母連勝，何 勇，羅 岸，朱志炎，田志宏

(長(zhǎng)江大學(xué) 生命科學(xué)學(xué)院/主要糧食作物產(chǎn)業(yè)化湖北省協(xié)同創(chuàng)新中心，湖北 荊州 434025)

質(zhì)體基因工程在植物育種中的應(yīng)用研究進(jìn)展

母連勝，何 勇，羅 岸，朱志炎，田志宏*

(長(zhǎng)江大學(xué) 生命科學(xué)學(xué)院/主要糧食作物產(chǎn)業(yè)化湖北省協(xié)同創(chuàng)新中心，湖北 荊州 434025)

與傳統(tǒng)的核轉(zhuǎn)化相比，質(zhì)體遺傳轉(zhuǎn)化作為外源基因表達(dá)更精確、安全和高效的新一代轉(zhuǎn)基因技術(shù)對(duì)作物品質(zhì)改良和產(chǎn)量提高做出了極大的貢獻(xiàn)，也給人們提供了植物育種的新思路。綜述了質(zhì)體遺傳轉(zhuǎn)化技術(shù)、篩選標(biāo)記(體系)及其在植物抗性性狀改良、產(chǎn)量提高、品質(zhì)改良、雜種優(yōu)勢(shì)利用中的應(yīng)用研究進(jìn)展，以期為質(zhì)體基因工程在植物遺傳改良，尤其是在單子葉植物遺傳改良中的應(yīng)用提供理論依據(jù)。

質(zhì)體； 葉綠體； 遺傳轉(zhuǎn)化； 基因工程

1988年，首次在單細(xì)胞真核生物衣藻中實(shí)現(xiàn)的葉綠體遺傳轉(zhuǎn)化[1]使人們意識(shí)到：作為植物體內(nèi)除細(xì)胞核以外含遺傳信息的細(xì)胞器之一的葉綠體，不僅是植物體進(jìn)行光合作用的重要場(chǎng)所，同時(shí)也具有作為植物遺傳轉(zhuǎn)化新工具的潛力。1990年，煙草(NicotianatabacumL.)質(zhì)體轉(zhuǎn)化成功[2]，標(biāo)志著高等植物質(zhì)體基因工程的開始。由于傳統(tǒng)核轉(zhuǎn)化所創(chuàng)制的轉(zhuǎn)基因植物存在潛在的環(huán)境安全性問題[3-4]，質(zhì)體基因工程以其外源基因表達(dá)量高[5]、基因表達(dá)無位置效應(yīng)和基因沉默現(xiàn)象[5-7]、可避免核轉(zhuǎn)化系統(tǒng)中由于花粉逃逸所帶來的環(huán)境安全性問題[8]及單次轉(zhuǎn)化事件中可實(shí)現(xiàn)多個(gè)基因同時(shí)轉(zhuǎn)化與表達(dá)[9-11]等特點(diǎn)而備受關(guān)注。近20 a來，人們已成功應(yīng)用質(zhì)體遺傳轉(zhuǎn)化手段將抗除草劑[12]、抗病[13]、抗蟲[5,14]、抗旱[7]、耐鹽漬[15]及綜合抗性[16]等相關(guān)外源基因?qū)氲綗煵輀13,16-18]、擬南芥(ArabidopsisthalianaL.)[19]、矮牽牛(PetuniahybridaL.)[20]、花椰菜(BrassicaoleraceaL.)[21]、結(jié)球甘藍(lán)(BrassicaoleraceaL.)[22]、馬鈴薯(SolanumtuberosumL.)[23]、萵苣(LactucasativaL.)[24-27]、番茄(SolanumlycopersicumL.)[28-30]、胡蘿卜(DaucuscarotaL.)[15]、棉花(GossypiumhirsutumL.)[31]、油菜(BrassicanapusL.)[32]、大豆(GlycinemaxL.)[33]、水稻(OryzasativaL.)[34-38]和野甘草(ScopariadulcisL.)[39]等20多種植物中，并在作物農(nóng)藝性狀改良和生物反應(yīng)器應(yīng)用方面取得了一些進(jìn)展，對(duì)作物品質(zhì)改良和產(chǎn)量提高做出了極大的貢獻(xiàn)，也給人們提供了植物育種的新思路。綜述了質(zhì)體遺傳轉(zhuǎn)化技術(shù)、篩選標(biāo)記(體系)及其在植物抗性性狀改良、產(chǎn)量提高、品質(zhì)改良、雜種優(yōu)勢(shì)利用中的應(yīng)用研究進(jìn)展，以期為質(zhì)體基因工程在植物遺傳改良，尤其是在單子葉植物遺傳改良中的應(yīng)用提供理論依據(jù)。

1 質(zhì)體的特點(diǎn)及其遺傳轉(zhuǎn)化技術(shù)

