在煙草中過(guò)量表達(dá)大豆SGF14a增強(qiáng)轉(zhuǎn)基因煙草對(duì)鋁脅迫的耐受性

楊志麗, 郭傳龍, 劉蕾, 武孔煥, 王琳, 李昆志, 趙艷, 陳麗梅

(昆明理工大學(xué)生命科學(xué)與技術(shù)學(xué)院生物工程技術(shù)研究中心,昆明650500)

摘要為了驗(yàn)證鋁耐受型丹波黑大豆根尖14-3-3a基因(soybean 14-3-3a,SGF14a)在植物應(yīng)答鋁脅迫中的作用,本研究利用35S組成型啟動(dòng)子和大豆SGF14a的編碼區(qū)構(gòu)建植物表達(dá)載體pK-35S-SGF14a,在野生型(wild type,WT)煙草中過(guò)量表達(dá)SGF14a獲得3個(gè)轉(zhuǎn)基因株系(S11、S19和S23);并用50 μmol/L鋁處理WT和轉(zhuǎn)基因煙草,分析過(guò)量表達(dá)SGF14a對(duì)煙草鋁耐受性的影響.結(jié)果表明:過(guò)量表達(dá)SGF14a的轉(zhuǎn)基因煙草經(jīng)鋁脅迫處理后根的相對(duì)生長(zhǎng)量比WT高約30%～40%;此外,在沒(méi)有鋁脅迫的正常生長(zhǎng)條件下,3株轉(zhuǎn)基因煙草根中可溶性蛋白的含量都比WT高,而用50 μmol/L鋁脅迫24 h后WT煙草和3株轉(zhuǎn)基因煙草根中可溶性蛋白的含量都下降,但轉(zhuǎn)基因煙草根中可溶性蛋白含量仍顯著高于WT,并且過(guò)量表達(dá)SGF14a還可顯著提高過(guò)氧化物酶、過(guò)氧化氫酶以及抗壞血酸過(guò)氧化物酶的活性,降低鋁脅迫下煙草根中過(guò)氧化氫(H2O2)的積累以及氧化脅迫的水平;同時(shí),轉(zhuǎn)基因煙草在低酸土壤中的生長(zhǎng)狀況也明顯優(yōu)于WT。這說(shuō)明過(guò)量表達(dá)SGF14a可增強(qiáng)煙草對(duì)鋁脅迫的耐受性及其適應(yīng)酸性土壤生長(zhǎng)的能力。

關(guān)鍵詞14-3-3蛋白; 過(guò)量表達(dá); 轉(zhuǎn)基因煙草; 鋁脅迫; 耐鋁性

中圖分類號(hào)Q 786文獻(xiàn)標(biāo)志碼A

基金項(xiàng)目:國(guó)家自然科學(xué)基金(30970263)。

收稿日期(Received):2014-09-28;接受日期(Accepted):2015-02-03;網(wǎng)絡(luò)出版日期(Published online):2015-05-19

Overexpression of soybeanSGF14aenhanced tolerance of transgenic tobacco plants to aluminum stress. Journal of ZhejiangUniversity(Agric. & LifeSci.), 2015,41(3):285-292

Yang Zhili, Guo Chuanlong, Liu Lei, Wu Konghuan, Wang Lin, Li Kunzhi, Zhao Yan, Chen Limei*(BiotechnologyResearchCenter,FacultyofLifeScienceandTechnology,KunmingUniversityofScienceandTechnology,Kunming650500,China)

SummaryApproximately 30% of the global arable land in the world is acidic soils (pH<5) and China’s acidic soil accounts for more than 21% of the area. Aluminum (Al) toxicity is a major limiting factor which limited crop production in acid soils. Al firstly inhibits the growth and development of plant roots. Consequently, it decreased the absorption of water and nutrients, which results in poor growth and production of plants. Therefore, in recent years, many researchers dedicated to study Al tolerance mechanisms and bred high Al-tolerant and acid soil-resistant crops by genetic engineering. 14-3-3 proteins are a group of highly conserved regulatory proteins found in eukaryotic cells, and have roles in regulating plant development and stress responses. A lot of 14-3-3 gene family members are isolated in different plants. 14-3-3 proteins regulate various physiological activities and functions by interacting with phosphorylated or non-phosphorylated target proteins in plants. The expression levels of certain 14-3-3 gene isoforms can be adjusted directly by environmental stimuli.

