fl(2)d單等位基因的敲除顯著降低小菜蛾的生殖力和育性

李飛飛，王貝貝，賴穎芳，楊菲穎，尤民生，何瑋毅

單等位基因的敲除顯著降低小菜蛾的生殖力和育性

李飛飛，王貝貝，賴穎芳，楊菲穎，尤民生，何瑋毅

閩臺(tái)作物有害生物生態(tài)防控國(guó)家重點(diǎn)實(shí)驗(yàn)室/福建農(nóng)林大學(xué)應(yīng)用生態(tài)研究所/福建農(nóng)林大學(xué)教育部害蟲(chóng)生態(tài)防控國(guó)際合作聯(lián)合實(shí)驗(yàn)室/福建農(nóng)林大學(xué)農(nóng)業(yè)農(nóng)村部閩臺(tái)作物有害生物綜合治理重點(diǎn)實(shí)驗(yàn)室/海峽兩岸特色作物安全生產(chǎn)省部共建協(xié)同創(chuàng)新中心，福州 350002

【】RNA甲基化是基因轉(zhuǎn)錄后水平表觀修飾的主要形式，參與了眾多重要的細(xì)胞學(xué)過(guò)程。小菜蛾（）是危害十字花科蔬菜的重要寡食性害蟲(chóng)，與RNA甲基化相關(guān)基因的功能尚未見(jiàn)報(bào)道。本研究通過(guò)克隆小菜蛾的RNA甲基化蛋白同源基因，鑒定其表達(dá)模式，并敲除該基因以探究其生物學(xué)功能。通過(guò)小菜蛾基因組網(wǎng)站查找基因序列，PCR擴(kuò)增其蛋白質(zhì)編碼序列（CDS）；采用實(shí)時(shí)熒光定量PCR（qRT-PCR）技術(shù)，檢測(cè)小菜蛾不同發(fā)育階段個(gè)體以及成蟲(chóng)生殖腺中的相對(duì)表達(dá)量；運(yùn)用CRISPR/Cas9結(jié)合卵的顯微注射技術(shù)，對(duì)小菜蛾進(jìn)行編輯；將被編輯過(guò)的成蟲(chóng)與野生型成蟲(chóng)雜交，并對(duì)其產(chǎn)生的后代進(jìn)行近交，篩選突變品系；觀測(cè)并比較突變體與野生型個(gè)體遺傳特性、生物學(xué)參數(shù)和表型的差異，明確的功能?？寺〉玫介L(zhǎng)度為912 bp的CDS，在雌蛹、雌成蟲(chóng)和卵中的表達(dá)量較高，雄成蟲(chóng)和雄蛹的表達(dá)量較低，幼蟲(chóng)期的表達(dá)量最低，成蟲(chóng)卵巢中表達(dá)量顯著高于精巢。通過(guò)向小菜蛾的卵注射靶向的向?qū)NA（sgRNA）和Cas9蛋白的混合物，對(duì)所產(chǎn)生的陽(yáng)性后代進(jìn)行10代的單對(duì)近交篩選，獲得3種雜合的移碼突變品系，分別缺失了4個(gè)（Δ213-4）、5個(gè)（Δ213-5）和7個(gè)（Δ214-7）堿基。在上述品系的篩選過(guò)程中，發(fā)現(xiàn)了6只缺失4個(gè)堿基的純合突變個(gè)體，2只缺失5個(gè)堿基的純合突變個(gè)體；缺失4個(gè)堿基的純合個(gè)體成功配成了兩對(duì)，近交未產(chǎn)卵；剩余的2只缺失4個(gè)堿基的雄性純合個(gè)體和2只缺失5個(gè)堿基的雄性純合個(gè)體分別與同世代的雌性雜合突變個(gè)體近交后仍未產(chǎn)卵。說(shuō)明純合突變的個(gè)體存活率極低，且可能無(wú)法產(chǎn)生后代。通過(guò)分析后代基因型的分離比，發(fā)現(xiàn)雜合突變個(gè)體近交以及雜合突變個(gè)體與野生型個(gè)體雜交產(chǎn)生的后代中，雜合突變個(gè)體與野生型個(gè)體的比例分別略小于2和1，說(shuō)明雜合突變會(huì)影響小菜蛾正常的生長(zhǎng)發(fā)育，并導(dǎo)致部分個(gè)體死亡。雜合突變體后代中含有突變的雌雄個(gè)體比例接近1﹕1（＜0.05），推測(cè)小菜蛾可能與性別決定無(wú)關(guān)。只要是有突變品系小菜蛾所參與的交配，雌成蟲(chóng)產(chǎn)卵量和卵的孵化率均顯著低于野生型（＜0.01），所產(chǎn)的卵多數(shù)發(fā)育異常，表現(xiàn)為失水皺縮、不能正常孵化。通過(guò)對(duì)成蟲(chóng)的生殖腺進(jìn)行解剖，發(fā)現(xiàn)在野生型雌成蟲(chóng)與突變體雄成蟲(chóng)交配后，卵巢內(nèi)卵的附著量較未交配的個(gè)體明顯減少；未交配的突變體雌成蟲(chóng)卵巢內(nèi)卵的附著量亦少于野生型，而突變體雄成蟲(chóng)的精巢未見(jiàn)明顯異常。部分能夠孵化的雜合突變個(gè)體在整個(gè)發(fā)育過(guò)程會(huì)發(fā)生不同程度的畸變，導(dǎo)致不能正常完成整個(gè)世代；另外一些雜合突變個(gè)體未見(jiàn)異常，可以將突變類(lèi)型遺傳給后代。根據(jù)上述發(fā)現(xiàn)，提出了基于的小菜蛾遺傳防控模型。參與小菜蛾的生殖過(guò)程和胚胎發(fā)育，突變后顯著影響后代種群數(shù)量，是開(kāi)展小菜蛾遺傳控制的理想靶標(biāo)。