作為質(zhì)體基因工程研究主體的質(zhì)體是植物細(xì)胞內(nèi)半自主的、原核生物起源的內(nèi)共生細(xì)胞器，是由雙層膜包裹，與碳水化合物的合成、貯藏密切相關(guān)的一類細(xì)胞器的總稱，由前質(zhì)體分化而來，根據(jù)其所含色素的不同分為葉綠體、有色體和白色體(造粉體、造蛋白體、造油體)。通常認(rèn)為光合真核生物質(zhì)體(葉綠體)中含有120～220 kb的環(huán)狀雙鏈DNA分子，其基因組只含100～250個(gè)基因(種子植物中約含130個(gè)基因)，具有高度多倍性，結(jié)構(gòu)高度保守，有自己特有的核酸和蛋白質(zhì)合成機(jī)制[40-43]。大多數(shù)陸生高等植物都具有20～30 kb序列相同的反向重復(fù)區(qū)域A和B(IRA和IRB)，將質(zhì)體基因組的大拷貝區(qū)(LSC)和小拷貝區(qū)(SSC)隔開，這決定了質(zhì)體，尤其是作為植物光合作用重要場(chǎng)所的葉綠體，可以被用于植物遺傳轉(zhuǎn)化研究。

質(zhì)體基因工程是利用攜帶外源基因的載體的側(cè)翼序列與質(zhì)體DNA同源區(qū)段之間發(fā)生同源重組[44-45]而實(shí)現(xiàn)的，轉(zhuǎn)化過程中外源DNA首先整合到1個(gè)或多個(gè)質(zhì)體基因組中，再在適合的選擇壓下進(jìn)行20～30次細(xì)胞分裂，以去除未轉(zhuǎn)化的質(zhì)體來實(shí)現(xiàn)質(zhì)體的同質(zhì)化[44,46]。目前，人們常采用基因槍法[13,17,24]、PEG介導(dǎo)法[47-49]、農(nóng)桿菌介導(dǎo)法[50]、顯微注射法[51]、激光注射法[52]、花粉管導(dǎo)入法[53]和轉(zhuǎn)運(yùn)肽介導(dǎo)的葉綠體間接轉(zhuǎn)化法[54]等將外源DNA導(dǎo)入質(zhì)體基因組中。由于外源DNA進(jìn)入葉綠體必須突破細(xì)胞壁、原生質(zhì)體膜和葉綠體膜的障礙，目前，質(zhì)體基因工程中常采用基因槍法和PEG介導(dǎo)法進(jìn)行遺傳轉(zhuǎn)化，PEG介導(dǎo)法相對(duì)基因槍法更為經(jīng)濟(jì)，但PEG介導(dǎo)法中原生質(zhì)體的分離和培養(yǎng)難度較大[55]。因此，目前質(zhì)體遺傳轉(zhuǎn)化仍以基因槍法為主。

2 質(zhì)體轉(zhuǎn)化篩選標(biāo)記(體系)

質(zhì)體基因組的高拷貝數(shù)決定了質(zhì)體轉(zhuǎn)化過程中外源基因不可能同時(shí)轉(zhuǎn)化所有的質(zhì)體基因組。因此，為保證整合到受體植物質(zhì)體基因組中的外源基因的遺傳穩(wěn)定性，必須利用篩選標(biāo)記基因并結(jié)合相應(yīng)的篩選底物在選擇培養(yǎng)過程中逐步去除未轉(zhuǎn)化的質(zhì)體基因組，以實(shí)現(xiàn)質(zhì)體基因組的同質(zhì)化。與核轉(zhuǎn)化中所使用的篩選標(biāo)記類似，目前，用于質(zhì)體轉(zhuǎn)化的篩選標(biāo)記主要包括2類，“正向”選擇標(biāo)記：可選擇性地將轉(zhuǎn)化細(xì)胞從非轉(zhuǎn)化細(xì)胞中篩選出來并促進(jìn)其生長(zhǎng)；“負(fù)向”選擇標(biāo)記：可抑制轉(zhuǎn)化細(xì)胞的生長(zhǎng)[56]。

目前，在雙子葉植物質(zhì)體遺傳轉(zhuǎn)化中所使用的選擇標(biāo)記多是基于抗生素抗性基礎(chǔ)的，包括：帶壯觀霉性和鏈霉素抗性的基因，如葉綠體基因組16S rDNA和23S rDNA不同位點(diǎn)的點(diǎn)突變基因[2,57-58]、編碼氨基糖苷-3′-腺苷酸轉(zhuǎn)移酶的aadA基因[59]；編碼新霉素磷酸轉(zhuǎn)移酶、具有新霉素和卡那霉素抗性的nptⅡ(neo)基因[60]；編碼氨基糖苷-3′-轉(zhuǎn)移酶、具有卡那霉素抗性的aphA-6基因[31,61]；編碼氨基糖苷乙酰轉(zhuǎn)移酶(6′)-Ie/氨基糖苷磷酸轉(zhuǎn)移酶(2″)-Ia、具有氨基糖苷類抗生素廣譜抗性的aac6-aph2雙功能抗性基因[62-63]。其中，壯觀霉素具有高度特異性且對(duì)植物細(xì)胞無毒害作用，在雙子葉植物質(zhì)體遺傳轉(zhuǎn)化中常作為篩選劑使用，aadA基因也因此成為最常用的標(biāo)記基因。研究表明，高等植物尤其是禾谷類單子葉植物雖然對(duì)鏈霉素具有一定的敏感性[64]，但其天然具有壯觀霉素抗性[65]，所以aadA標(biāo)記對(duì)部分具抗生素抗性的雙子葉植物和單子葉植物選擇效率低[66]，在一定程度上限制了該標(biāo)記的應(yīng)用。最近，研究發(fā)現(xiàn)，編碼氯霉素乙酰轉(zhuǎn)移酶、具氯霉素抗性的cat基因在使用過程中不會(huì)使植物產(chǎn)生自發(fā)的抗生素抗性突變，可擴(kuò)大質(zhì)體轉(zhuǎn)化禾谷類作物的范圍[67]。Maliga[46]認(rèn)為，潮霉素B也可用于葉綠體轉(zhuǎn)化篩選，篩選過程中應(yīng)注意劑量效應(yīng)：不適合只含少量整合有hpt基因拷貝的葉綠體基因組的轉(zhuǎn)化細(xì)胞的篩選；當(dāng)轉(zhuǎn)化細(xì)胞中含大量整合有hpt基因拷貝的葉綠體基因組時(shí)，篩選才會(huì)有效，但最終未見其成功報(bào)道。李丁等[35-37]以hpt基因作為篩選標(biāo)記進(jìn)行了水稻葉綠體轉(zhuǎn)化研究，在T0代轉(zhuǎn)化植株中檢測(cè)到了hpt基因的表達(dá)，后續(xù)試驗(yàn)中采用TALENs輔助手段將轉(zhuǎn)化效率提高了2倍，但仍未獲得同質(zhì)化植株。