Our previous study showed that Al stress induced the expression of 14-3-3a (SGF14a) in the Al resistant soybean (Glycinemax) root tips. In order to further validate the role ofSGF14ain response to Al stress in plants, plant expression vectors ofSGF14awere constructed. Now, the 35S constitutive promoter was used to overexpressSGF14a. We constructed pK-35S-SGF14aplant expression vectors using gateway technology, then introduced theSGF14aexpression vectors intoAgrobacteriumpMP105, which was transformed into tobacco plants via transformation method. The plants were selected by genomic polymerase chain reaction (PCR), reverse transcriptase-PCR and Western blot analysis. Finally we got three transgenic lines (S11, S19, and S23). The wild type (WT) and transgenic tobacco plants (S11, S19, S23) were treated with 50 μmol/L Al to analyze the effect ofSGF14aoverexpression on the Al-resistance of tobacco. Changes of the relative root growth, H2O2, malondialdehyde (MDA), soluble protein contents as well as antioxidant enzyme activities in transgenic plants were compared.

The results showed that the relative root growth of the transgenic plants increased approximately 1.5-fold as compared with the WT. Moreover, the soluble protein contents in the transgenic tobacco roots increased significantly compared with the WT plants, and antioxidant enzyme activities (peroxidase, catalase, and ascorbate peroxidase) in roots of transgenic tobacco plants also increased when exposed to 50 μmol/L Al. Moreover, the H2O2accumulation and oxidative stress level in transgenic tobacco roots were reduced under the Al stress. The growth status of the transgenic tobacco was better than that of WT when grown in acidic soil.

In summary, the evidences suggest that overexpression ofSGF14aan enhance the Al-tolerance of transgenic tobacco and its ability to adapt to acidic soil.

Key words14-3-3 protein; overexpression; transgenic tobacco; aluminum stress; aluminum tolerance

14-3-3蛋白能與磷酸化的靶蛋白相互作用從而調(diào)節(jié)很多生物過(guò)程,如代謝、生長(zhǎng)、發(fā)育和信號(hào)傳導(dǎo)途徑[1]。很多研究結(jié)果證明植物14-3-3蛋白通過(guò)參與赤霉素(gibberellin)、脫落酸(abscisic acid)和乙烯(ethylene)等激素信號(hào)通路來(lái)調(diào)控植物生長(zhǎng)發(fā)育過(guò)程[1]。14-3-3蛋白對(duì)植物體內(nèi)物質(zhì)代謝也有重要的調(diào)控作用,它通過(guò)調(diào)節(jié)硝酸還原酶(nitrate reductase)、蔗糖磷酸合成酶(sucrose phosphate synthase)和谷氨酰胺合成酶(glutamine synthetase)等代謝酶的活性來(lái)調(diào)控植物體內(nèi)碳氮代謝過(guò)程[2]。此外,14-3-3蛋白還通過(guò)調(diào)節(jié)質(zhì)膜中的K+泵和質(zhì)膜H+-ATP酶(plasma membrane H+-ATPase,PM H+-ATPase)的活性來(lái)維持細(xì)胞內(nèi)外電化學(xué)梯度,調(diào)控植物體內(nèi)的物質(zhì)運(yùn)輸和氣孔開(kāi)閉等[1]。

很多研究發(fā)現(xiàn)環(huán)境脅迫的刺激能直接改變14-3-3基因的某一個(gè)特異異構(gòu)型的表達(dá)水平,因此14-3-3蛋白能夠參與植物對(duì)鹽、磷缺乏、干旱、冷害和重金屬等多種非生物脅迫和病原菌侵染等生物脅迫的應(yīng)答[3]。ukaszewicz等[4]通過(guò)基因工程的手段改變14-3-3蛋白的表達(dá)來(lái)研究其功能,結(jié)果表明在馬鈴薯中過(guò)量表達(dá)14-3-3蛋白能夠改變脂類、氨基酸和礦物質(zhì)的組成,并且提高其抗氧化脅迫的能力,而抑制14-3-3蛋白的表達(dá)則出現(xiàn)相反的結(jié)果。在水稻中過(guò)量表達(dá)玉米的14-3-3基因(ZmGF14-6)后能夠增強(qiáng)水稻的抗旱能力[5],在棉花中過(guò)量表達(dá)擬南芥的14-3-3基因(GF14λ)后能夠使棉花保持“常青”的表型并增強(qiáng)棉花對(duì)干旱脅迫的耐受性[6]。通過(guò)反義技術(shù)抑制擬南芥14-3-3蛋白的表達(dá),可促進(jìn)葉片中淀粉的積累并增加轉(zhuǎn)基因植物的生長(zhǎng)[7]。