小菜蛾；；雜合突變體；生殖力；育性

0 引言

【研究意義】小菜蛾（）屬鱗翅目（Lepidoptera）菜蛾科（Plutellidae），是危害十字花科蔬菜的重要害蟲(chóng)，全球每年用于其防治的費(fèi)用及其造成的損失高達(dá)40—50億美元[1]。在我國(guó)南方菜區(qū)，小菜蛾可終年發(fā)生危害，世代重疊嚴(yán)重、抗藥性強(qiáng)，促使研究人員不斷探索控制其種群增長(zhǎng)的方法[2-3]。隨著有害生物綜合治理（integrated pest management，IPM）理念的提出，人們不再拘泥于傳統(tǒng)的防治策略，而是致力于開(kāi)發(fā)綠色、環(huán)保、有利于生態(tài)平衡的可持續(xù)治理方法[4-5]。當(dāng)前，害蟲(chóng)災(zāi)變機(jī)理的研究已邁入組學(xué)時(shí)代，基于害蟲(chóng)遺傳控制的原理和方法，研發(fā)小菜蛾綠色防控技術(shù)顯示出了巨大的潛力和優(yōu)勢(shì)[6-8]。篩選合適的靶標(biāo)基因，研究配套的遺傳操作系統(tǒng)，將進(jìn)一步提升害蟲(chóng)遺傳控制的效率和穩(wěn)定性[9-10]。RNA甲基化是基因轉(zhuǎn)錄后水平表觀修飾的主要形式，rRNA、mRNA、tRNA甚至是非編碼RNA都會(huì)出現(xiàn)超過(guò)100多種的修飾類(lèi)型，其中以m6A（6-腺苷酸甲基化）最為常見(jiàn)[11-12]。m6A甲基化存在于絕大多數(shù)生物體內(nèi)，包括哺乳動(dòng)物、昆蟲(chóng)、細(xì)菌和病毒。m6A影響生物體基因表達(dá)、mRNA穩(wěn)定性、RNA可變剪接、蛋白翻譯效率和X染色體失活等重要的細(xì)胞學(xué)過(guò)程[13-14]。參與m6A甲基化過(guò)程蛋白的失活會(huì)影響生物體正常的生命活動(dòng)，探明小菜蛾中m6A甲基化蛋白成員female- lethal-2-d（fl(2)d）的生物學(xué)功能，對(duì)今后以該基因作為靶標(biāo)建立小菜蛾遺傳防控系統(tǒng)具有重要意義。【前人研究進(jìn)展】近年來(lái)，人們將RNA甲基化修飾抗體富集技術(shù)和高通量測(cè)序技術(shù)相結(jié)合，實(shí)現(xiàn)了對(duì)m6A甲基化在轉(zhuǎn)錄組水平的分布和豐度檢測(cè)[15-16]。在人類(lèi)和小鼠的基因組中，m6A甲基化位點(diǎn)高度保守，大多集中在終止密碼子附近和長(zhǎng)的內(nèi)部外顯子，說(shuō)明這種修飾在基因表達(dá)的表觀遺傳調(diào)控中發(fā)揮了重要作用[17]。參與m6A代謝和信號(hào)途徑的蛋白有三大類(lèi)，分別是RNA甲基化蛋白（writers）、去甲基化蛋白（erasers）和甲基化閱讀蛋白（readers），它們均以復(fù)合物的形式發(fā)揮功能[18]。一旦哺乳動(dòng)物體內(nèi)的這些蛋白出現(xiàn)異常，就會(huì)引發(fā)一系列的疾病，包括腫瘤、神經(jīng)性疾病和發(fā)育遲緩等[15,19-20]。Writers主要包括methyltransferase-like 3（METTL3）、methyltransferase-like 14（METTL14）、fl(2)d、vir-Like m6A methyltransferase associated（KIAA1429/VIRMA）和RNA-binding motif protein 15（RBM15）[18,21-22]，在昆蟲(chóng)中的報(bào)道較少。家蠶（）中發(fā)現(xiàn)，和在滯育品系中的表達(dá)量高于非滯育品系，說(shuō)明m6A甲基化介導(dǎo)的轉(zhuǎn)錄后表觀遺傳調(diào)控可能參與了家蠶滯育的發(fā)生[23]；在細(xì)胞水平利用RNA干擾（RNAi）技術(shù)沉默和，會(huì)導(dǎo)致家蠶細(xì)胞周期進(jìn)程停止、染色體聯(lián)會(huì)和分離失敗[24]。、和的表達(dá)水平以及m6A含量在工蜂和蜂王幼蟲(chóng)階段不同時(shí)期發(fā)生顯著變化，說(shuō)明m6A甲基化影響了蜜蜂的幼蟲(chóng)發(fā)育和級(jí)型分化[25]。的研究常見(jiàn)于果蠅[26-28]，該基因主要有3個(gè)靶基因：（）、和，前兩個(gè)基因是性別決定通路的上游基因，主要通過(guò)影響這兩個(gè)基因前體mRNA的可變剪接影響果蠅的性別決定、劑量補(bǔ)償、卵子形成與分化等[29-32]。也可以調(diào)控第3個(gè)靶基因的可變剪接，但對(duì)雌、雄兩性的影響并無(wú)差異[33]。此外，在果蠅胚胎和卵巢中高表達(dá)，在卵子形成的早期，fl(2)d蛋白在不同的細(xì)胞核間移動(dòng)，說(shuō)明它是一種影響卵子發(fā)生的關(guān)鍵核蛋白[34]?！颈狙芯壳腥朦c(diǎn)】昆蟲(chóng)RNA甲基化的研究目前僅見(jiàn)于家蠶、蜜蜂和果蠅，在小菜蛾上還未見(jiàn)報(bào)道。通過(guò)參與RNA甲基化過(guò)程影響了眾多生物學(xué)過(guò)程，是開(kāi)展害蟲(chóng)遺傳控制潛在的靶標(biāo)。由于鱗翅目昆蟲(chóng)中同源基因不具有決定昆蟲(chóng)性別的功能[35-36]，利用基因編輯技術(shù)clustered regular interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）探明小菜蛾同源基因是否參與性別決定、生殖及其他重要生長(zhǎng)發(fā)育過(guò)程，可為今后利用該基因進(jìn)行害蟲(chóng)的遺傳防控打下理論基礎(chǔ)?！緮M解決的關(guān)鍵問(wèn)題】小菜蛾的序列及其表達(dá)模式；建立小菜蛾突變品系；突變品系遺傳特性、生物學(xué)參數(shù)和表型的變化；在小菜蛾生長(zhǎng)發(fā)育中的功能。

1 材料與方法

試驗(yàn)于2019—2020年在福建農(nóng)林大學(xué)閩臺(tái)作物有害生物生態(tài)防控國(guó)家重點(diǎn)實(shí)驗(yàn)室和應(yīng)用生態(tài)研究所完成。