為了避免抗生素抗性基因使用過程中存在的潛在危險(xiǎn)，一些關(guān)于非抗生素篩選標(biāo)記的研究已獲得一定進(jìn)展。其中一類是基于除草劑抗性建立的篩選標(biāo)記，包括psbA突變基因[68]、AHAS突變基因[69]、編碼草丁膦乙酰轉(zhuǎn)移酶的bar基因[70-71]、編碼EPSP合成酶的EPSPS基因[12,72]、編碼4-羥基苯丙酮酸雙加氧酶的HPPD基因[73-74]、編碼乙酰乳酸合成酶的ALS基因[75]，但同hpt基因類似，這些標(biāo)記基因也不適合初期篩選，當(dāng)轉(zhuǎn)化細(xì)胞中含大量整合有這些標(biāo)記基因拷貝的葉綠體基因組時(shí)，篩選才會(huì)有效。另一類是基于代謝途徑相關(guān)基因建立的，包括編碼甜菜堿醛脫氫酶的badh基因[15,76-78]、編碼鄰氨基苯甲酸合成酶的ASA2基因[79-80]、編碼D-氨基酸氧化酶的dao基因[81]、編碼D-絲氨酸脫氨酶的dsdA基因[82]，這在一定程度上促進(jìn)了天然對(duì)壯觀霉素有抗性的一些重要經(jīng)濟(jì)作物，尤其是禾谷類作物的質(zhì)體基因工程的發(fā)展。同時(shí)，基于編碼胞嘧啶脫氨酶的codA基因的“負(fù)向”選擇體系也已建立[83-84]，現(xiàn)多用于驗(yàn)證生長(zhǎng)于添加5-FC的培養(yǎng)基上的帶codA基因葉綠體轉(zhuǎn)化植株是否通過P1噬菌體位點(diǎn)特異性重組酶CRE-lox作用刪除了codA基因[85]。

為方便在利用選擇壓篩選轉(zhuǎn)化細(xì)胞之前，對(duì)轉(zhuǎn)化組織進(jìn)行可視化人工篩選含轉(zhuǎn)化細(xì)胞的組織，一些可視化的選擇標(biāo)記和報(bào)告基因也得到相應(yīng)開發(fā)，如編碼PSI主要亞基的基因的突變體psaA、psaB和rbcL等[86-87]、來自大腸桿菌的編碼葡萄糖苷酶的uidA基因[88-89]、編碼綠色熒光蛋白的gfp基因[25,90-92]、編碼熒光素酶的lux基因[93-94]，但這些可視化的標(biāo)記大多不能單獨(dú)使用，必須結(jié)合抗生素或除草劑抗性基因才能發(fā)揮作用。

雖然大部分作物的質(zhì)體遺傳遵循母系遺傳規(guī)律，但選擇標(biāo)記基因向野生型植物[95]或微生物[96]轉(zhuǎn)移的可能性也不能夠完全排除[97]?；诖?，同核轉(zhuǎn)化一樣，從轉(zhuǎn)化的質(zhì)體基因組中去除標(biāo)記基因是避免選擇標(biāo)記基因向野生型植物或微生物轉(zhuǎn)移的最簡(jiǎn)單易行的一種方法。當(dāng)前，正向重復(fù)序列介導(dǎo)的同源重組刪除體系[71,74,86,98]、噬菌體位點(diǎn)特異性重組酶介導(dǎo)的刪除體系[99-102]、標(biāo)記基因的瞬時(shí)共整合體系[98]和共轉(zhuǎn)化分離體系[70,72,103-104]已經(jīng)在質(zhì)體基因工程中得到發(fā)展。