全世界大約有30%的可耕地屬于酸性土壤,鋁毒是酸性土壤中限制作物生長(zhǎng)的主要因素之一[8]。鋁毒早期最明顯的癥狀是抑制根尖細(xì)胞生長(zhǎng)和細(xì)胞分裂,從而抑制根的生長(zhǎng),導(dǎo)致根系損傷,影響水分和養(yǎng)分的吸收,限制植物的生長(zhǎng)。低磷和鋁脅迫均誘導(dǎo)擬南芥、羽扇豆、蠶豆和大豆中某些14-3-3異構(gòu)型的表達(dá)[9-11]。本課題組[11]前期研究表明鋁脅迫可誘導(dǎo)耐鋁型丹波黑大豆(resistant soybean,RB)根尖14-3-3a基因(SGF14a)的表達(dá),為了驗(yàn)證SGF14a在植物應(yīng)答鋁脅迫中的作用,本研究通過(guò)通路克隆技術(shù)(Gateway)構(gòu)建SGF14a的植物表達(dá)載體,轉(zhuǎn)化煙草產(chǎn)生SGF14a過(guò)量表達(dá)株系,考察過(guò)量表達(dá)大豆SGF14a對(duì)煙草鋁耐受性的影響,為提高植物耐鋁能力的基因工程手段操作提供基因資源和操作策略。

1材料與方法

1.1煙草的培養(yǎng)

野生型(wild type,WT)煙草種子(Nicotianatabacumcv. Xanthi)消毒后,播種于MS固體培養(yǎng)基(Murashige-Skoog media)中,待種子發(fā)芽后進(jìn)行繼代培養(yǎng),獲得無(wú)菌煙草幼苗用于轉(zhuǎn)基因操作。

1.2植物表達(dá)載體的構(gòu)建

本研究利用通路(Gateway)克隆技術(shù)構(gòu)建目的基因SGF14a的植物表達(dá)載體,構(gòu)建策略如圖1所示。首先根據(jù)GenBank數(shù)據(jù)庫(kù)上發(fā)表的大豆14-3-3a基因(SGF14a)的cDNA編碼區(qū)序列設(shè)計(jì)上游特異引物P1:5′-AAGCTTATGTCGGATTCTTCTC GGGAGGAG-3′(含HindⅢ酶切位點(diǎn))和下游特異引物P2:5′-CTCGAGCTATTCACCTGGTTGTT GCTTAGAT-3′(含XhoⅠ酶切位點(diǎn)),然后提取RB大豆根總RNA,反轉(zhuǎn)錄為cDNA。以反轉(zhuǎn)錄的cDNA為模板,用帶有合適酶切位點(diǎn)的SGF14a基因上、下游引物進(jìn)行PCR擴(kuò)增,獲得SGF14a基因cDNA編碼區(qū)全長(zhǎng)DNA片段,然后亞克隆于TA克隆載體pMD18-T(大連寶生物有限公司)中獲得pMD18T-SGF14a載體,對(duì)獲得的陽(yáng)性克隆進(jìn)行測(cè)序,檢測(cè)外源基因SGF14a是否發(fā)生突變,再用HindⅢ和XhoⅠ雙酶切pMD18T-SGF14a和Gateway的入門載體pENTR(購(gòu)于美國(guó)Invitrogen公司),將SGF14a基因cDNA編碼區(qū)亞克隆到pENTR載體上,產(chǎn)生入門克隆載體pENTR-SGF14a。最后進(jìn)行LR反應(yīng),(含有attL的入門克隆和attR位點(diǎn)的目的載體DNA分子發(fā)生重組反應(yīng)),在LR重組酶的作用下,入門克隆載體pENTR-SGF14a和表達(dá)載體pK2GW7(購(gòu)自比利時(shí)VIB/Gent公司)進(jìn)行LR反應(yīng),發(fā)生重組反應(yīng)產(chǎn)生SGF14a的植物表達(dá)載體,簡(jiǎn)稱pK-35S-SGF14a。LR反應(yīng)按照LR反應(yīng)試劑盒(LR ClonaseTMplus enzyme mix)(Invitrogen公司)說(shuō)明書(shū)進(jìn)行,反應(yīng)混合液混勻后于25 ℃反應(yīng)過(guò)夜,轉(zhuǎn)化大腸埃希菌感受態(tài)細(xì)胞DH5(購(gòu)于天根生化科技有限公司),在含有50 μg/mL大觀霉素(spectinomycin,Spe)的LB平板上篩選重組克隆,獲得植物表達(dá)載體pK-35S-SGF14a。