1.1 試驗(yàn)材料與試劑

蟲(chóng)源為本實(shí)驗(yàn)室長(zhǎng)期保存的人工飼料飼養(yǎng)的小菜蛾品系（AD），飼養(yǎng)溫度為（25±1）℃，光周期為16 h﹕8 h（L﹕D），相對(duì)濕度為70%—80%?；蚩寺∷镁隇榇竽c桿菌DH5。

福州尚亞生物科技有限公司提供引物及測(cè)序服務(wù)?？俁NA提取試劑盒、GoScriptTMReverse Transcription System逆轉(zhuǎn)錄試劑盒、GoTaq qPCR Master Mix實(shí)時(shí)定量PCR（qRT-PCR）試劑盒和無(wú)核酸酶水購(gòu)于Promega；2×Hieff Canace? PCR Master Mix高保真酶購(gòu)于翊圣生物科技有限公司；DNA提取試劑盒購(gòu)于天根生化科技有限公司；膠回收試劑盒購(gòu)于Omega；克隆載體PJET1.2和RNA體外轉(zhuǎn)錄試劑盒MEGAscript RNAi kit購(gòu)于ThermoFisher；敲除試驗(yàn)的相關(guān)試劑購(gòu)于金斯瑞生物科技有限公司。

1.2 RNA提取和cDNA合成

使用RNA提取試劑盒并按照說(shuō)明書(shū)提取不同樣品小菜蛾的總RNA，使用分光光度計(jì)檢測(cè)RNA的質(zhì)量，用1%（質(zhì)量體積分?jǐn)?shù)）的瓊脂糖凝膠電泳檢測(cè)RNA的完整性。取2 μg總RNA，參照逆轉(zhuǎn)錄試劑盒說(shuō)明書(shū)合成第一條單鏈cDNA，-20℃保存?zhèn)溆谩?/p>

1.3 基因克隆和序列分析

以小菜蛾卵的cDNA為模板，根據(jù)小菜蛾基因組數(shù)據(jù)庫(kù)（http://iae.fafu.edu.cn/DBM）[8,37]提供的基因序列信息設(shè)計(jì)引物（表1），擴(kuò)增蛋白質(zhì)編碼序列（CDS）區(qū)域。PCR程序：98℃ 3 min；98℃ 30 s，58℃ 30 s，72℃ 1 min，30個(gè)循環(huán)；72℃ 10 min。PCR擴(kuò)增后進(jìn)行膠回收純化，將基因片段連接到克隆載體上并轉(zhuǎn)化至大腸桿菌DH5感受態(tài)細(xì)胞中，挑取陽(yáng)性克隆進(jìn)行測(cè)序，獲得目的基因序列信息。將獲得的CDS序列與對(duì)應(yīng)基因組序列進(jìn)行比對(duì)，界定外顯子與內(nèi)含子區(qū)域。

表1 本研究所用引物序列

下劃線標(biāo)記的序列表示sgRNA，斜體部分的序列表示T7啟動(dòng)子

The sequences underlined indicate the sgRNAs, and the sequences in italic denote the T7 promoter

1.4 實(shí)時(shí)熒光定量PCR

收集小菜蛾不同發(fā)育階段的個(gè)體和成蟲(chóng)的生殖腺，每一樣品各設(shè)置4個(gè)生物學(xué)重復(fù)，提取總RNA并逆轉(zhuǎn)錄成cDNA。其中收集交配過(guò)的雌蟲(chóng)所產(chǎn)2 h以內(nèi)的卵，0.5 g/重復(fù)；4齡幼蟲(chóng)，5只/重復(fù)；化蛹后第2天的蛹，5只/重復(fù)；初羽化未交配的成蟲(chóng)（分雌雄），5只/重復(fù)，并分別解剖相應(yīng)的卵巢和精巢，50個(gè)/重復(fù)。液氮處理后，-80℃保存?zhèn)溆?。以小菜蛾核糖體蛋白基因ribosomal protein L32（）為內(nèi)參[38]，設(shè)計(jì)實(shí)時(shí)熒光定量PCR引物（表1）。參照試劑盒說(shuō)明書(shū)配制20 μL反應(yīng)體系，在實(shí)時(shí)熒光定量PCR系統(tǒng)（ABI QuantStudioTM6 Flex）上進(jìn)行反應(yīng)，反應(yīng)程序：95℃ 10 min；95℃ 15 s，60℃ 30 s，40個(gè)循環(huán)；95℃ 15 s，60℃ 1 min；95℃ 15 s。采用2-ΔΔCt的方法計(jì)算的相對(duì)表達(dá)量。

1.5 sgRNA設(shè)計(jì)及體外轉(zhuǎn)錄

參照Chen等[38]的方法，按照GGN19GG原則，在靠近5′端的外顯子區(qū)域設(shè)計(jì)2條向?qū)NA（sgRNA），同時(shí)利用Cas9脫靶效應(yīng)網(wǎng)站（http://www. rgenome.net/cas-offinder）進(jìn)行檢驗(yàn)。在2條sgRNA的兩端設(shè)計(jì)另外2條檢測(cè)引物，用于后續(xù)突變位點(diǎn)的檢測(cè)（表1）。

上游引物CRISPR-F由三部分組成：T7聚合酶結(jié)合位點(diǎn)、sgRNA序列以及sgRNA骨架質(zhì)粒的部分序列，下游引物CRISPR-R由與sgRNA骨架質(zhì)?；パa(bǔ)的部分序列組成，擴(kuò)增模板是sgRNA骨架質(zhì)粒。對(duì)擴(kuò)增后的PCR產(chǎn)物進(jìn)行膠收純化，使用試劑盒對(duì)PCR產(chǎn)物進(jìn)行體外轉(zhuǎn)錄，最后用氯仿異戊醇純化sgRNA。-80℃保存?zhèn)溆谩?/p>

