRT-PCR結(jié)果表明，葉綠素合成基因（如、和）在突變體中顯著下調(diào)，而、和等葉綠素合成基因在野生型與突變體之間無(wú)顯著差異。酵母雙雜交系統(tǒng)篩選水稻葉片酵母cDNA文庫(kù)，發(fā)現(xiàn)YGL3與RNA編輯因子MORF8存在互作。水稻黃綠葉突變體的表型是由突變導(dǎo)致，該基因與已報(bào)道的水稻黃綠葉基因等位。在葉片中高度表達(dá)，同時(shí)YGL3與MORF8在酵母中互作。

水稻（L.）；黃綠葉；YGL3；CRISPR-Cas9；葉綠體發(fā)育

0 引言

【研究意義】水稻（L.）是研究植物功能基因的模式作物，也是世界三分之二人口的口糧。加強(qiáng)水稻分子生物學(xué)研究，對(duì)水稻分子設(shè)計(jì)育種具有重要意義。葉綠體是植物主要的光合器官，是脂肪、淀粉、激素等代謝產(chǎn)物的合成場(chǎng)所，對(duì)植物產(chǎn)量的獲得具有十分重要的作用[1-2]。葉色變異是一種常見(jiàn)的突變性狀，突變基因影響葉綠素的合成、降解以及葉綠體的發(fā)育。多數(shù)水稻葉色變異突變體光合作用效率下降，植株生長(zhǎng)發(fā)育受阻，造成水稻減產(chǎn)[3-4]。葉色變異通常出現(xiàn)在苗期，也會(huì)出現(xiàn)在分蘗期和抽穗期，葉色表型易于識(shí)別鑒定。近年來(lái)，水稻葉色變異相關(guān)基因的鑒定與克隆，豐富了水稻葉色形成的分子機(jī)制，為水稻高光效育種提供基因資源。此外，黃化、白條紋、白化轉(zhuǎn)綠等葉色變異表型可以作為形態(tài)標(biāo)記，用于保證雜交稻制種和不育系繁殖的純度[5-6]?！厩叭搜芯窟M(jìn)展】水稻葉色變異可以分為條紋、白化、黃化、斑點(diǎn)葉、白化轉(zhuǎn)綠和持綠等。植物葉綠素合成以谷氨酰-tRNA為底物，最終生成葉綠素a和葉綠素b，涉及27個(gè)基因編碼的15種酶[7]。目前，水稻中已經(jīng)克隆了14個(gè)直接參與葉綠素合成的基因，這些基因突變都會(huì)引起葉色異常[8-10]。編碼谷氨酰胺tRNA還原酶，其突變體表現(xiàn)出淡綠葉的表型[8]。編碼葉綠素合成酶，突變植株表現(xiàn)為葉片黃化[9]。編碼鎂原卟啉Ⅸ單酯環(huán)化酶的催化亞基，參與葉綠素合成[10]。目前，水稻中已鑒定并克隆葉色變異基因超過(guò)120個(gè)，多數(shù)涉及葉綠素的生物合成、降解以及葉綠體的發(fā)育[11-12]。此外，質(zhì)-核信號(hào)轉(zhuǎn)導(dǎo)途徑受阻[13]、轉(zhuǎn)錄后修飾[14]、質(zhì)體轉(zhuǎn)錄復(fù)合物形成受阻[15]、葉綠體蛋白酶調(diào)控受阻[16-17]、葉綠體蛋白轉(zhuǎn)運(yùn)受阻[18]、ATP轉(zhuǎn)運(yùn)蛋白基因[19]、轉(zhuǎn)錄因子與表觀遺傳[20-21]等突變機(jī)制也被鑒定出來(lái)。【本研究切入點(diǎn)】目前，約120個(gè)水稻葉色相關(guān)基因已被克隆，但水稻葉色形成調(diào)控網(wǎng)絡(luò)極其復(fù)雜，需要鑒定更多的涉及葉色變異的功能基因，而利用CRISPR-Cas9技術(shù)創(chuàng)制突變體庫(kù)，從中篩選鑒定葉色變異突變體，是鑒定葉色控制基因的有效途徑?！緮M解決的關(guān)鍵問(wèn)題】本研究從中花11 CRISPR- Cas9突變體庫(kù)中篩選獲得黃綠葉突變體，對(duì)突變體進(jìn)行表型鑒定、光合色素含量測(cè)定和葉綠體形態(tài)結(jié)構(gòu)觀察，并克隆，進(jìn)行的組織表達(dá)分析、蛋白氨基酸序列分析以及互作蛋白的篩選，以期解析調(diào)控葉綠體發(fā)育的分子機(jī)理，為在水稻分子育種上的應(yīng)用奠定基礎(chǔ)。