pMD18-T:TA克隆載體;pENTR:入門載體;pK2GW7:植物表達(dá)載體;(HindⅢ,XhoⅠ,XmnⅠ,SalⅠ,BamHⅠ,KpnⅠ,ExoRⅠ,NotⅠ,SalⅠ,EcoRⅤ):限制性酶切位點(diǎn);(Amp r,Km r,Spe r):篩選標(biāo)記基因;LacZ:報(bào)告基因;Origin:起始位點(diǎn);35S:組成型啟動(dòng)子;(attR1、attR2和attL1、attL2):附著點(diǎn)(attachment site,att)的重組位點(diǎn);ccdB *:負(fù)選擇細(xì)菌病毒性基因;(RB和LB):右邊界序列和左邊界序列. 　　pMD18-T: TA cloning vector; pENTR: Entry vector; pK2GW7: Plant expression vector; (HindⅢ, XhoⅠ, XmnⅠ,SalⅠ, BamHⅠ, KpnⅠ, ExoRⅠ, NotⅠ, SalⅠ, EcoRⅤ): Restriction enzyme sites; (Amp r, Km r, Spe r): Marker genes for plant selection; LacZ: Reporter gene; Origin: Initiation sites; 35S: Constitutive promoter; (attR1,attR2 and attL1, attL2): att recombination sites; ccdB *: Bacterial toxin gene for negative selection; (RB and LB): Right border sequence and left border sequence, respectively. 圖1　植物表達(dá)載體pK-35S-SGF14a構(gòu)建示意圖 Fig.1　Diagram of the construction of plant expression vector pK-35S-SGF14a

1.3煙草的轉(zhuǎn)化及轉(zhuǎn)基因煙草的篩選

通過(guò)電轉(zhuǎn)化法將植物表達(dá)載體pK-35S-SGF14a轉(zhuǎn)入農(nóng)桿菌中,在含有Spe的平板上篩選得到陽(yáng)性克隆,經(jīng)菌液PCR檢測(cè)證實(shí)含有pK-35S-SGF14a的陽(yáng)性克隆用于煙草的轉(zhuǎn)化。通過(guò)農(nóng)桿菌介導(dǎo)的葉盤轉(zhuǎn)化法轉(zhuǎn)染W(wǎng)T煙草,將轉(zhuǎn)染后的葉片轉(zhuǎn)移到含有卡那霉素(kanamycin,Km)和頭孢噻肟鈉(cefotaxine,Cef)的芽誘導(dǎo)培養(yǎng)基(MS4)上誘導(dǎo)外植體發(fā)芽,約15 d繼代1次。待芽長(zhǎng)大后從外植體上切下轉(zhuǎn)入含Km和Cef的生根培養(yǎng)基上誘導(dǎo)根的生長(zhǎng),得到抗Km的煙草植株。

1.4轉(zhuǎn)基因煙草的基因組PCR和逆轉(zhuǎn)錄PCR檢測(cè)

用CTAB法從煙草葉片中提取基因組DNA作為模板,分別用SGF14a的上、下游特異引物進(jìn)行PCR擴(kuò)增,檢測(cè)SGF14a在轉(zhuǎn)基因煙草基因組中的整合情況。用試劑TRIzol?提取煙草根的總RNA,取3 μg的總RNA用逆轉(zhuǎn)錄酶(moloney murine leukemia virus reverse transcriptase,M-MLVRT)反轉(zhuǎn)錄合成cDNA,用SGF14a的上、下游特異引物進(jìn)行逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction,RT-PCR),以檢測(cè)SGF14a的轉(zhuǎn)錄水平(TRIzol?試劑和M-MLVRT均購(gòu)于大連寶生物有限公司)。