1.6 卵的顯微注射

注射前，采用以下方法和體系進(jìn)行Cas9蛋白和sgRNA的預(yù)處理：1 μL Cas9蛋白（200 ng·μL-1），2 μL sgRNA（250 ng·μL-1）、0.5 μL 10×Cas Nuclease Reaction Buffer，加水補(bǔ)足至5 μL，37℃條件下孵育15 min。選擇小菜蛾雌成蟲(chóng)交配后第2天產(chǎn)的卵進(jìn)行注射，將卵卡置于成蟲(chóng)產(chǎn)卵盒中30 min后拿出，然后使用顯微注射系統(tǒng)（IM 300 Programmable Cell Microinjector）將Cas9蛋白和sgRNA混合物注射入卵。將注射完成的卵置于培養(yǎng)皿中，在25℃、相對(duì)濕度70%的條件下進(jìn)行孵化，隨后將孵化的幼蟲(chóng)轉(zhuǎn)移至人工飼料上。

1.7 突變體篩選

將注射后的卵發(fā)育成的后代稱為G0代，待其發(fā)育到成蟲(chóng)后，分別將單只與性別相反的野生型小菜蛾成蟲(chóng)雜交。產(chǎn)生子一代（G1）后，提取G0代成蟲(chóng)基因組DNA，利用突變體檢測(cè)引物進(jìn)行PCR擴(kuò)增，對(duì)PCR產(chǎn)物進(jìn)行測(cè)序。通過(guò)觀察PAM結(jié)構(gòu)附近有無(wú)突變，確定產(chǎn)生突變的群體。自G1代起，均采用同種群內(nèi)隨機(jī)近交的方式篩選突變體，待產(chǎn)出后代后，檢測(cè)親本的突變類(lèi)型。對(duì)檢測(cè)為突變的親代所對(duì)應(yīng)的后代進(jìn)行保存，剔除親本均為野生型的后代，直至篩選出2條染色體均被編輯的純合突變品系（圖1）。

圓圈表示細(xì)胞核，圈中長(zhǎng)方形表示編輯的目標(biāo)染色體，空白填充表示野生型，不同顏色的填充表示不同類(lèi)型的突變

1.8 拍照記錄及數(shù)據(jù)分析

用數(shù)碼顯微系統(tǒng)（VHX-2000C，Keyence）對(duì)突變品系中發(fā)育異常的卵進(jìn)行拍照觀察，并與野生型的卵進(jìn)行比較。分別解剖15—20只雜合突變品系初羽化未交配的雌成蟲(chóng)卵巢和雄成蟲(chóng)精巢，以及與突變體雄成蟲(chóng)交配后12 h內(nèi)未產(chǎn)卵的野生型雌成蟲(chóng)卵巢進(jìn)行拍照觀察，觀察完成后提取DNA進(jìn)行PCR檢測(cè)，用于確定是否為突變個(gè)體，并與野生型初羽化未交配成蟲(chóng)的卵巢和精巢進(jìn)行比較。產(chǎn)卵量數(shù)據(jù)的收集采用觀察法，野生型觀察了25對(duì)，除了缺失4個(gè)堿基的突變體類(lèi)型觀察了3—5對(duì)，其他突變體類(lèi)型為10—18對(duì)。記錄單對(duì)小菜蛾成蟲(chóng)交配后2 d內(nèi)產(chǎn)的卵為第一次數(shù)據(jù)，換一次卵卡后交配2 d內(nèi)產(chǎn)的卵為第二次數(shù)據(jù)，兩次的數(shù)據(jù)相加即為最終產(chǎn)卵量。在每次收集卵卡的當(dāng)天開(kāi)始統(tǒng)計(jì)孵化幼蟲(chóng)的數(shù)量，連續(xù)統(tǒng)計(jì)3 d，兩次統(tǒng)計(jì)得到的孵化幼蟲(chóng)總和除以產(chǎn)卵總數(shù)即為孵化率。采用Microsoft Office Excel（2016）軟件對(duì)野生型和突變品系成蟲(chóng)的產(chǎn)卵量及卵的孵化率進(jìn)行差異顯著性檢測(cè)并繪圖；采用卡方（Chi-square）檢驗(yàn)檢測(cè)不同突變類(lèi)型的遺傳比率以及突變后代的性比。

2 結(jié)果

2.1 小菜蛾fl(2)d的鑒定及表達(dá)模式

克隆得到的CDS長(zhǎng)度為912 bp（圖2-A），預(yù)測(cè)編碼304個(gè)氨基酸。通過(guò)與基因組序列進(jìn)行比對(duì)，發(fā)現(xiàn)含有4個(gè)外顯子，5個(gè)內(nèi)含子。利用國(guó)際生物技術(shù)信息中心（NCBI）上的保守結(jié)構(gòu)域數(shù)據(jù)庫(kù)（CDD）網(wǎng)站（https://www.ncbi. nlm.nih.gov/Structure/ cdd）分析，發(fā)現(xiàn)其CDS在第85—237個(gè)氨基酸位置處有一個(gè)WTAP超家族保守功能域[31,39]（圖2-B）。qRT-PCR結(jié)果顯示，在雌蛹、雌成蟲(chóng)和卵中的表達(dá)量較高，雄成蟲(chóng)和雄蛹的表達(dá)量較低，不同性別幼蟲(chóng)的表達(dá)量均最低（圖2-C），卵巢中表達(dá)量顯著高于精巢（圖2-D），說(shuō)明該基因可能與小菜蛾的生殖過(guò)程和胚胎發(fā)育有關(guān)。

A：fl(2)d CDS序列的克隆CDS cloning of fl(2)d；M：DNA分子標(biāo)準(zhǔn)DNA marker。B：預(yù)測(cè)的fl(2)d蛋白質(zhì)序列Predicted protein sequence offl(2)d；紅色字母：保守功能域Red letters indicate the conserved domain。C：fl(2)d在不同發(fā)育階段的表達(dá)Expression of fl(2)d in different developmental stages；E：卵Egg；FL：雌幼蟲(chóng)female larva；ML：雄幼蟲(chóng)male larva；FP：雌蛹Female pupa；MP：雄蛹Male pupa；FA：雌成蟲(chóng)Female adult；MA：雄成蟲(chóng)Male adult；采用單因素方差分析法進(jìn)行差異顯著性檢驗(yàn)，使用Tukey法進(jìn)行多重比較，不同字母表示差異顯著（P＜0.05）Significant difference analysis was performed using one-way ANOVA followed by a Tukey’s HSD post hoc test. Different letters indicate significant difference at P＜0.05 level。D：fl(2)d在成蟲(chóng)生殖腺的表達(dá)Expression of fl(2)d in adult gonads；O：卵巢Ovary；T：精巢Testis；采用t檢驗(yàn)進(jìn)行差異顯著性分析，**表示檢驗(yàn)性水平P＜0.01 Significant difference analysis was performed using t test. Double asterisks indicate significant difference of P＜0.01

