百日草離體葉肉細(xì)胞分化為管狀分子概述

李 娜，白 蘭，郭學(xué)民

(河北科技師范學(xué)院生命科技學(xué)院，河北 秦皇島，066600)

百日草離體葉肉細(xì)胞分化為管狀分子概述

李 娜，白 蘭，郭學(xué)民*

(河北科技師范學(xué)院生命科技學(xué)院，河北 秦皇島，066600)

概述了植物細(xì)胞分化的模式系統(tǒng)——管狀分子分化實(shí)驗(yàn)系統(tǒng)的建立以及基于該系統(tǒng)的管狀分子分化的生理學(xué)、細(xì)胞學(xué)、生物化學(xué)和分子生物學(xué)等方面的研究進(jìn)展，并對(duì)今后的研究方向進(jìn)行了展望。

百日草；次生細(xì)胞壁；細(xì)胞分化；管狀分子

管狀分子(Traeheary Elements，TEs)是維管植物木質(zhì)部?jī)?nèi)導(dǎo)管和管胞的總稱，皆為長(zhǎng)柱狀細(xì)胞，次生壁木質(zhì)化，成熟后均缺乏原生質(zhì)體，其功能是輸導(dǎo)水分、礦質(zhì)元素和機(jī)械支持作用。導(dǎo)管和管胞差別是，管胞無(wú)穿孔，管胞間壁僅有具緣紋孔，借以實(shí)現(xiàn)物質(zhì)轉(zhuǎn)運(yùn)；而導(dǎo)管分子間的某些區(qū)域具有穿孔，多個(gè)導(dǎo)管分子通過(guò)末端的穿孔連接形成一個(gè)長(zhǎng)的管道，即導(dǎo)管，與管胞相比，其輸導(dǎo)能力大大增強(qiáng)。管狀分子來(lái)源于形成層，由形成層細(xì)胞經(jīng)過(guò)細(xì)胞擴(kuò)增、次生壁沉積、胞內(nèi)物質(zhì)自溶、形成端壁穿孔等步驟而形成[1, 2]。

管狀分子分化已被作為植物細(xì)胞分化的模式系統(tǒng)，在形態(tài)結(jié)構(gòu)、生化和分子組成、發(fā)育及生理功能上都具有明顯的特點(diǎn)，是植物解剖學(xué)、發(fā)育生物學(xué)和細(xì)胞生物學(xué)的研究熱點(diǎn)之一。多年來(lái)，各國(guó)學(xué)者建立了整體實(shí)驗(yàn)系統(tǒng)和離體實(shí)驗(yàn)系統(tǒng)，對(duì)管狀分子的形態(tài)解剖學(xué)、生理學(xué)以及分子機(jī)制進(jìn)行了一定的研究。其中，百日草(ZinniaelegansJacq.)葉肉細(xì)胞離體培養(yǎng)系統(tǒng)是目前分化效果最好的實(shí)驗(yàn)系統(tǒng)，人們利用該系統(tǒng)開(kāi)創(chuàng)性地研究了離體條件下管狀分子分化的基本過(guò)程，并對(duì)管狀分子分化的細(xì)胞學(xué)、生理學(xué)、生物化學(xué)和分子生物學(xué)進(jìn)行了卓有成效的研究。筆者擬對(duì)30多年來(lái)人們利用百日草葉肉細(xì)胞離體培養(yǎng)系統(tǒng)的研究成果進(jìn)行概述，為進(jìn)一步闡明管狀分子分化機(jī)制提供基礎(chǔ)。

1 離體實(shí)驗(yàn)系統(tǒng)的建立

因?yàn)楣軤罘肿有螒B(tài)隨著分化進(jìn)程而發(fā)生顯著變化，其中包括環(huán)紋、螺紋和網(wǎng)紋次生細(xì)胞壁(SCW)的形成以及自溶作用。所以，管狀分子分化被認(rèn)為是植物細(xì)胞分化的模式系統(tǒng)。然而，大多數(shù)早期關(guān)于分化的研究，采用的是多細(xì)胞系統(tǒng)，這樣的系統(tǒng)包含幾種作為起始材料的細(xì)胞類型，為追蹤單個(gè)細(xì)胞分化過(guò)程帶來(lái)了困難。Kohlenbach和Schmidt[3]發(fā)現(xiàn)，以機(jī)械法分離的單個(gè)百日草葉肉細(xì)胞可直接分化為管狀分子，這促使Fukuda和Komamine[4]在此基礎(chǔ)上，建立了一個(gè)有效的、高頻分化的實(shí)驗(yàn)系統(tǒng)。該系統(tǒng)已被全世界許多實(shí)驗(yàn)室廣泛應(yīng)用，有時(shí)根據(jù)特定情況僅僅對(duì)一些細(xì)節(jié)進(jìn)行了修改[5～10]。