3 質(zhì)體基因工程在植物育種中的應(yīng)用

目前，傳統(tǒng)的雜交育種和現(xiàn)代的分子育種在作物育種中都起著舉足輕重的作用，尤其是分子育種大大加快了育種的速度，但基于對(duì)傳統(tǒng)核轉(zhuǎn)化產(chǎn)品的安全性考慮，質(zhì)體遺傳轉(zhuǎn)化也已用于作物育種并取得了一定成效。

3.1 抗性性狀改良

植物在生長(zhǎng)發(fā)育過程中，隨時(shí)都面臨著各種各樣的生物和非生物脅迫，嚴(yán)重影響著植物的生長(zhǎng)發(fā)育，尤其是作物的產(chǎn)量和品質(zhì)。然而，通過傳統(tǒng)的核轉(zhuǎn)化將抗性基因?qū)胫参锞哂袧撛诘娘L(fēng)險(xiǎn)：抗性基因可通過花粉逃逸，與雜草遠(yuǎn)緣雜交而使其獲得相應(yīng)抗性，從而產(chǎn)生“超級(jí)雜草”使得農(nóng)藥和殺蟲劑失效。雖然通過基因沉默可顯著降低這種風(fēng)險(xiǎn)，但其增加了運(yùn)行成本。因此，通過無遠(yuǎn)緣雜交風(fēng)險(xiǎn)的質(zhì)體基因工程提高植物對(duì)常見脅迫因子的抗性成為研究熱點(diǎn)，進(jìn)而提高作物產(chǎn)量、減少農(nóng)藥和殺蟲劑的使用量、提高作物在逆境下的生存潛力。

3.1.1 生物脅迫抗性 通過質(zhì)體遺傳轉(zhuǎn)化提供除草劑抗性最早見于Daniell等[12]將牽?；ǖ腅PSPS基因成功整合到煙草葉綠體基因組中并表達(dá)草甘膦抗性。Ye等[103]在后續(xù)的煙草葉綠體轉(zhuǎn)化試驗(yàn)中證明，來自原核生物的EPSPS基因也可用于葉綠體遺傳轉(zhuǎn)化，但其產(chǎn)物的積累量和草甘膦抗性強(qiáng)弱無相關(guān)性。隨后，相關(guān)學(xué)者通過在葉綠體中表達(dá)bar基因[70-71]、HPPD基因[73-74]、ALS基因[75]和編碼D-氨基酸氧化酶的dao基因[81]陸續(xù)獲得了帶草銨膦、磺草酮、異惡唑草酮、咪唑啉酮、磺脲和D-氨基酸抗性的植株。

病蟲害是影響作物產(chǎn)量的主要因素之一，其化學(xué)防治會(huì)增加環(huán)境危害和作物生產(chǎn)成本。雖然通過核轉(zhuǎn)化引入相關(guān)抗性基因能夠降低風(fēng)險(xiǎn)，但葉綠體的多拷貝性更大程度地提高了外源基因在葉綠體中的表達(dá)量，特別是在某些抗性強(qiáng)弱與抗性蛋白在細(xì)胞內(nèi)的表達(dá)水平直接相關(guān)的時(shí)候。McBride等[105]將蘇云金芽孢桿菌(Bacillusthuringiensis)Bt蛋白的編碼基因crylAc導(dǎo)入煙草葉綠體基因組中，得到了能抗谷實(shí)夜蛾(Helicoverpazea)、煙芽夜蛾(Heliothisvirescens)和甜菜夜蛾(Spodopteraexigua)幼蟲的轉(zhuǎn)化植株，最早實(shí)現(xiàn)了質(zhì)體抗蟲基因工程。隨后，Cry2Aa2[5]、Cry1Ab[106-107]、Cry1Ia5[108]和Cry9Aa2[109]等Bt毒性蛋白分別通過葉綠體遺傳轉(zhuǎn)化應(yīng)用于煙草[106,108]、大豆[5]、結(jié)球甘藍(lán)[107]和馬鈴薯[109]等作物，Cry2Aa2蛋白在煙草葉綠體轉(zhuǎn)化株成熟葉片中的表達(dá)量最高可達(dá)到細(xì)胞總可溶性蛋白的45%。DeGray等[110]通過向煙草葉綠體基因組中導(dǎo)入抗菌肽MSI-99的編碼序列，最早證實(shí)了質(zhì)體抗病基因工程的可行性，轉(zhuǎn)化植株能顯著抑制黃曲霉(Aspergillusflavus)、串珠鐮刀菌(Fusariummoniliforme)、大麗輪枝菌(Verticilliumdahliae)、煙草假單胞桿菌(Pseudomonassyringaepv.tabaci)的生長(zhǎng)，最近發(fā)現(xiàn)其也可抑制稻瘟病的發(fā)生[111]。Retrocyclin-101和Protegrin-1抗菌多肽在煙草葉綠體中的高效表達(dá)(Retrocyclin-101在煙草葉綠體轉(zhuǎn)化株中的表達(dá)量可以達(dá)到細(xì)胞總可溶性蛋白的32%～38%)，同樣使轉(zhuǎn)化植株能夠抵御細(xì)菌或病毒感染[112]。為了培育具有抗病和抗蟲復(fù)合性狀的轉(zhuǎn)基因植物材料，利用多基因表達(dá)元件，Chen等[16]在煙草中表達(dá)了來自芋頭(Colocasiaesculenta)的半胱氨酸蛋白酶抑制劑、甘薯的貯藏蛋白和爪哇擬青霉的幾丁質(zhì)酶編碼基因cystatin、sporamin和chitinase，結(jié)果表明，外源基因的表達(dá)能夠減輕胡蘿卜軟腐果膠桿菌胡蘿卜亞種(Pectobacteriumcarotovorumsubsp.carotovorum)導(dǎo)致的軟腐病和煙草赤星病菌(Alternariaalternata)導(dǎo)致的葉斑病，并能導(dǎo)致甜菜夜蛾和斜紋夜蛾(Spodopteralitura)幼蟲攝入葉片后生長(zhǎng)遲緩和死亡，同時(shí)也提高了轉(zhuǎn)化植株對(duì)滲透脅迫的抗性；Jin等[113]在煙草葉綠體中表達(dá)具有復(fù)合抗性的半夏凝集素(pinellia ternata agglutinin，pta)基因，獲得的轉(zhuǎn)化植株不僅能夠有效抗鱗翅目和同翅目害蟲，而且具有明顯的抗菌和抗病毒活性。