2.2 CRISPR/Cas9介導(dǎo)的fl(2)d編輯及其突變類(lèi)型

采用CRISPR/Cas9基因編輯技術(shù)，在的2號(hào)外顯子上設(shè)計(jì)2條sgRNA，對(duì)小菜蛾卵進(jìn)行Cas蛋白和sgRNA的混合注射。共注射了160個(gè)小菜蛾G0代卵，成功羽化64只。將成功羽化的成蟲(chóng)與野生型小菜蛾進(jìn)行兩兩配對(duì)，待其產(chǎn)生后代后，提取G0代的64只成蟲(chóng)及3只干癟蛹的基因組DNA，利用檢測(cè)引物進(jìn)行PCR擴(kuò)增。對(duì)產(chǎn)物進(jìn)行測(cè)序和比對(duì)分析，發(fā)現(xiàn)成功引起突變的sgRNA只有1條（圖3-A），發(fā)生突變的成蟲(chóng)只有3只（突變率為1.88%，圖3-B）、干癟蛹1只。對(duì)發(fā)生突變的3只成蟲(chóng)產(chǎn)生的后代分別進(jìn)行保種，將每個(gè)種群的成蟲(chóng)隨機(jī)近交，篩選突變純合體。經(jīng)過(guò)10代的近交篩選，只獲得了3種不同的雜合突變類(lèi)型，分別缺失4個(gè)（Δ213-4）、5個(gè)（Δ213-5）和7個(gè)（Δ214-7）堿基（圖3-C），均導(dǎo)致的移碼突變，后續(xù)試驗(yàn)所用突變品系的小菜蛾均為這3種雜合類(lèi)型。

A：fl(2)d基因結(jié)構(gòu)及sgRNA靶點(diǎn)示意圖Schematic diagram of the fl(2)d gene structure and sgRNA target site。B：G0代野生型（上）和突變體（下）成蟲(chóng)fl(2)d測(cè)序峰圖；紅色框是sgRNA，藍(lán)色框是PAM結(jié)構(gòu)Sequencing chromatograms of fl(2)d of wild-type (top) and mutant (bottom) adults; The edited site is indicated by a red rectangle and the protospacer adjacent motif (PAM) sequence is showed by a blue rectangle。C：突變序列，虛線表示缺失的序列Mutant sequences, the dashed lines represent the deleted bases caused by CRISPR/Cas9

2.3 小菜蛾fl(2)d基因突變的遺傳效應(yīng)

通過(guò)分析雜合突變個(gè)體近交后代共362只個(gè)體，在G3代和G4代中各獲得了3只Δ213-4突變品系的純合小菜蛾個(gè)體，且成功配成兩對(duì)，但同為純合突變個(gè)體近交后無(wú)卵產(chǎn)生。將剩余的兩只雄性純合突變個(gè)體，分別與同代雌性雜合突變個(gè)體近交后也無(wú)卵產(chǎn)生。同時(shí)，在G3代獲得了2只Δ213-5突變品系的雄性純合小菜蛾，分別與同代雌性突變個(gè)體近交后，仍無(wú)卵產(chǎn)生。除了在G3代和G4代出現(xiàn)了純合突變品系，在10代的近交過(guò)程中均未再出現(xiàn)純合突變個(gè)體。因此推測(cè)，純合突變個(gè)體的存活率極低，且可能表現(xiàn)為不育，純合突變的類(lèi)型無(wú)法遺傳。由于G1代近交產(chǎn)生的突變類(lèi)型較多，且大多數(shù)突變類(lèi)型或無(wú)法遺傳或?yàn)橥x突變，因此從G2代起，對(duì)篩選過(guò)程中各個(gè)世代3種突變類(lèi)型的親本和后代共992個(gè)個(gè)體的基因型開(kāi)展分析。發(fā)現(xiàn)不同比例的基因型分離情況符合孟德?tīng)栠z傳定律，經(jīng)卡方檢驗(yàn)達(dá)到顯著水平（表2）。值得注意的是，親本為雜合突變個(gè)體與野生型的近交后代中，雜合突變個(gè)體和野生型的比值略低于1，親本均為雜合突變個(gè)體的近交后代中，雜合突變個(gè)體和野生型比值略低于2，說(shuō)明突變基因型的存在可能影響了后代的存活率。

2.4 小菜蛾fl(2)d的生物學(xué)功能

通過(guò)統(tǒng)計(jì)單只配對(duì)的雌成蟲(chóng)產(chǎn)卵和卵孵化情況，發(fā)現(xiàn)無(wú)論是哪種突變類(lèi)型，其親本或均為雜合突變體，亦或親本只有一方為雜合突變體，產(chǎn)卵量和孵化率顯著低于野生型（＜0.01，圖4-A、4-B），產(chǎn)卵量和孵化率較野生型相比分別減少了49.17%—93.22%和69.49%—100%。值得注意的是，當(dāng)雌雄親本均為缺失4個(gè)堿基的雜合突變品系時(shí)，孵化率明顯低于其他交配組合，推測(cè)其移碼突變產(chǎn)生的新蛋白可能對(duì)小菜蛾胚胎正常的生長(zhǎng)發(fā)育具有負(fù)面效應(yīng)。同時(shí)，統(tǒng)計(jì)了突變親本產(chǎn)生的后代中突變個(gè)體的性別比例，發(fā)現(xiàn)在G2到G10代的試驗(yàn)過(guò)程中，每一代突變個(gè)體的雌雄比例及G2至G10代的突變個(gè)體總和的雌雄比例均接近1﹕1，且經(jīng)卡方檢驗(yàn)達(dá)到顯著水平（圖4-C，＜0.05，2=1.30），說(shuō)明該基因可能不影響小菜蛾的性別決定。