1 材料與方法

1.1 試驗(yàn)材料

從中花11 CRISPR-Cas9突變體庫(kù)中鑒定到2份葉片黃化突變體與。2個(gè)黃綠葉突變體經(jīng)多代自交，黃綠葉能夠穩(wěn)定遺傳。

1.2 光合色素含量測(cè)定

選取生長(zhǎng)一致的野生型和突變體各10株，參考Porra等[22]方法測(cè)定苗期葉片的光合色素含量。稱取0.2 g葉片，剪碎放入含有5 mL 95%無(wú)水乙醇的離心管中，搖勻黑暗處理48h，每隔12 h搖勻一次，設(shè)置3次生物重復(fù)。用雙通道紫外分光光度計(jì)（TU1901，北京）測(cè)定在663、645和470 nm波長(zhǎng)下的吸光值。

1.3 透射電鏡觀察

選取二葉期的野生型和突變體葉片，置于含有2.5%戊二醛固定液的離心管中，抽真空1 h，室溫放置24 h。再用四氧化鋨固定，而后經(jīng)過(guò)脫水、置換、包埋和切片，以JEM-1200Ex型透射電鏡觀察拍照。

1.4 CRISPR-Cas9敲除位點(diǎn)鑒定

利用Primer Premier 5軟件設(shè)計(jì)CRISPR-Cas9敲除位點(diǎn)測(cè)序引物（表1）。分別取50 mg野生型和突變體葉片剪碎，加入30 μL DNA提取液（北京聚合美生物科技有限公司；MF848），用200 μL槍頭將葉片搗碎，置于Eppendorf Mastercycler nexusX2梯度PCR儀中95℃5 min，然后離心1 min。吸取上清液2 μL作為模板進(jìn)行PCR擴(kuò)增；PCR反應(yīng)體系為DNA 2 μL、引物2 μL、10×Buffer 25 μL、dNTP（10 mmol·L-1）10 μL、KOD 0.1 μL和ddH2O 10.9 μL。PCR反應(yīng)程序?yàn)?4℃2 min；98℃10 s，55℃ 30 s，68℃ 30 s，33個(gè)循環(huán)；68℃5 min，4℃5 min。PCR產(chǎn)物進(jìn)行測(cè)序，利用BioXM 2.6軟件進(jìn)行DNA序列比對(duì)，確定突變位點(diǎn)。

1.5 氨基酸序列比對(duì)

利用YGL3蛋白氨基酸序列在NCBI（http://www. ncbi.nlm.nih.gov/）中查找不同物種中YGL3的同源蛋白，下載不同物種的氨基酸序列，使用BioXM 2.6軟件對(duì)氨基酸序列進(jìn)行比對(duì)分析。

1.6 相關(guān)基因的表達(dá)分析

利用康為世紀(jì)RNApure Plant Kit（Cat.#CW0559）提取中花11不同組織和突變體RNA，使用PrimeScriptRT Reagent Kit（TaKaRa）反轉(zhuǎn)錄合成cDNA。熒光定量PCR反應(yīng)體系為8.8 μL模板、1.2 μL引物和10 μL SYBR Green，在Bio-rad CFX96熒光定量PCR儀上進(jìn)行擴(kuò)增，引物序列參考劉喜等[23]。