1.5蛋白質(zhì)印跡法(Western blot)分析

用蛋白抽提液Tris-HCl 50 mmol/L,10%甘油,β-巰基乙醇10 mmol/L,苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF)1 mmol/L,乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)2 mmol/L,10%不溶性聚乙烯吡咯烷酮(polyvinyl pyrrolidone,PVP)提取煙草根的可溶性蛋白,通過(guò)考馬斯亮藍(lán)法(Bradford)測(cè)定蛋白含量,通過(guò)聚丙烯酰胺凝膠電泳(SDS-PAGE:12%分離膠和4%濃縮膠,每個(gè)泳道上樣量為50g)分離蛋白,用半干式轉(zhuǎn)膜儀將SDS-PAGE分離的蛋白轉(zhuǎn)移到聚偏二氟乙烯膜(polyvinylidene fluoride,PVDF)上,用5%的脫脂奶粉常溫封閉1 h,然后用磷酸鹽緩沖液(phosphate buffer saline,PBS)清洗PVDF膜3次,每次10 min;接著加入10L大豆14-3-3a蛋白兔抗體,與PVDF膜在常溫下孵育2～3 h;用PBS清洗PVDF膜3次后,加入偶聯(lián)有辣根過(guò)氧化物酶的羊抗兔二抗,常溫孵育1 h;最后用高靈敏度化學(xué)發(fā)光檢測(cè)試劑盒顯示結(jié)果(羊抗兔二抗和高靈敏度化學(xué)發(fā)光檢測(cè)試劑盒均購(gòu)于康為世紀(jì)生物科技有限公司)。

1.6煙草的處理和根相對(duì)生長(zhǎng)量測(cè)定

鋁對(duì)植物的毒害作用最典型的癥狀表現(xiàn)在對(duì)根生長(zhǎng)的抑制作用,因此通過(guò)測(cè)定根的生長(zhǎng)量來(lái)分析煙草的鋁耐受能力。WT和轉(zhuǎn)基因煙草的組培苗水培2周,用pH 4.3的0.5 mmol/L CaCl2預(yù)處理過(guò)夜,記錄根的長(zhǎng)度,然后置于0和50 μmol/L的AlCl3溶液(含有0.5 mmol/L CaCl2,pH 4.3)中處理24 h,記錄處理后的根長(zhǎng)度,每個(gè)株系6個(gè)重復(fù)。根相對(duì)生長(zhǎng)量/%=(處理后根長(zhǎng)度-處理前根長(zhǎng)度)/(對(duì)照處理后的根長(zhǎng)度-對(duì)照處理前的根長(zhǎng)度)×100。

1.7丙二醛和H2O2含量的測(cè)定

收集鋁脅迫處理后煙草根組織,在液氮中磨碎后用Tirs-HCl(1 mol/L,pH 7.5)抽提,離心收集上清液。丙二醛(malondialdehyde,MDA)含量的測(cè)定參考Gurel等[12]的方法;H2O2含量的測(cè)定參考Gay等[13]的方法。

1.8抗氧化酶活性的測(cè)定

凍存的根尖用液氮充分研磨后,用蛋白抽提緩沖液(50 mmol/L Tris-HCl pH 7.4,甘油10%,巰基乙醇10 mmol/L,PMSF 1 mmol/L, EDTA 2 mmol/L,不溶性PVP 10%)提取可溶性蛋白,采用考馬斯亮藍(lán)法測(cè)定可溶性蛋白的含量。過(guò)氧化物酶(peroxidase,POD)活性測(cè)定參照Britton等[14]的愈創(chuàng)木酚方法。過(guò)氧化氫酶(catalase,CAT)活性測(cè)定按照Abei[15]的方法進(jìn)行?？箟难徇^(guò)氧化物酶(ascorbate peroxidase,APX)活性的測(cè)定采用Nakano等[16]的方法。

1.9煙草植株在低酸性土壤中的生長(zhǎng)情況分析

將生長(zhǎng)狀況一致的WT和轉(zhuǎn)基因煙草幼苗移栽到裝有1 000 g(干質(zhì)量)的酸性土壤(pH 4.8)中,每周澆水2次,在光照時(shí)間為12 h、光照強(qiáng)度約為1 200mol/(m2·s)的溫室中培養(yǎng)3個(gè)月后觀察生長(zhǎng)狀況并拍照。

1.10數(shù)據(jù)分析

所有的生理生化指標(biāo)分析均進(jìn)行3次重復(fù)。用ANOVA SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)和差異顯著性分析。