3.1.2 非生物脅迫抗性 隨著人類的開發(fā)利用和極端天氣出現(xiàn)頻次的增加，為了保障植物的生長(zhǎng)和產(chǎn)量，對(duì)植物耐旱、耐極端溫度和耐鹽能力的要求越來越高。滲透保護(hù)劑通過在滲透調(diào)節(jié)過程中穩(wěn)定膜和蛋白質(zhì)能有效提高植物的耐鹽和耐旱能力，但大多數(shù)植物不能直接利用，在植物體內(nèi)引入編碼滲透保護(hù)劑的相關(guān)基因是一個(gè)可行的辦法[114]。Khan等[114]通過質(zhì)體遺傳轉(zhuǎn)化在煙草葉綠體基因組中導(dǎo)入編碼阿拉伯糖醇脫氫酶的ArDH基因,獲得了能正常生長(zhǎng)于含350 mmol/L NaCl土壤中的轉(zhuǎn)化植株。Kumar等[15]利用質(zhì)體轉(zhuǎn)化技術(shù)在胡蘿卜中引入并表達(dá)了編碼甜菜堿脫氫酶的BADH基因，發(fā)現(xiàn)在含100 mmol/L NaCl的培養(yǎng)基上，轉(zhuǎn)化植株BADH基因的表達(dá)量比非轉(zhuǎn)化植株提高了50～54倍，有效提高了轉(zhuǎn)化株的耐鹽能力。另外，通過增加不飽和脂肪酸含量來增強(qiáng)植物的抗氧化防御能力，或利用大腸桿菌panD基因編碼的L-天冬氨酸脫羧酶將L-天冬氨酸分解為巴拉寧和CO2，可有效提高植物對(duì)高溫脅迫的耐受性[115]。

3.2 產(chǎn)量提高

隨著世界人口的快速增加，為了保障國(guó)家糧食安全，對(duì)作物產(chǎn)量的提高要求越來越迫切。目前，質(zhì)體轉(zhuǎn)基因作物產(chǎn)量的提高都是基于改進(jìn)光合作用機(jī)制來提高光合作用效率實(shí)現(xiàn)的。葉綠體基質(zhì)上的1,5-二磷酸核酮糖羧化酶/加氧酶(RuBisco)能通過參與CO2固定和初級(jí)生產(chǎn)進(jìn)而提高作物產(chǎn)量，它包括葉綠體基因編碼的大亞基(rbcL)和核基因編碼并定向進(jìn)入葉綠體的小亞基(rbcS)，其催化活性緩慢且低效，常受到CO2和O2濃度及光照強(qiáng)度等因素的影響，在不同的植物之間也存在差異。因此，通過基因工程對(duì)RuBisco的表達(dá)進(jìn)行調(diào)節(jié)有可能提高作物光合作用和生產(chǎn)率，進(jìn)而提高作物產(chǎn)量，尤其是在CO2濃度、溫度和水氮供給等條件動(dòng)態(tài)變化的環(huán)境中[116]。但研究發(fā)現(xiàn)，將rbcS基因?qū)氩⒄系饺~綠體基因組中并不能提高植物的光合效率[117]，核轉(zhuǎn)化反而會(huì)限制RuBisco的活性[118]；用其他物種來源的rbcL基因替換煙草葉綠體基因組中的rbcL基因可能會(huì)導(dǎo)致RuBisco功能缺失[119]，而特殊設(shè)計(jì)的載體(含rbcL和aadA)用于質(zhì)體遺傳轉(zhuǎn)化卻不會(huì)影響RuBisco的活性[120]。將來源于C4植物的rbcL基因?qū)氩⒄系紺3植物葉綠體基因組中[121]，或?qū)牟煌锓N中篩選得到的光合作用效率較高的RuBisco的編碼基因引入植物葉綠體基因組中[122]，都有可能提高植物的光合作用效率。

