全 文 ：文章编号: 1007-0435( 2006) 03-0298-03 ·博士论文摘要·
柱花草SgNCED1基因的克隆及功能分析
博士生:杨锦芬　导师:郭振飞
(华南农业大学生命科学学院草业生物技术实验室, 广州　510642)
　　脱落酸( ABA)作为一种调控生长发育的激素, 对
植物多种生理过程起着调控作用。9-顺式环氧类胡萝
卜素双加氧酶 ( 9-cis-epoxycar otenoid diox ygenase,
N CED )是高等植物 ABA 生物合成途径的关键酶。
N CED 基因受逆境胁迫诱导,导致逆境A BA 的积累。
研究N CED 基因克隆、表达,调控ABA 的生物合成,
是提高植物抗逆性的重要途径。柱花草( Sty losanthes
guianensi s)是一种热带和亚热带地区广泛种植的耐旱
草种。本文研究了柱花草NCED 基因的克隆、表达及
功能分析,主要结果如下:
1. 柱花草S gN CED1基因的克隆
根据已报道的3种豆科植物的N CED 基因同源序列
设计简并引物,用反转录PCR扩增( RT -PCR)结合cD-
NA 末端快速扩增( RACE)以及嵌套PCR的方法,从水
分胁迫的柱花草叶片中克隆了S gN CED1基因,其 cD-
NA 全长由2241 碱基对( bp)组成, 其开放阅读框架为
1809 bp,编码602个氨基酸。Southern 杂交结果表明,该
基因在柱花草基因组中以单拷贝形式存在。氨基酸序列
同源比对和系统进化分析表明 S gN CED1 与花生
( A rachis hyp ogaea)的A hN CED1高度同源( 92% ) ,与
另外三种豆科植物的NCED 也有很高的同源性( 72%～
75%) , 与其他科已报道的N CED 蛋白序列同源性在
68%以上。亚细胞定位预测结果表明: SgN CED 1蛋白的
N-末端具有叶绿体转运肽。
2. 几种逆境诱导的S gN CED1基因表达分析及
ABA 含量变化
Norther n杂交的结果表明, S gN CED1基因在柱
花草叶片和根部均有表达, 在干旱胁迫下的ABA 合成
调控中起重要作用。SgN CED1基因在短时间内就能
强烈响应脱水和盐胁迫的诱导, 低温胁迫也能诱导
SgN CED1的表达。在干旱、脱水和盐胁迫下, 柱花草
的内源ABA 均大量积累, ABA 的合成与S gN CED1
基因的表达基本同步。低温也诱导SgN CED1基因的
表达,但研究结果表明,它是由于低温导致叶片水分胁
迫而引起的。S gN CED 1基因编码的蛋白是柱花草
ABA 合成途径中的关键酶, 调控逆境胁迫下ABA 的
合成。
3. S gN CED1基因转化烟草及功能分析
构建了S gN CED1基因的表达载体pBI-SgNCED 1,
并用农杆菌介导的方法转化烟草( N icot iana tabacum )。
Southern和Northern 杂交以及RT -PCR的结果表明,
SgN CED1基因插入了植株编号为5、S6、S8、S16、S24
和P54的烟草基因组中,并且得到了表达。对转基因烟
草的分析证明, S gN CED 1基因的表达提高了烟草内
源ABA 的含量, 为野生型的1. 7～3. 52 倍, 提高了转
基因烟草的抗旱和耐盐性, 在一定程度上降低了气孔
导度和蒸腾速率, 但光合速率维持在正常水平, 转基因
烟草的生长并未受到明显抑制。
　　4. 转S gN CED1基因烟草的抗强光和抗氧化胁迫
分析和非光化学猝灭系数( N PQ )
SgN CED1基因的过量表达提高了转基因烟草对
强光和氧化胁迫的抗性。强光处理引起了野生型对照
植株和转基因植物的最大光化学效率( Fv / Fm )和光
系统Ⅱ电子传递量子效率( 5 PSⅡ)降低, 而转基因烟草
的 F v / Fm 和5 PSⅡ维持在较高水平;在百草枯处理下,
野生型对照植株的 Fv / Fm、5 PSⅡ、光化学猝灭系数
( qP )和N PQ降低,而转基因烟草的Fv / Fm、5 PSⅡ、qP
和N PQ维持在较高水平, 表明转基因烟草具有较强
的热耗散能力,能够减缓强光和氧化胁迫对光合机构
的伤害,维持较高的光合效率。转基因烟草能够维持膜
的稳定性,减缓百草枯处理下氧化胁迫对质膜的伤害,
但是转基因烟草的超氧物歧化酶和过氧化氢酶活性在
非胁迫条件下无明显提高, 内源ABA 的提高可能通过
其他途径提高了烟草的抗氧化胁迫能力。
关键词: 柱花草; ABA; NCED; 逆境胁迫; 转基因烟草
中图分类号: S812; Q943　　　文献标识码: A
完成时间: 2006年4月
收稿日期: 2006-06-22; 修回日期: 2006-07-10
基金项目:广东省自然科学基金重点项目( 04105978)和国家转基因植物研究专项( JY03-B-32-02)
作者简介:杨锦芬( 1978-) ,广东广州人,博士研究生,主要从事草业生物技术研究
导师简介:郭振飞( 1964-) ,男,研究员,研究方向为草业生物技术, E-mail: zhfguo@ scau. edu. cn
第14卷　第 3期
　Vo l. 14　 No . 3
草　地　学　报
ACT A AGRESTIA SIN ICA
　　　2006年　 9 月
　Sep. 　　 2006
Cloning and Functional Analysis of a 9-Cis-Epoxycarotenoid Dioxygenase