2結(jié)果與分析

2.1SGF14a過(guò)量表達(dá)產(chǎn)生的轉(zhuǎn)基因煙草株系

M:DNA分子標(biāo)志物;NC:負(fù)對(duì)照(PCR體系中用去離子水進(jìn)行PCR擴(kuò)增);PC:正對(duì)照(以pK-35S-SGF14a質(zhì)粒作為PCR模板);WT:野生型煙草;(S4、S9、S11、S12、S13、S19、S23):獲得的不同轉(zhuǎn)基因煙草株系編號(hào);18S rRNA:內(nèi)參基因. 　　M: DNA marker; NC: Negative control (with deionized water as template for PCR ); PC: Positive control (with pK-35S-SGF14a as template for PCR); WT: Wild type tobacco plants; (S4， S9， S11， S12， S13， S19, S23): Serial numbers of transgenic tobacco plants; 18S rRNA: Internal reference gene. 圖2　轉(zhuǎn)基因煙草基因組PCR(A)、RT-PCR(B)和蛋白質(zhì)印跡法(C)檢測(cè)SGF14a基因的插入情況和轉(zhuǎn)錄水平 Fig.2　Genomic PCR (A), RT-PCR (B) and Western blot (C) analysis for detection of SGF14a integration and transcriptional levels in transgenic and wild type tobacco lines

利用大豆SGF14a的編碼區(qū)構(gòu)建植物表達(dá)載體pK-35S-SGF14a(圖1),用pK-35S-SGF14a轉(zhuǎn)化煙草獲得具有Km抗性的轉(zhuǎn)基因株系23個(gè),以基因組DNA為模板,用SGF14a基因的特異性引物進(jìn)行基因組PCR擴(kuò)增，檢測(cè)SGF14a基因在轉(zhuǎn)基因煙草中的插入情況(圖2A)。結(jié)果表明，在7個(gè)(S4、S9、S11、S12、S13、S19和S23)具有Km抗性的轉(zhuǎn)基因煙草株系中能夠擴(kuò)增出與正對(duì)照相同的DNA片段(0.8 ku),而在負(fù)對(duì)照和WT中不能擴(kuò)增出與正對(duì)照相同的DNA片段,說(shuō)明在這7個(gè)轉(zhuǎn)基因株系的基因組中有SGF14a基因插入。RT-PCR分析結(jié)果(圖2B)表明，在WT中不能擴(kuò)增出0.8 ku的SGF14acDNA片段,而在6個(gè)轉(zhuǎn)基因株系中除S9株系外都擴(kuò)增出0.8 ku的SGF14acDNA,說(shuō)明在這5個(gè)轉(zhuǎn)基因植株(S4、S11、S13、S19和S23)中SGF14a能夠正常轉(zhuǎn)錄。選擇3個(gè)(S11、S19和S23)轉(zhuǎn)基因株系提取根的可溶性蛋白進(jìn)行蛋白印跡法分析(Western blot)(圖2C),結(jié)果在WT煙草中也檢測(cè)出14-3-3蛋白的條帶。這可能是因?yàn)閃T煙草中本身的14-3-3蛋白與大豆14-3-3蛋白的同源性非常高,所以大豆14-3-3蛋白的抗體也能識(shí)別煙草的14-3-3蛋白所致。3個(gè)轉(zhuǎn)基因株系14-3-3蛋白的表達(dá)量明顯比WT煙草高,其中S11的表達(dá)量最高,S19和S23的表達(dá)量次之,說(shuō)明在這3個(gè)轉(zhuǎn)基因株系中有SGF14a的過(guò)量表達(dá)。

2.2過(guò)量表達(dá)SGF14a對(duì)轉(zhuǎn)基因煙草鋁耐受性的影響

為了考察轉(zhuǎn)基因煙草對(duì)鋁脅迫的耐受性,用50 μmol/L AlCl3處理S11、S19、S23轉(zhuǎn)基因株系和WT無(wú)菌苗的根24 h后分析煙草根相對(duì)生長(zhǎng)量(圖3A),結(jié)果顯示3個(gè)轉(zhuǎn)基因煙草根相對(duì)生長(zhǎng)量比WT的高30%～40%,說(shuō)明SGF14a基因的過(guò)量表達(dá)能夠增加煙草對(duì)鋁脅迫的耐受性?？扇苄缘鞍椎暮磕軌蚍从持参锬褪墉h(huán)境非生物脅迫的程度,植物中可溶性蛋白含量越高,說(shuō)明其耐受脅迫能力越強(qiáng)。從圖3B可以看出：在沒(méi)有鋁脅迫的正常生長(zhǎng)條件下,3個(gè)轉(zhuǎn)基因煙草根中可溶性蛋白含量都比WT高,這說(shuō)明過(guò)量表達(dá)SGF14a能夠增加煙草根中可溶性蛋白的積累；在50 μmol/L鋁脅迫24 h后,WT煙草和3個(gè)轉(zhuǎn)基因煙草根中可溶性蛋白含量都下降,但3個(gè)轉(zhuǎn)基因煙草根中可溶性蛋白含量仍然顯著高于WT,說(shuō)明在煙草中過(guò)量表達(dá)SGF14a能夠增加可溶性蛋白的積累。