因此，深入了解RuBisco的裝配和活性調(diào)節(jié)因子、與其他生物技術(shù)協(xié)同作用提高光合作用的碳同化和開發(fā)新技術(shù)才能通過基因工程進(jìn)一步實(shí)現(xiàn)RuBisco活性的提高并增加作物的產(chǎn)量。在此基礎(chǔ)上，Lin等[123]將藍(lán)藻中RuBisco的大亞基(Se-rbcL)、小亞基(Se-rbcS)和組裝分子伴侶(Se-RbcX)的編碼基因?qū)霟煵莸娜~綠體基因組中，有效提高了轉(zhuǎn)化株的CO2固定效率。Whitney等[124]在煙草葉綠體中共表達(dá)擬南芥RuBisco的大亞基編碼基因AtL和RuBisco累積因子1基因(AtRAF1)獲得了高CO2固定效率的轉(zhuǎn)化株，其CO2固定效率比單獨(dú)表達(dá)AtL的轉(zhuǎn)化株高2倍，而且生長(zhǎng)速度明顯加快，這進(jìn)一步說明通過質(zhì)體遺傳轉(zhuǎn)化增強(qiáng)RuBisco活性對(duì)作物產(chǎn)量的提高是有效的。

3.3 品質(zhì)改良

隨著人類生活水平的不斷提高，人們對(duì)作物品質(zhì)提出了更高的要求，功能性食品的開發(fā)利用也得到發(fā)展。作物品質(zhì)改良可通過加強(qiáng)農(nóng)業(yè)生產(chǎn)過程管理、常規(guī)育種或用包括質(zhì)體遺傳轉(zhuǎn)化在內(nèi)的不同基因工程方法改變植物代謝實(shí)現(xiàn)。與健康問題相關(guān)的最重要的飲食成分是食品中的大量元素和微量元素，然而，這些組分被認(rèn)為有可能會(huì)導(dǎo)致某些不耐受癥、存在潛在毒性或致敏性或隨著營(yíng)養(yǎng)物質(zhì)的吸收而被干擾，但可以對(duì)其進(jìn)行修改以提高食品的功能性[125]。一些以作物為基礎(chǔ)的植物營(yíng)養(yǎng)組分或含量經(jīng)過改良的轉(zhuǎn)基因植物已經(jīng)被用于減輕與飲食相關(guān)的疾病，如增加馬鈴薯中蛋白質(zhì)含量、增加幾種作物中氨基酸(玉米和水稻中的賴氨酸、苜蓿中的蛋氨酸)、膽堿、葉酸、黃酮、花青素、維生素E、胡蘿卜素、鐵和鋅的含量[126]。當(dāng)然，這些也可以通過質(zhì)體基因工程實(shí)現(xiàn)。研究表明，抗壞血酸在葉綠體基質(zhì)中濃度達(dá)到300 mmol/L時(shí)能參與抗氧化保護(hù)[127-128]；除了增加對(duì)脅迫條件的耐受性以外，抗壞血酸代謝經(jīng)過改良的葉綠體轉(zhuǎn)化植物中維生素C的含量也可能會(huì)增加[15]。被認(rèn)為是一種前瞻性甜味劑的Monellin是一種甜味蛋白，通過分子藥物開發(fā)技術(shù)從根本上增強(qiáng)了Monellin的表達(dá)，可以用于改進(jìn)質(zhì)體轉(zhuǎn)化植株產(chǎn)品的味道[129]?；谥参矬w內(nèi)異戊二烯生物合成的細(xì)胞質(zhì)中甲羥戊酸和質(zhì)體中丙酮酸磷酸甘油醛(MEP)代謝途徑，人們發(fā)展了提高相關(guān)物質(zhì)生物合成前體的利用率、修飾生物合成途徑相關(guān)酶或調(diào)節(jié)機(jī)制和代謝途徑向合成新物質(zhì)成分轉(zhuǎn)移等改進(jìn)作物品質(zhì)的方法。Hasunuma等[130]在煙草葉綠體基因組中過表達(dá)生物合成異戊二烯前體物質(zhì)異戊烯基焦磷酸(IPP)的1-脫氧木酮糖-5-還原異構(gòu)酶(DXR)發(fā)現(xiàn)，其下游產(chǎn)物葉綠素a、β-胡蘿卜素、葉黃素、玉米黃素、茄尼醇和β-谷甾醇的含量得到有效提高。Kumar等[131]利用多基因策略將整個(gè)細(xì)胞質(zhì)的甲羥戊酸途徑(MEV)成功導(dǎo)入煙草葉綠體基因組中，不僅增加了轉(zhuǎn)化株體內(nèi)類胡蘿卜素的含量，而且也提高了甲羥戊酸、甾醇、角鯊烯和三?；视王サ暮?，進(jìn)一步證實(shí)了異戊二烯在植物代謝中的中樞作用及合成的復(fù)雜性。在番茄的質(zhì)體遺傳轉(zhuǎn)化技術(shù)成功建立之后[132]，人們將編碼番茄紅素β-環(huán)化酶的Lyc(來自水仙)[133]和crtY(來自草生歐文菌)[134]基因?qū)敕奄|(zhì)體基因組中，成功促進(jìn)了番茄紅素轉(zhuǎn)化為β-胡蘿卜素并進(jìn)一步提高了維生素A含量，極大程度地提高了番茄果實(shí)的營(yíng)養(yǎng)價(jià)值。