表1 本研究所用引物序列

1.7 酵母雙雜交

將的全長(zhǎng)CDS克隆融合到pGBKT7載體中，將的全長(zhǎng)CDS克隆融合到pGADT7載體中，采用Clontech酵母轉(zhuǎn)化試劑盒（Cat.#630489）將不同組合的質(zhì)粒共轉(zhuǎn)化酵母菌AH109，涂布于二缺培養(yǎng)基（SD/-Leu/-Trp，DDO），30℃生化培養(yǎng)箱培養(yǎng)2—3 d。然后，挑取二缺培養(yǎng)基上的3個(gè)單克隆置于四缺培養(yǎng)基（SD/-Leu/-Trp/-His/-Ade，QDO），30℃生化培養(yǎng)箱培養(yǎng)5 d。酵母轉(zhuǎn)化步驟參照Clontech說(shuō)明書(shū)。

2 結(jié)果

2.1 突變體表型考察及CRISPR-Cas9敲除位點(diǎn)的鑒定

從中花11 CRISPR-Cas9突變體庫(kù)中篩選獲得2份穩(wěn)定遺傳黃綠葉突變體與。與野生型相比，苗期突變體與均表現(xiàn)出黃葉表型（圖1-A）。光合色素含量測(cè)定結(jié)果表明，苗期突變體的葉綠素a、葉綠素b、總色素含量和類(lèi)胡蘿卜素含量均顯著低于野生型（圖1-B），表明突變體黃葉表型主要由光合色素含量降低導(dǎo)致。CRISPR-Cas9編輯靶標(biāo)位點(diǎn)測(cè)序發(fā)現(xiàn)在野生型和突變體間存在單堿基插入，導(dǎo)致編碼的蛋白翻譯提前終止，即是功能缺失型突變體（圖1-C—圖1-D）。利用qRT-PCR分析在野生型和突變體中的表達(dá)水平，結(jié)果表明，與野生型相比，在突變體中表達(dá)水平顯著降低（圖1-E）。

A：苗期野生型和突變體ygl3的表型；B：苗期野生型和突變體ygl3光合色素含量測(cè)定；C：CRISPR-Cas9敲除位點(diǎn)的鑒定；D：野生型和突變體氨基酸序列比對(duì)；E：LOC_Os01g73450表達(dá)分析

2.2 葉綠體形態(tài)結(jié)構(gòu)觀察

為探究野生型和突變體葉綠體發(fā)育的差異，選取苗期突變體和野生型的葉片進(jìn)行透射電鏡觀察。結(jié)果表明，野生型葉綠體結(jié)構(gòu)正常，規(guī)則地貼壁分布，葉綠體內(nèi)基粒結(jié)構(gòu)正常，類(lèi)囊體片層結(jié)構(gòu)有序排列（圖2-A和圖2-B），而突變體葉綠體形態(tài)異常，類(lèi)囊體片層顯著減少，無(wú)序排列（圖2-C和圖2-D）。以上結(jié)果說(shuō)明，與野生型相比，突變體的葉綠體發(fā)育異常。

2.3 YGL3的組織表達(dá)分析

為研究的表達(dá)模式，在Rice eFP Browser（http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi）網(wǎng)站上對(duì)的表達(dá)模式進(jìn)行預(yù)測(cè)，結(jié)果顯示，在各個(gè)組織中都有表達(dá)，呈組成型表達(dá)，葉片中表達(dá)水平最高。為了驗(yàn)證預(yù)測(cè)結(jié)果，選取中花11植株的根、葉鞘、葉、穗以及種子提取RNA，反轉(zhuǎn)錄成cDNA用于qRT-PCR分析。所得結(jié)果和預(yù)測(cè)結(jié)果基本一致，證實(shí)呈組成型表達(dá)（圖3）。