2.5 小菜蛾fl(2)d突變個(gè)體的生殖表型與利用

注射Cas9蛋白和sgRNA后的G0代卵，自孵化出幼蟲(chóng)起至成蟲(chóng)產(chǎn)卵后死亡，整個(gè)世代過(guò)程無(wú)明顯異樣。但通過(guò)使突變品系的成蟲(chóng)進(jìn)行單對(duì)交配，即讓雜合突變個(gè)體與野生型個(gè)體雜交或雜合突變個(gè)體近交，發(fā)現(xiàn)雌成蟲(chóng)產(chǎn)卵2 d后的總卵量明顯少于野生型（圖5-A、5-B），且部分卵隨著發(fā)育時(shí)間的延長(zhǎng)逐漸失水皺縮，無(wú)法孵化出幼蟲(chóng)（圖5-C、5-D）。同時(shí)，還有部分卵在產(chǎn)出5 d后，雖然表型與野生型的初孵卵無(wú)異，但仍無(wú)法正常孵化出健康幼蟲(chóng)（圖5-E、5-F）。通過(guò)解剖發(fā)現(xiàn)，未交配突變體雌成蟲(chóng)卵巢內(nèi)卵的附著量比野生型顯著下降，因此導(dǎo)致突變體雌成蟲(chóng)所參與的交配組合產(chǎn)卵量的下降。與突變體雄成蟲(chóng)交配過(guò)的野生型雌成蟲(chóng)，其卵巢內(nèi)卵的附著量也明顯少于交配前的數(shù)量（圖5-G、5-H）。未交配的突變品系雄成蟲(chóng)生殖腺與野生型比較，無(wú)明顯異常。試驗(yàn)過(guò)程中發(fā)現(xiàn)部分突變品系的個(gè)體在發(fā)育至幼蟲(chóng)、蛹及成蟲(chóng)階段均會(huì)發(fā)生不同程度的畸變，暗示可能參與了小菜蛾的生長(zhǎng)發(fā)育。另外一些雜合突變個(gè)體未見(jiàn)異常，可以將突變類(lèi)型遺傳給后代。

A：突變品系的產(chǎn)卵量The fecundity of mutant strains。B：突變品系的孵化率The hatchability of mutant strains。WT：野生型成蟲(chóng)雜交Hybridization between wild-type adults；F-4：雜合雌性Δfl(2)d213-4成蟲(chóng)與野生型雜交Hybridization of heterozygous female adult of Δfl(2)d213-4 with wild-type；M-4：雜合雄性Δfl(2)d213-4成蟲(chóng)與野生型雜交Hybridization of heterozygous male adult of Δfl(2)d213-4 with wild-type；-4：雜合Δfl(2)d213-4成蟲(chóng)近交Inbreeding between heterozygous adults of Δfl(2)d213-4；F-5：雜合雌性Δfl(2)d213-5成蟲(chóng)與野生型雜交Hybridization of heterozygous female adult of Δfl(2)d213-5 with wild-type；M-5：雜合雄性Δfl(2)d213-5成蟲(chóng)與野生型雜交Hybridization of heterozygous male adult of Δfl(2)d213-5 with wild-type；-5：雜合Δfl(2)d213-5成蟲(chóng)近交Inbreeding between heterozygous adults of Δfl(2)d213-5；F-7：雜合雌性Δfl(2)d214-7成蟲(chóng)與野生型雜交Hybridization of heterozygous female adult of Δfl(2)d214-7 with wild-type；M-7：雜合雄性Δfl(2)d214-7成蟲(chóng)與野生型雜交Hybridization of heterozygous male adult of Δfl(2)d214-7 with wild-type；-7：雜合Δfl(2)d214-7成蟲(chóng)近交Inbreeding between heterozygous adults of Δfl(2)d214-7。C：性別比例The sex ratios of mutant offspring；采用t檢驗(yàn)進(jìn)行差異顯著性分析，**表示顯著性水平P＜0.01 Significant difference analysis was performed using t test. Double asterisks indicate significant difference of P＜0.01

表2 G2—G10代fl(2)d基因型分離情況

斜杠表示未檢測(cè)。親本只統(tǒng)計(jì)基因型，不考慮性別The slash denotes the missing data. Only genotypes of the parents were recorded regardless of the sexuality。A：野生型基因型Wild-type genotype；a：突變型基因型Mutant genotype

A：野生型雌成蟲(chóng)產(chǎn)卵情況The fecundity of wild-type female adult。B：突變體雌成蟲(chóng)產(chǎn)卵情況The fecundity of mutant female adult。C、D：失水皺縮的卵The shrinking and dehydrated eggs。E：未能正常孵化的卵Eggs that do not hatch successfully。F：未能正常出卵的幼蟲(chóng)The larvae that fail to come out of eggs。G：未交配野生型雌成蟲(chóng)卵巢Ovary of the wild-type virgin female adult。H：與突變體雄成蟲(chóng)交配后野生型雌成蟲(chóng)卵巢Ovary of the wild-type female adult mated with the mutant male adult。除A圖外，所觀察的卵均為突變體雌成蟲(chóng)與野生型雄成蟲(chóng)單對(duì)交配后所產(chǎn)的后代，突變體類(lèi)型為Δfl(2)d213-5 Except for figure A, the presented phenotypes of the eggs are all produced from the single-pair mating of mutant female adult and wild-type male adult, and the type of mutation is Δfl(2)d213-5

鑒于單等位基因的突變顯著影響了小菜蛾的產(chǎn)卵量及孵化率但不影響性別比例的特性，筆者提出了一個(gè)防控模型（圖6）：首先構(gòu)建兩種基因組被編輯的小菜蛾品系，一種為被綠色熒光蛋白（GFP）基因插入單個(gè)基因座并發(fā)生堿基移碼突變的小菜蛾，另一種為被紅色熒光蛋白（RFP）基因插入到雌性特有的W染色體的小菜蛾。將兩種編輯品系的小菜蛾進(jìn)行雜交，通過(guò)觀察后代卵的熒光類(lèi)型，只保留發(fā)單一綠色熒光的小菜蛾雄性卵，大量飼養(yǎng)至成蟲(chóng)進(jìn)行田間釋放。從理論上講，只需要周期性釋放被編輯的小菜蛾雄成蟲(chóng)就能夠達(dá)到預(yù)期的防控目的。