Hasunuma等[135]將海洋短波單胞菌屬(Brevundimonas)細(xì)菌的β-胡蘿卜素酮酶(crtW)和β-胡蘿卜素環(huán)化酶(crtZ)的編碼基因?qū)霟煵萑~綠體基因組中，轉(zhuǎn)化株葉片中類胡蘿卜素的總量提高了2.1倍，而且合成了一種新的類胡蘿卜素4-茴香酸。隨后，Harada等[24]將crtW、crtZ和來自海洋副球菌屬(Paracoccus)細(xì)菌的異戊烯基焦磷酸異構(gòu)酶編碼基因(idi)導(dǎo)入并整合到生菜的葉綠體基因組中，轉(zhuǎn)化株以消耗本體類胡蘿卜素為代價(jià)，在其葉片中富集了大量的游離蝦青素和不同的蝦青素脂肪酸酯等氧化類胡蘿卜素。利用多順反子間基因表達(dá)元件(IIEE)[136]，Lu等[137]在番茄質(zhì)體基因組中整合并表達(dá)了維生素E合成途徑中的3個(gè)關(guān)鍵酶尿黑酸葉綠基轉(zhuǎn)移酶(HPT)、生育酚環(huán)化酶(TCY)和生育酚甲基轉(zhuǎn)移酶(TMT)的編碼基因，有效提高了番茄果實(shí)中維生素E的含量。

Madoka等[138]利用改進(jìn)的質(zhì)體accD操縱子在煙草中過表達(dá)了乙酰輔酶A羧化酶(ACCase)，延緩了轉(zhuǎn)化株的葉片衰老，降低了淀粉含量，提高了脂肪酸和脂質(zhì)含量，并提高了脂肪酸中不飽和脂肪酸的含量，對(duì)種子中脂肪酸和脂質(zhì)含量及組成無影響，種子的產(chǎn)量提高了2倍，間接提高了其單位產(chǎn)油量。Craig等[139]在煙草葉綠體基因組中導(dǎo)入來自美洲野生馬鈴薯(Solanumcommersonii)或藍(lán)藻(Anacystisnidulans)的Δ9去飽和酶編碼基因des，有效改變了轉(zhuǎn)化株葉片和種子中脂肪酸的組成，提高了不飽和脂肪酸含量，并提高了其耐寒性。Dunne等[140]利用細(xì)菌來源的異戊烯轉(zhuǎn)移酶編碼基因ipt建立了基于細(xì)胞分裂素選擇的質(zhì)體轉(zhuǎn)化系統(tǒng)，在煙草中表達(dá)了負(fù)責(zé)直鏈和支鏈脂肪酸生物合成起始的3-酮脂酰-?；d體蛋白合成酶Ⅲ(KasⅢ)，改變了轉(zhuǎn)化株葉片中脂肪酸的組成，不飽和脂肪酸含量得到一定提高。

3.4 雜種優(yōu)勢(shì)利用

一直以來，雜種優(yōu)勢(shì)利用是提高作物產(chǎn)量的重要途徑。高純度F1代雜種的制備通常需要消耗大量的人力物力，隨著對(duì)植物雄性不育的深入研究，人們發(fā)現(xiàn)利用植物的雄性不育可大幅度降低雜交制種的成本。因此，雄性不育資源的獲得是雜種優(yōu)勢(shì)利用的關(guān)鍵環(huán)節(jié)。但長(zhǎng)期以來，雄性不育系的傳統(tǒng)選育周期過長(zhǎng)、操作不便且不育基因過于單一，不能很好地滿足育種的需要，極大程度地影響了雜種優(yōu)勢(shì)的利用。

雄性不育主要分為傳統(tǒng)的雄性核不育和細(xì)胞質(zhì)核互作雄性不育2種類型。傳統(tǒng)的雄性核不育多由1對(duì)隱性基因控制，能夠滿足植物雄性不育系的最佳選育要求，但目前很難通過傳統(tǒng)的雜交育種方式獲得100%不育的水稻等作物的普通雄性不育系[141]。細(xì)胞質(zhì)核互作雄性不育是由細(xì)胞質(zhì)基因和細(xì)胞核基因互作控制的雄性不育，核質(zhì)互作不育系是目前水稻等作物雜種優(yōu)勢(shì)利用的主要工具，但受遺傳背景影響，可供利用的水稻優(yōu)良核質(zhì)互作不育系十分有限，微效恢復(fù)基因的存在增加了核質(zhì)互作不育系選育的難度。伴隨著生物技術(shù)的發(fā)展，通過植物基因工程手段，利用細(xì)胞毒素基因barnase和TA29啟動(dòng)子的特異表達(dá)[142]、反義技術(shù)和擾亂線粒體等細(xì)胞器[143-144]的正常功能來獲得植物雄性不育系極大地加快了植物雄性不育系選育的進(jìn)程，也已成為當(dāng)前利用基因工程手段創(chuàng)制植物雄性不育系的主要方法。在此基礎(chǔ)之上，從轉(zhuǎn)基因安全角度考慮，也有學(xué)者提出可利用質(zhì)體轉(zhuǎn)化將普通雄性不育基因或可育基因?qū)胭|(zhì)體基因組來獲得理想的植物雜種優(yōu)勢(shì)株系的方案[145]，但目前尚未見報(bào)道。Ruiz等[145]在煙草葉綠體基因組中表達(dá)編碼β-酮硫解酶的phaA基因，研究了其光調(diào)節(jié)效應(yīng)，并評(píng)估了其在不同光周期下的表達(dá)，除正常可育的轉(zhuǎn)基因株系外，還發(fā)現(xiàn)了缺少花粉的雄性不育表型，而且雄性不育表型可通過改變光照條件恢復(fù)其育性，證實(shí)了通過質(zhì)體基因工程手段創(chuàng)制細(xì)胞質(zhì)雄性不育系的可行性。隨后，李丁等[146]提出了利用葉綠體轉(zhuǎn)基因技術(shù)機(jī)械化生產(chǎn)非轉(zhuǎn)基因雜交稻種子的策略，進(jìn)一步豐富了質(zhì)體基因工程在雜種優(yōu)勢(shì)利用上的應(yīng)用。