MDA含量反映了植物受脅迫后質(zhì)膜的氧化程度,H2O2含量反映了脅迫后細(xì)胞內(nèi)活性氧的積累。從圖3D可以看出：在沒(méi)有鋁脅迫的正常生長(zhǎng)條件下,WT煙草和3個(gè)轉(zhuǎn)基因煙草根中MDA含量沒(méi)有明顯差別；50 μmol/L鋁脅迫24 h使WT煙草和轉(zhuǎn)基因煙草根中MDA含量上升,但3個(gè)轉(zhuǎn)基因煙草根中MDA含量仍然低于WT煙草。從圖3C可以看出：在正常生長(zhǎng)條件下，轉(zhuǎn)基因煙草根中H2O2的含量明顯低于WT煙草,在鋁脅迫后WT煙草和轉(zhuǎn)基因煙草根中H2O2的積累都有所增加,但轉(zhuǎn)基因煙草根中H2O2含量明顯低于WT煙草。這說(shuō)明在煙草中過(guò)量表達(dá)SGF14a可顯著降低煙草根中H2O2的含量,減輕在鋁脅迫下煙草根中H2O2的積累和膜脂肪過(guò)氧化程度。

-Al:無(wú)鋁處理組;+Al:50 μmol/L鋁處理組。柱狀圖上不同小寫字母表示在P<0.05水平差異有統(tǒng)計(jì)學(xué)意義. 　　-Al: Without Al treatment; +Al: 50 μmol/L Al treatment. Different lowercase letters above bar graph indicate statistically significant differences at the 0.05 probability level． 圖3　鋁脅迫下WT和轉(zhuǎn)基因煙草根中根相對(duì)生長(zhǎng)量、可溶性蛋白、H 2O 2和MDA含量 Fig.3　Relative root growth and the contents of soluble protein, H 2O 2 and MDA in WT and transgenic tobacco lines under Al stress

-Al:無(wú)鋁處理組;+Al:50 μmol/L鋁處理組。柱狀圖上不同小寫字母表示在P<0.05水平差異有統(tǒng)計(jì)學(xué)意義. 　　-Al:Without Al treatment; +Al: 50 μmol/L Al treatment. Different lowercase letters above bar graph indicate statistically significant differences at the 0.05 probability level． 圖4　鋁脅迫下WT和轉(zhuǎn)基因煙草根中POD、CAT和APX活性 Fig.4　Activity of POD, CAT and APX in WT and transgenic tobacco lines under Al stress

2.3過(guò)量表達(dá)SGF14a對(duì)轉(zhuǎn)基因煙草抗氧化酶活性的影響

14-3-3蛋白對(duì)抗氧化酶活性的調(diào)控作用已經(jīng)在研究中得到證實(shí)[17],POD、CAT和APX是清除活性氧最重要的抗氧化物酶類,在植物抗氧化系統(tǒng)中發(fā)揮著重要作用。為考察過(guò)量表達(dá)SGF14a在鋁脅迫下轉(zhuǎn)基因煙草抗氧化酶活性的影響,測(cè)定了50 μmol/L鋁脅迫24 h后轉(zhuǎn)基因和WT煙草根中POD(圖4A)、CAT(圖4B)、APX(圖4C)的活性。結(jié)果表明，在沒(méi)有鋁脅迫條件下,SGF14a過(guò)表達(dá)植株S11、S19、S23根中POD、CAT、APX活性均高于WT。鋁脅迫導(dǎo)致WT和3個(gè)轉(zhuǎn)基因株系根中POD、CAT、APX活性都升高,但3個(gè)轉(zhuǎn)基因株系中這3種抗氧化酶活性都顯著高于WT,這說(shuō)明過(guò)量表達(dá)SGF14a能顯著增加轉(zhuǎn)基因煙草在鋁脅迫下根中抗氧化酶的活性。