圖6 基于fl(2)d的小菜蛾遺傳防控模型

3 討論

盡管已經(jīng)在許多物種中開(kāi)展了與RNA甲基化相關(guān)的研究，但對(duì)昆蟲(chóng)的研究?jī)H局限于其影響家蠶的滯育、蜜蜂的幼蟲(chóng)發(fā)育和級(jí)型分化以及果蠅的生殖過(guò)程、胚胎發(fā)育和性別決定[23,25,28]。本研究選擇參與m6A甲基化進(jìn)程的writers蛋白fl(2)d，通過(guò)qRT-PCR分析了該基因在小菜蛾體內(nèi)的時(shí)空表達(dá)，發(fā)現(xiàn)表達(dá)量在雌蛹、雌成蟲(chóng)及卵期高于其他發(fā)育階段，這與Lence等[28]的研究結(jié)果一致，表明該基因可能參與了小菜蛾的生殖發(fā)育。通過(guò)基因編輯技術(shù)CRISPR/ Cas9敲除，發(fā)現(xiàn)含有單個(gè)突變的等位基因的小菜蛾其生殖力和育性顯著低于野生型，這種影響與編輯類(lèi)型和突變親本的性別無(wú)關(guān)，說(shuō)明參與了小菜蛾的生殖過(guò)程和胚胎發(fā)育。在哺乳動(dòng)物中，缺乏的小鼠胚胎表現(xiàn)出顯著的細(xì)胞增殖缺陷，影響了內(nèi)胚層和中胚層的形成，從而導(dǎo)致胚胎在發(fā)育早期便死亡[40-42]。筆者發(fā)現(xiàn)，當(dāng)突變雌成蟲(chóng)與野生型雄成蟲(chóng)雜交以及突變成蟲(chóng)之間自交，雌成蟲(chóng)產(chǎn)卵量和卵孵化率的下降是由于卵巢發(fā)育受到了突變的影響。同樣地，參與m6A甲基化過(guò)程的另一writers蛋白對(duì)果蠅生殖能力也會(huì)產(chǎn)生影響，突變后雌成蟲(chóng)表現(xiàn)出卵巢異常且不育[43]或卵室數(shù)量減少且無(wú)法完成發(fā)育[44]。然而，當(dāng)突變體小菜蛾雄成蟲(chóng)與野生型雌成蟲(chóng)雜交后，其產(chǎn)卵量和孵化率同含有突變體雌成蟲(chóng)的交配組合結(jié)果類(lèi)似，但交配前突變體雄成蟲(chóng)精巢與野生型的并無(wú)明顯差異。這一結(jié)果引發(fā)了筆者新的思考：在突變體雄成蟲(chóng)精巢與野生型精巢表型無(wú)差異的基礎(chǔ)上，解剖與突變體雄成蟲(chóng)交配后野生型雌成蟲(chóng)的卵巢，發(fā)現(xiàn)卵巢內(nèi)卵的附著量比未交配的個(gè)體顯著減少，說(shuō)明突變體雄成蟲(chóng)可能在交配過(guò)程中向雌蟲(chóng)卵巢提供了一些不利于卵子發(fā)育的物質(zhì)。已有研究表明，雄成蟲(chóng)的精液蛋白能以多種方式促進(jìn)后代的繁殖，包括促進(jìn)精子儲(chǔ)存和雌性產(chǎn)卵[45-47]。筆者推測(cè)，突變體雄成蟲(chóng)可能產(chǎn)生了異常的精液蛋白，導(dǎo)致精子的活力降低，在與野生型雌成蟲(chóng)交配的過(guò)程中，由于大多數(shù)精子不能與卵子發(fā)生正常的精卵結(jié)合，導(dǎo)致卵的發(fā)育受阻[48-50]，其具體機(jī)理還有待進(jìn)一步的研究。在篩選突變品系的試驗(yàn)過(guò)程中，發(fā)現(xiàn)有相當(dāng)比例的突變個(gè)體在幼蟲(chóng)、蛹和成蟲(chóng)階段均會(huì)出現(xiàn)畸形的現(xiàn)象，不能正常完成整個(gè)世代，說(shuō)明該基因可能還參與了小菜蛾的生長(zhǎng)發(fā)育。果蠅中的研究發(fā)現(xiàn)突變m6A甲基化過(guò)程中相關(guān)的writers蛋白，無(wú)論是純合還是雜合突變體，其雌、雄成蟲(chóng)均表現(xiàn)出明顯的行為缺陷，包括移動(dòng)及飛行能力的下降和翅不能合攏于背部[28,44]。此外，本研究對(duì)小菜蛾突變品系后代中的突變個(gè)體進(jìn)行了9個(gè)世代的性別比例統(tǒng)計(jì)，發(fā)現(xiàn)每一代中被編輯的雌雄個(gè)體比例及G2到G10代所有突變個(gè)體的雌雄比例基本都符合1﹕1的理論值，說(shuō)明該基因可能與小菜蛾的性別決定無(wú)關(guān)。然而，果蠅的基因突變后，雌性的活力和受精能力顯著下降，而雄性卻不受影響[51]。在果蠅中，同時(shí)缺失writers基因和性別決定通路關(guān)鍵基因的單等位基因也會(huì)影響種群的性別比例，具體表現(xiàn)為雌性的存活率嚴(yán)重下降，而雄性不受影響，這可能是由于劑量補(bǔ)償通路受損所導(dǎo)致[28]。