2.4 YGL3及其同源蛋白氨基酸序列的比對(duì)

編碼一個(gè)含有351個(gè)氨基酸定位于質(zhì)體的尿嘧啶核苷酸激酶。在水稻基因組中尿嘧啶核苷酸激酶基因只有1個(gè)拷貝。通過(guò)對(duì)水稻、玉米、高粱、擬南芥、漸尖二型花以及野生稻等物種中尿嘧啶核苷酸激酶進(jìn)行氨基酸序列進(jìn)行比對(duì)，發(fā)現(xiàn)水稻YGL3與玉米、高粱的尿嘧啶核苷酸激酶氨基酸序列有約60%的相似性（圖4），尿嘧啶核苷酸激酶在結(jié)構(gòu)域上較為保守，N端差異較大。

A、B：野生型葉綠體結(jié)構(gòu)；C、D：突變體ygl3葉綠體結(jié)構(gòu)。Bar=5 μm（A、C）和Bar=2 μm（B、D）

圖3 YGL3的組織表達(dá)分析

2.5 葉綠素合成基因表達(dá)分析

與野生型相比，突變體的色素含量顯著降低，暗示的突變影響葉綠素合成途徑。因此，通過(guò)對(duì)野生型和突變體中葉綠素合成基因進(jìn)行qRT-PCR分析。與野生型相比，突變體中葉綠素合成基因（編碼谷氨酰-tRNA合成酶）、（編碼尿卟啉原氧化脫羧酶）和（編碼尿卟啉原脫羧酶）的轉(zhuǎn)錄水平顯著降低（圖5）。葉綠素合成基因、、、的表達(dá)水平在突變體中上調(diào)或無(wú)差異。因此，葉綠素合成途徑相關(guān)基因表達(dá)異常可能是導(dǎo)致突變體葉綠素含量降低的原因之一。

2.6 YGL3與MORF8在酵母中互作

為進(jìn)一步闡明YGL3的分子功能，構(gòu)建pGBKT7- YGL3融合表達(dá)載體，利用水稻葉片酵母cDNA文庫(kù)，進(jìn)行酵母雙雜交篩選YGL3的互作蛋白。鑒定獲得4個(gè)與YGL3互作的克隆，均為RNA編輯因子MORF8的片段（圖6-A）。隨后，構(gòu)建pGADT7-MORF8融合表達(dá)載體，進(jìn)行YGL3與MORF8的互作驗(yàn)證。與陰性對(duì)照相比，YGL3-BD與MORF8-AD組合可以在缺陷培養(yǎng)基（SD-Leu/-Trp/-His/-Ade）上生長(zhǎng)，表明在酵母體內(nèi)YGL3與MORF8存在互作（圖6-B）。

3 討論

葉綠體是綠色植物特有的細(xì)胞器，正常的葉綠體對(duì)植物生長(zhǎng)發(fā)育至關(guān)重要[24]。葉綠素生物合成和葉綠體發(fā)育的調(diào)控網(wǎng)絡(luò)極其復(fù)雜，需要鑒定更多的基因完善葉綠體發(fā)育的調(diào)控路徑。目前，已克隆的水稻葉色相關(guān)基因超過(guò)120個(gè)。在已克隆的基因中，有直接編碼葉綠素合成與降解的基因如[25]、[26]和[27]，有調(diào)控葉綠體發(fā)育的基因如[28]、[29]和[30]。本研究鑒定葉色突變體在苗期植株葉片為黃化，與野生型相比，突變體葉片葉綠素a、葉綠素b、總色素和類(lèi)胡蘿卜素含量均顯著降低。葉綠體透射電鏡觀察結(jié)果表明，與野生型相比，突變體葉綠體形態(tài)異常，類(lèi)囊體片層結(jié)構(gòu)排列無(wú)序。水稻黃葉突變體葉綠體基粒片層顯著降低[9]；黃綠葉突變體的葉綠體形態(tài)不規(guī)則，有較多空的囊泡狀結(jié)構(gòu)[31]。本研究中葉綠體形態(tài)異常，類(lèi)囊體片層減少，分布不規(guī)則，推測(cè)突變體黃葉的表型可能由葉綠體發(fā)育異常和光合色素含量下降引起。