2.4過(guò)量表達(dá)SGF14a對(duì)煙草適應(yīng)酸性土壤能力的影響

鋁毒是酸性土壤中植物生長(zhǎng)的重要限制因素,將S11、S19、S23轉(zhuǎn)基因株系和WT的無(wú)菌苗移栽到酸性土壤中栽培,觀察轉(zhuǎn)基因植物生長(zhǎng)表型的變化,結(jié)果發(fā)現(xiàn)3個(gè)轉(zhuǎn)基因株系在酸性壤上的生長(zhǎng)情況明顯優(yōu)于WT(圖5)。在酸性土壤上生長(zhǎng)的早期,3個(gè)轉(zhuǎn)基因煙草植株的高度比WT高20%(圖5A),在接近開(kāi)花期,3個(gè)轉(zhuǎn)基因煙草植株的高度比WT高25%～30%(圖5B),說(shuō)明在煙草中過(guò)量表達(dá)SGF14a可增加轉(zhuǎn)基因煙草適應(yīng)酸性土壤生長(zhǎng)的能力。

圖5　WT煙草和轉(zhuǎn)基因煙草在酸性土壤中生長(zhǎng)30 d(A)和90 d(B)的狀況 Fig.5　Growth performance of WT and transgenic tobacco lines after 30 days (A) and 90 days (B) in the acidic soil

3討論

以往的研究[7]表明大麥葉片14-3-3蛋白結(jié)合蛋白中有谷胱甘肽還原酶、抗氧化酶Mg-SOD、POD、APX等。在高等植物中,14-3-3蛋白與抗氧化酶APX的互作在植物應(yīng)答鹽脅迫過(guò)程中發(fā)揮重要作用,如在擬南芥中過(guò)量表達(dá)番茄的14-3-3蛋白TFT7可提高轉(zhuǎn)基因植物APX的活性,在鹽脅迫下轉(zhuǎn)基因植物H2O2和MDA的含量顯著低于WT,轉(zhuǎn)基因植物抗鹽能力顯著增強(qiáng)[18]。在轉(zhuǎn)基因水稻中通過(guò)RNAi干擾技術(shù)沉默14-3-3基因GF14e的表達(dá),使抗氧化酶基因POX22.3的表達(dá)增強(qiáng),過(guò)氧化物酶相關(guān)防御反應(yīng)被激活,細(xì)胞壁物質(zhì)的交聯(lián)作用增強(qiáng),限制病原體侵染,因而提高水稻對(duì)白葉枯病菌的抗性[19],這說(shuō)明14-3-3蛋白對(duì)抗氧化酶的表達(dá)和活性有調(diào)控作用。植物體內(nèi)H2O2含量的變化與抗氧化酶的表達(dá)水平或活性及抗氧化物質(zhì)的含量有關(guān),本研究在轉(zhuǎn)基因煙草中過(guò)量表達(dá)RB的14-3-3蛋白使鋁脅迫下轉(zhuǎn)基因煙草根中3種清除H2O2的抗氧化酶活性顯著高于WT,因此H2O2和MDA含量顯著低于WT。這說(shuō)明在轉(zhuǎn)基因煙草根中過(guò)量表達(dá)RB的SGF14a可通過(guò)調(diào)控抗氧化酶的活性來(lái)降低根中H2O2的含量,由此提高煙草抗鋁脅迫的能力。

本研究還觀察到過(guò)量表達(dá)SGF14a可影響轉(zhuǎn)基因煙草根中可溶性蛋白的含量。在無(wú)鋁脅迫下,過(guò)量表達(dá)大豆SGF14a的轉(zhuǎn)基因煙草根中積累的可溶性蛋白顯著高于WT。Schoonheim等[7]的研究表明在大麥葉片14-3-3蛋白的結(jié)合蛋白中有核糖體結(jié)合蛋白、蛋白質(zhì)翻譯的起始因子6和延伸因子1a等,說(shuō)明14-3-3蛋白參與了蛋白質(zhì)合成的調(diào)控?？扇苄缘鞍椎暮亢椭参锏哪弯X能力有關(guān),有些耐鋁植物在鋁脅迫下通過(guò)增加可溶性蛋白的合成來(lái)緩解鋁的毒性。過(guò)量表達(dá)14-3-3蛋白的轉(zhuǎn)基因煙草在鋁脅迫下根內(nèi)積累的可溶性蛋白顯著高于WT,這可能是轉(zhuǎn)基因煙草耐鋁能力增強(qiáng)的另一原因之一。

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*通信作者(Corresponding author):陳麗梅,Tel:+86-871-65920213;E-mail:chenlimeikm@126.com

第一作者聯(lián)系方式:韓雙,E-mail:sshuanghan@163.com

URL:http://www.cnki.net/kcms/detail/33.1247.s.20150519.1316.007.html