用液體介質(zhì)浸漬百日草葉片，研磨后，以小網(wǎng)眼篩過(guò)濾，液體介質(zhì)反復(fù)漂洗懸浮液，分離得到單個(gè)葉肉細(xì)胞。要注意：(1)材料要適當(dāng)。取幼苗第一對(duì)葉，而非成年植物最嫩的葉片。(2)條件要適當(dāng)。旋轉(zhuǎn)培養(yǎng)轉(zhuǎn)速為10 r/min，0.3～0.4 mol/L山梨醇調(diào)節(jié)滲透壓，生長(zhǎng)調(diào)節(jié)劑為0.1 mg/L a-萘乙酸(NAA)和1 mg/L 6-芐基腺嘌呤(6-BA)，起始細(xì)胞濃度為(0.4～3.8)×108細(xì)胞/L。這樣，幾乎30%分離葉肉細(xì)胞，在適當(dāng)?shù)碾x體培養(yǎng)條件下，可半同步地分化為管狀分子。

2 細(xì)胞學(xué)、生理學(xué)和生物化學(xué)方面的研究

百日草實(shí)驗(yàn)系統(tǒng)的建立促進(jìn)了管狀分子分化諸多方面的研究(圖1)。初步的細(xì)胞學(xué)和生理學(xué)研究表明，細(xì)胞分裂不是管狀分子分化的前提條件，而一些DNA合成在分化中發(fā)揮重要作用[11～21]；以肌動(dòng)蛋白依賴的微管重組，限定了次生細(xì)胞壁的特有格局[6，22～27]；分化過(guò)程是動(dòng)態(tài)的，細(xì)胞變化表現(xiàn)在次生細(xì)胞壁沉積前細(xì)胞器數(shù)量的增加，次生細(xì)胞壁沉積開(kāi)始不久次生細(xì)胞壁木質(zhì)化啟動(dòng)[28～31]，原生質(zhì)體逐漸自溶，初生壁非木質(zhì)化部分的局部水解，分化過(guò)程終止[28，32～34]。另外，百日草系統(tǒng)已清晰地證明，管狀分子分化受植物激素諸如生長(zhǎng)素、細(xì)胞分裂素[4]、油菜素內(nèi)酯[35，36]、赤霉素[37]、一氧化氮[38]、乙烯[39]、信號(hào)肽(例如CLE肽[40]、木質(zhì)素[41]、植物磺肽素[42])的調(diào)控；另外，大量生化和免疫學(xué)研究，揭示了細(xì)胞壁成分諸如纖維素、木聚糖、木質(zhì)素和其他次生壁特有分子的變化[29，30，43～50]，以及自溶過(guò)程中的各種事件，諸如蛋白質(zhì)和核酸的降解[51，52]等。

圖1 百日草葉肉細(xì)胞管狀分子分化模型

在生長(zhǎng)素、細(xì)胞分裂素、植物磺肽素、木質(zhì)素、油菜素內(nèi)酯、乙烯、赤霉素作用下，離體葉肉細(xì)胞可被誘導(dǎo)分化為管狀分子，但是CLE肽卻抑制分化。

3 相關(guān)基因的鑒定與描述

人們也用分子生物學(xué)方法，進(jìn)一步分析了百日草實(shí)驗(yàn)系統(tǒng)中管狀分子分化的分子機(jī)制(圖1)。運(yùn)用同源克隆法(例如，核酸酶ZEN1[53]，肉桂醇脫氫酶和過(guò)氧物酶[54]，b-微管蛋白[55]，油菜素內(nèi)酯合成酶[56]和Rho/Rac small GTPases[57])，差示篩選法(例如，分化標(biāo)記，管狀分子分化相關(guān)的(TED)2-4(Tracheary Element Differentiation-related(TED)2-4)[58～60]，果膠裂解酶[61])，消減雜交法(例如，核糖核酸酶[62]及分化標(biāo)記、蛋白酶和木質(zhì)素合成酶[10，63～65])、全面的轉(zhuǎn)錄組分析微陣列[66～68]和cDNA-AFLP[69]，分別鑒定了許多與編碼管狀分子特定事件相關(guān)蛋白質(zhì)的cDNA，和限定管狀分子分化特定階段的標(biāo)記蛋白。因?yàn)榘偃詹蒉D(zhuǎn)化方法尚不穩(wěn)定，所以其分離基因的功能分析受到很大制約(例如，果膠裂解酶ZePel[61]，TED4[70]，過(guò)氧物酶ZPO-C[54])。然而，最近，通過(guò)基因槍法和電穿孔轉(zhuǎn)染法，瞬間將基因或雙鏈RNAs導(dǎo)入百日草細(xì)胞，成功地描述了其他基因的功能[57，71～73]，這可能為管狀分子分化相關(guān)基因功能的分析提供了有益的線索。