NP_188498.1：擬南芥；NP_001137013.1：玉米；OEL23965.1：漸尖二型花；XP_002456998.1：高粱；XP_015693434.1：野生稻；YGL3：水稻。完全或部分保守的氨基酸分別是藍(lán)色和粉色標(biāo)示

HEMA：編碼谷氨酰-tRNA合成酶；HEME：編碼尿卟啉原脫羧酶；HEML：編碼谷氨酰-1-半醛轉(zhuǎn)氨酶；HEMB：編碼膽色素原合酶；HEMC：編碼羥甲基后膽色素原合酶；HEMF：編碼糞卟啉Ⅲ氧化酶；URO-D：編碼尿卟啉原氧化脫羧酶。**：在0.01水平差異極顯著

目前，水稻研究進(jìn)入后基因組學(xué)時(shí)代，越來(lái)越多的等位基因被克隆，等位基因的發(fā)現(xiàn)有助于進(jìn)一步闡明該基因的分子功能。因此，收集鑒定更多的水稻葉色突變體，將會(huì)推動(dòng)對(duì)葉色調(diào)控分子路徑的認(rèn)識(shí)。本研究中的黃葉基因是水稻第1染色體核基因編碼的尿嘧啶核苷酸激酶OsUMPK。Zhu等[32]通過(guò)圖位克隆鑒定到的等位基因，突變體在整個(gè)生育期均表現(xiàn)出黃綠葉。此外，Chen等[33]通過(guò)甲基磺酸乙酯誘變蜀恢527鑒定到的等位突變體，研究中分離鑒定的發(fā)生單堿基替換，產(chǎn)生新的截?cái)噢D(zhuǎn)錄本。與野生型相比，突變體葉片黃綠色，成熟期突變體矮化。本研究是通過(guò)CRISPR-Cas9技術(shù)編輯中花11獲得，突變位點(diǎn)在第一外顯子上，與、的突變位點(diǎn)不同，3個(gè)等位突變體葉片表現(xiàn)為黃化，葉片色素含量降低，但下降的幅度均不一致，可能是遺傳背景導(dǎo)致的。

DDO：基本營(yíng)養(yǎng)缺陷培養(yǎng)基SD/-Leu/-Trp；QDO：選擇營(yíng)養(yǎng)缺陷培養(yǎng)基SD-Leu/-Trp/-His/-Ade

在擬南芥中存在一個(gè)同源基因，其T-DNA插入突變體表現(xiàn)出矮化、葉色異常的表型，PUMPKIN參與質(zhì)體基因-UCC、-UAC、、以及的內(nèi)含子剪切[34]。目前，水稻已經(jīng)報(bào)道了17個(gè)PPR蛋白、1個(gè)MORF蛋白以及3個(gè)剪切因子參與質(zhì)體基因轉(zhuǎn)錄后調(diào)控。編碼一個(gè)PLS-DYW亞家族的PPR蛋白，其突變導(dǎo)致植株白化致死，參與質(zhì)體基因轉(zhuǎn)錄本編輯和內(nèi)含子剪切[35]。PLS-DYW亞家族的PPR蛋白DUA1調(diào)控低溫下與的RNA編輯[36]。編碼一個(gè)P家族PPR蛋白，參與轉(zhuǎn)錄本編輯和4個(gè)質(zhì)體基因（、、和）內(nèi)含子剪切[37]。編碼一個(gè)葉綠體內(nèi)含子剪切促進(jìn)因子，影響多個(gè)質(zhì)體基因內(nèi)含子剪切[38]。本研究利用酵母雙雜交系統(tǒng)篩選到一個(gè)YGL3的互作蛋白MORF8。MORF家族蛋白在擬南芥已經(jīng)被證實(shí)參與質(zhì)體基因轉(zhuǎn)錄后調(diào)控，如RNA編輯、RNA剪切[39-40]。然而，在水稻中，只鑒定出一個(gè)MORF蛋白WSP1的功能，突變體表現(xiàn)出葉片白條紋和白穗的表型，參與多個(gè)質(zhì)體基因（、、和）RNA編輯以及內(nèi)含子的剪切[41]。的突變是否參與質(zhì)體基因轉(zhuǎn)錄后調(diào)控，將是下一步研究的重點(diǎn)。