4 展望

質(zhì)體轉(zhuǎn)化較核轉(zhuǎn)化具有更大的優(yōu)勢(shì)，20多年來被廣泛應(yīng)用于藥物、疫苗、抗原和酶的生產(chǎn)及作物農(nóng)藝性狀改良等方面。雖然在實(shí)驗(yàn)室研究中已經(jīng)取得了顯著的成就，也有部分轉(zhuǎn)化材料進(jìn)入了田間試驗(yàn)階段，但基于質(zhì)體遺傳轉(zhuǎn)化技術(shù)的轉(zhuǎn)基因材料的商業(yè)化應(yīng)用仍未獲得成功。目前，眾多的研究在利用高通量克隆方法構(gòu)建葉綠體表達(dá)載體[147-148]和尋找新的選擇標(biāo)記[67,149]上做出了較大的貢獻(xiàn)，轉(zhuǎn)基因植物的蛋白質(zhì)純化[150]和在絕對(duì)安全的轉(zhuǎn)基因控制條件下生物反應(yīng)器中植物組織的生長(zhǎng)[151-152]等下游技術(shù)也得到了相應(yīng)發(fā)展，但葉綠體表達(dá)載體的構(gòu)建、外源基因在葉綠體基因組中的整合和同質(zhì)化株系的獲得仍是一個(gè)相對(duì)簡(jiǎn)單但漫長(zhǎng)的過程。

另外，通過基因工程手段來增加主要糧食作物，尤其是提高單子葉植物光合作用效率，長(zhǎng)期以來都被認(rèn)為是提高作物產(chǎn)量的一種行之有效的改良策略。但截至目前，在單子葉植物中，尤其是水稻和玉米等主要糧食作物的質(zhì)體轉(zhuǎn)化技術(shù)仍不成熟，僅在水稻中有少量報(bào)道[34-38]，同質(zhì)化問題仍是限制其發(fā)展的主要問題。因此，在雙子葉植物質(zhì)體基因工程的框架基礎(chǔ)上，開發(fā)可用于單子葉植物質(zhì)體轉(zhuǎn)化的有效方法，真正建立起成熟的單子葉植物質(zhì)體基因工程體系，才能進(jìn)一步解決傳統(tǒng)核遺傳轉(zhuǎn)化的弊端，一旦獲得突破性的進(jìn)展，將推動(dòng)質(zhì)體基因工程的新發(fā)展，進(jìn)而開啟植物分子育種的新篇章。

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Progress on Application of Plastid Genetic Engineering in Plant Breeding

MU Liansheng,HE Yong,LUO An,ZHU Zhiyan,TIAN Zhihong*

(College of Life Science,Yangtze University/Hubei Collaborative Innovation Center for Grain Industry,Jingzhou 434025,China)

Compared with traditional nuclear transformation system,plastid genetic engineering that performs much safer,more precise and efficient expression of foreign genes has made great achievements in crop quality and yield improvement,even offers new ideas for plant breeding.This paper reviewed the plastid transformation techniques,screening markers(systems) and their applications in the improvement of plant resistance traits,yield and quality and heterosis utilization，so as to provide theoretical reference for the application of plastid genetic engineering in plant breeding,especially in monocotyledonous plants.

plastid; chloroplast; genetic transformation; genetic engineering

2017-03-02

主要糧食作物產(chǎn)業(yè)化湖北省協(xié)同創(chuàng)新中心開放基金項(xiàng)目(2015MS006)

母連勝(1991-)，男，湖北鄖西人，在讀碩士研究生，研究方向：水稻基因工程。 E-mail:593603883@qq.com。何勇為共同第一作者

*通訊作者：田志宏(1966-)，男，湖北監(jiān)利人，教授，博士，主要從事植物遺傳與分子生物學(xué)及水稻分子育種研究。 E-mail:zhtian@yangtzeu.edu.cn

時(shí)間：2017-05-09 08：40：00

Q943.2

A

1004-3268(2017)06-0001-12

網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/41.1092.S.20170509.0840.001.html