4 其他植物的研究

在利用百日草實(shí)驗(yàn)系統(tǒng)，研究管狀分子分化調(diào)控的基礎(chǔ)上，建立了擬南芥人工培養(yǎng)細(xì)胞的管狀分子分化系統(tǒng)，該系統(tǒng)在油菜素內(nèi)酯調(diào)控下，有30%～50%繼代細(xì)胞分化為管狀分子[74，75]。后續(xù)的基因芯片分析表明，多數(shù)擬南芥基因在管狀分子分化期間特異表達(dá)[74]，這些基因包括編碼植物特定NAC-domain轉(zhuǎn)錄因子VND1-7的基因家族，借以最終揭示長(zhǎng)久以來(lái)尋找的管狀分子分化的轉(zhuǎn)錄開(kāi)關(guān)[74]。在某些植物和人工培養(yǎng)細(xì)胞中，VND基因被作為管狀分子異常分化的有效誘導(dǎo)物[76～79]。

5 待解決的問(wèn)題和未來(lái)研究展望

管狀分子分化百日草葉肉細(xì)胞離體實(shí)驗(yàn)系統(tǒng)的研究在草本植物中廣泛展開(kāi)。該系統(tǒng)為誘導(dǎo)和促進(jìn)管狀分子分化的研究建立了有效的平臺(tái)，并獲得了一些關(guān)鍵基因和調(diào)節(jié)因子，初步揭示了管狀分子分化的機(jī)制，為在單細(xì)胞水平上認(rèn)識(shí)細(xì)胞分化和轉(zhuǎn)分化途徑、分化過(guò)程影響因素提供了可能，但復(fù)雜、精確的分子機(jī)制的構(gòu)建仍需進(jìn)一步研究。(1)管狀分子分化具有復(fù)雜的時(shí)空特異性，調(diào)控網(wǎng)絡(luò)綜合而龐大，雖然生長(zhǎng)素和細(xì)胞分裂素是管狀分子分化的基本調(diào)節(jié)激素，已有學(xué)者對(duì)2者進(jìn)行了大量研究并取得一定的成果，但是其他激素諸如赤霉素、乙烯、脫落酸、油菜素內(nèi)酯等作用效果研究資料極少，所以，植物激素作用機(jī)制的研究尚未明確。(2)管狀分子分化是植物細(xì)胞凋亡的典型例子，成熟的管狀分子喪失了細(xì)胞核和細(xì)胞內(nèi)容物，成為死的、中空的管狀細(xì)胞。目前對(duì)管狀分子細(xì)胞凋亡的信號(hào)轉(zhuǎn)導(dǎo)途徑知之甚少，需要進(jìn)一步探索。(3)以草本植物百日草，甚至擬南芥為材料得到的管狀分子分化的初步機(jī)制，是否適合木本植物，尚需驗(yàn)證。(4)雖然百日草系統(tǒng)較為成熟，管狀分子分化率和同步性較高，但隨著研究的深入，仍需進(jìn)一步改進(jìn)和優(yōu)化游離單細(xì)胞的離體培養(yǎng)方法，摸索分化的最佳條件，提高分化率和同步性。(5)未來(lái)需要綜合利用分子生物學(xué)、細(xì)胞生物學(xué)、生物信息學(xué)和系統(tǒng)生物學(xué)的研究方法，從多角度、多層次、多學(xué)科開(kāi)展研究工作，結(jié)合模式植物擬南芥實(shí)驗(yàn)系統(tǒng)和實(shí)驗(yàn)植物分子遺傳學(xué)，在不久的將來(lái)有可能全面闡明管狀分子分化的機(jī)制。

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(責(zé)任編輯：朱寶昌)

Progress on Differentiation of Isolated Mesophyll Cells into Tracheary Elements in Common Zinnia (ZinniaelegansJacq.)

LI Na, BAI Lan, GUO Xuemin

(College of Life Science & Technology, Hebei Normal University of Science & Technology, Qinhuangdao Hebei, 066600, China)

Differentiation of tracheary elements (TEs) has been regarded as a model system for cytodifferentiation in plants. Fukuda and Komamine established an efficient experimental system for TE differentiation from isolated single mesophyll cells ofZinniaelegans, which provided an excellent platform for the study of TEs differentiation at single cell level. The establishment of the system and the progress on physiology, cytology, biochemistry and molecular biology of the system-based TEs differentiation were summarized in this paper. At last, the future research direction of TEs differentiation was prospected.

Zinniaelegans; secondary cell wall; cytodifferentiation; tracheary element

10.3969/J.ISSN.1672-7983.2016.04.010

河北科技師范學(xué)院博士后啟動(dòng)基金項(xiàng)目(項(xiàng)目編號(hào)：2013YB021)。

2016-11-23;修改稿收到日期: 2016-12-21

S681.9

A

1672-7983(2016)04-0061-07

李娜(1991-)，女，碩士研究生。主要研究方向：植物發(fā)育細(xì)胞生物學(xué)。

*通訊作者，男，博士，教授。主要研究方向：植物結(jié)構(gòu)生理學(xué)。E-mail: xueminguo@126.com。