4 結(jié)論

黃綠葉突變體表型由基因突變導(dǎo)致。突變體苗期葉綠素a、葉綠素b、總色素和類(lèi)胡蘿卜素含量顯著減少，葉綠體發(fā)育異常，類(lèi)囊體片層減少。在水稻中呈組成型表達(dá)，且葉片中表達(dá)最高。YGL3與RNA編輯因子MORF8在酵母中存在互作。是已報(bào)道的的等位基因，因編碼框發(fā)生單堿基插入導(dǎo)致蛋白翻譯提前終止而引起黃葉表型。

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Identification and Gene Functional Analysis of Yellow Green Leaf Mutant

XU ZiYi, CHENG Xing, SHEN Qi, ZHAO YaNan, TANG JiaYu, LIU Xi

Huaiyin Normal University/Jiangsu Key Laboratory for Eco-agriculture Biotechnology Around Hongze Lake/Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environment Protection, Huaian 223300, Jiangsu

【】To enrich and deepen people’s understanding of the molecular mechanism of plant leaf color, the phenotype identification and gene cloning of the yellow green leaf mutant() were carried out to clarify the molecular function ofand lay the foundation for elucidating the molecular mechanism ofregulating rice leaf color.【】Two stable genetic allelic yellow green leaf mutants,and, were isolated from the CRISPR-Cas9 knockout mutant library of Zhonghua 11. The phenotype of the mutant was identified, and the chlorophyll contents of the wild-type andwere determined. The chloroplast structure of the wild-type andwas observed by transmission electron microscope. qRT-PCR was used to analyze the tissue expression of, and BioXM2.6 software was used for sequence alignment of YGL3 and its homologs. Yeast two hybrid was used to screen the interacting proteins of YGL3.【】Compared with the wild type, the leaves ofwere yellowing, and the contents of chlorophyll, carotenoid and total photosynthetic pigment at seedling stage inwere significantly decreased. Transmission electron microscopy showed that the chloroplast morphology ofwas abnormal, and the thylakoid lamellar structure was less, whereas the chloroplast morphology of the wild type was normal and the thylakoid lamellar structure was orderly arranged. CRISPR-Cas9 knock-out site identification showed that thegene had a single base insertion, which resulted in the early termination of protein translation. The gene encoding 351 amino acids was mutated into a truncated protein with 55 amino acids. Compared with the wild type, the expression level ofwas significantly down-regulated in the mutants. qRT-PCR showed thatwas expressed in roots, panicles, seeds, leaf sheaths and leaves.was highly expressed in leaves.encodes a plastid localized UMP kinase. The YGL3 protein was conserved in,and. YGL3 shared the high sequence homology (59.4% amino acid identity) to. qRT-PCR showed that chlorophyll synthesis genes, including,and,were significantly down-regulated in, whereas the expression levels of,andwere no significant difference between the wild type and. Yeast two hybrid screen showed that YGL3 interacted with RNA editing factor MORF8.【】The phenotype of the yellow leaf mutantresulted from themutation.was an allele of the yellow green leave genewas highly expressed in leaves, and YGL3 interacted with MORF8 in yeasts.

rice (L.); yellow green leaf;; CRISPR-Cas9; chloroplast development

10.3864/j.issn.0578-1752.2021.15.001

2020-12-14；

2021-02-10

江蘇省高等學(xué)校自然科學(xué)研究面上項(xiàng)目（19KJB180009）、淮陰師范學(xué)院大學(xué)生創(chuàng)新創(chuàng)業(yè)計(jì)劃（202019009XJ）

許子怡，E-mail：2631140968@qq.com。通信作者劉喜，E-mail：1240623244@qq.com

（責(zé)任編輯 李莉）