隨著生物技術(shù)的不斷發(fā)展，眾多與害蟲(chóng)種群控制相關(guān)的概念相繼被提出并運(yùn)用于生產(chǎn)實(shí)踐，其中就包括種群置換和種群抑制[52-53]。相較于種群置換，種群抑制并不影響靶標(biāo)害蟲(chóng)在生態(tài)系統(tǒng)中的生態(tài)位，只是將其種群數(shù)量控制在經(jīng)濟(jì)損失所允許的水平之下。本試驗(yàn)通過(guò)CRISPR/Cas9結(jié)合卵的顯微注射技術(shù)，對(duì)小菜蛾進(jìn)行了編輯并成功誘發(fā)突變，進(jìn)一步發(fā)現(xiàn)其純合突變個(gè)體存活率極低且不育，單等位基因雜合突變個(gè)體的成蟲(chóng)生殖力和育性則顯著下降，為作為潛在的靶標(biāo)應(yīng)用于小菜蛾的遺傳防控打下了理論基礎(chǔ)。由于該雜合突變品系的獲得不需要進(jìn)行多代的近交篩選，節(jié)約了大量的試驗(yàn)成本，一旦檢測(cè)出該基因被成功編輯，便可以迅速應(yīng)用于生產(chǎn)實(shí)踐。同時(shí)，考慮到昆蟲(chóng)雌性個(gè)體具有繁殖后代的特性及其在飼養(yǎng)和田間釋放方面所增加的經(jīng)濟(jì)成本，在發(fā)育的早期階段篩選雄性個(gè)體是開(kāi)展害蟲(chóng)遺傳控制的關(guān)鍵步驟[54]。由于小菜蛾在4齡幼蟲(chóng)以前無(wú)法肉眼辨別性別，因此可以采用基因敲入技術(shù)[55]，將不同的熒光蛋白基因分別整合到基因座和雌性特有的W染色體。理論上而言，前者可導(dǎo)致單等位基因的突變并產(chǎn)生一種熒光，用于區(qū)分后代中的野生型個(gè)體，而后者則可以使雌性個(gè)體產(chǎn)生另一種熒光。利用熒光顯微鏡篩選兩種編輯品系小菜蛾雜交后的蟲(chóng)卵，便可快速地篩選出雄性突變個(gè)體，并排除野生型和雌性突變個(gè)體[55]，以便進(jìn)行大規(guī)模的飼養(yǎng)和田間釋放。這種防治策略通過(guò)降低靶標(biāo)害蟲(chóng)的產(chǎn)卵量和孵化率，從而“柔和”地控制其種群數(shù)量并保證該物種在自然界可以繼續(xù)繁衍存在，不會(huì)像基因驅(qū)動(dòng)系統(tǒng)那樣“暴力”地降低種群數(shù)量，甚至可能導(dǎo)致該物種的滅絕并引起負(fù)面生態(tài)效應(yīng)[56-57]。

4 結(jié)論

參與了小菜蛾正常的生長(zhǎng)發(fā)育過(guò)程，主要表現(xiàn)在對(duì)生殖過(guò)程和胚胎發(fā)育的影響，但不影響小菜蛾的性別決定。單等位基因的突變顯著降低了小菜蛾的生殖力和育性，是開(kāi)展遺傳控制的理想靶標(biāo)。

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Knockout of single allele ofsignificantly decreases the fecundity and fertility in

LI FeiFei, WANG BeiBei, LAI YingFang, YANG FeiYing, YOU MinSheng, HE WeiYi

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops/Institute of Applied Ecology, Fujian Agriculture and Forestry/International Joint Research Laboratory of Ecological Pest Control, Ministry of Education, Fujian Agriculture and Forestry University/Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry/Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fuzhou 350002

【】RNA methylation is the main form of epigenetic modification at post-transcriptional level, which is involved in many important cellular processes. The diamondback moth,, is an important oligophagous insect pest, causing serious loss on the production of cruciferous vegetables. However, the function of RNA methylation-related genes inis still unclear. The present study aims to identify and clone the homologous, one of the members of the RNA methylation protein complex (writers), to determine the expression pattern of, and to knockoutusing CRISPR/Cas9 for the investigation of its biological functions in.【】The sequence of homologouswas identified in the genome database of, which was used for PCR amplification of the coding sequence (CDS). Quantitative real-time PCR (qRT-PCR) was used to study the relative expression levels ofin different developmental stages and adult gonads of. Thewas edited using CRISPR/Cas9 combined with egg injection. Each of the adults that developed from the injected eggs was used to pair with a wild-type adult for reproduction. Offspring of the same population was forced to inbreed by single-pair mating to establish the mutant strains. The differences of genetic characters, biological parameters and phenotypes between mutants and wild-type individuals were recorded and compared to decipher the function of.【】The CDS ofwith length of 912 bp was isolated, the expression of which was high in female pupa, adult and egg, moderate in male adult and pupa, the lowest in larva, and significantly higher in ovary than in testis of adult. The sgRNAs targetingand the Cas9 protein were mixed to inject eggs, and the offsprings carrying mutant alleles were screened for homozygous strains based on single-pair inbreeding for 10 generations. Three types of heterozygous mutant strains both predicted to cause frameshift of the CDS were obtained, with the deletion of 4 (Δ213-4), 5 (Δ213-5) and 7 (Δ213-7) bases. During the screening process, six and two homozygous mutants from Δ213-4 and Δ213-5 strains were identified, respectively. The homozygous mutants of Δ213-4 successfully mated in two pairs, but no eggs were produced. Meanwhile, each two male adults of homozygous mutants of either Δ213-4 or Δ213-5 were mated with the same type of female heterozygous mutant, and also no eggs were produced.The results indicated that individuals with homozygousmutation may have extremely low survival rate and not be able to produce offspring. Through analyzing separation ratio of the genotypes of offspring from the inbreeding of heterozygous mutants and the hybridization between heterozygous mutants and wild-type, it was found that the ratio of heterozygous mutant individuals to wild-type was slightly less than 2 and 1, respectively, indicating that heterozygous mutation ofwould affect the normal growth and development of, and in some cases would lead to death. The offsprings of mutant individuals, which carry a mutant allele, showed a sex ratio close to 1﹕1 (＜0.05). It was speculated that themight not be involved in sex determination in. For the mating consists of mutant adults, the fecundity and hatchability were significantly lower (＜0.01) than the mating between wild-type adults. Most of the eggs produced from the mutant parents look abnormal, and could not hatch normally due to water loss and shrinkage. Based on the dissection of adult gonads, it was found that the number of attached eggs on the ovary of the mutant female adult and the wild-type female adult that has mated with mutant male adult was less than that of the wild-type virgin female adult, while no obvious abnormality was found for the testis of mutant male adult. Some of the hatched heterozygous mutants showed different degrees of distortion during the whole developmental process, resulting in the failure to complete life cycle. A small part of the heterozygous mutant individuals could develop normally, and thus transmit the mutant allele to their offspring. According to our findings, a model of genetic control ofbased onwas proposed.【】Theis involved in the reproductive process and embryonic development of, mutation of which significantly affects the population size of the offspring, making it an ideal target for the genetic control of.

;; heterozygous mutant;fecundity; fertility

10.3864/j.issn.0578-1752.2021.14.009

2020-11-06；

2021-01-15

國(guó)家重點(diǎn)研發(fā)計(jì)劃（2017YFD0200400）、福建省自然科學(xué)基金（2019J21369）、福建省科技重大專(zhuān)項(xiàng)（2018NZ0002）

李飛飛，E-mail：lff0371@163.com。通信作者何瑋毅，E-mail：wy.he@fafu.edu.cn。通信作者尤民生，E-mail：msyou@fafu.edu.cn

（責(zé)任編輯 岳梅）