Gene (SgNCED1) from Stylosanthes Guianensis
Candidate: YANG Jin-fen　Advisor: GU O ZHen-fei
( Biotechnology Laboratory for T urfgrass and Forages, College of Life S cience , South Chin a Agricu lture Un ivers ity,
Gu angzhou, Guangdong Province 510642, Chin a)
　　Abscisic acid ( ABA ) plays important ro les in the adaptat ion of plants to a variety of env ir onmental stress-
es. T he oxidat ive cleavage of cis-epoxycaro te-noids catalyzed by 9-cis-epoxycarotenoid diox ygenase ( N CED ) is
the key step in the regulat ion of abscisic acid ( ABA) biosynthesis in higher plants. The environmental st resses
induce the expression of NCED and thereby r egulate the endogenous ABA level. Sty losanthes g uianensis is an
important drought-tolerant pasture legume of the t ropical and subtropical regions. The object iv es of this study
w er e to clone the N CED gene fr om S . guianensis and to analyze it s funct ion in response to abiot ic st resses as
w el l as to prov ide a method for the molecular breeding of st ress-to lerant crops. The results are summarized in
the fol low ing 4 sect ions.
1. Cloning o f SgN CED1 from S. guianensi s
Degenerate primers w er e synthesized based on the high homologous region among the N CED sequences of
thr ee legum inous plants, and w ere used for cloning of a N CED gene f rom the dehydr ated leaves of S . guianen-
si s by using r everse t ranscriptase-PCR ( RT -PCR) , rapid amplificat ion of cDNA ends ( RACE) and nested
PCR. A full length of N CED cDNA, namely: S gN CED1, w as cloned and sequenced. It w as 2241-bp long and
contained an open reading frame ( ORF) of 1809-bp which encodes a pept ide of 602 amino acids. Southern blot
analy sis show ed that SgN CED1 w as a single copy gene in the genome of S . guianensis. Mult iple sequence
alignments of the deduced amino sequences show ed that the SgN CED1 protein shared 92% identity with the
A hN CED1 fr om peanut and w as highly homologous ( 72%～75%) to the N CEDs o f the three leguminous
plants. A chloroplast t ransit pept ide was lo cated at the N-terminus of the S gNCED 1.
2. Analysis on the SgN CED1 expression and ABA bio synthesis in r esponse to abiot ic st resses
Norther n blot analy sis show ed that SgN CED1 expression was induced both in the leaves and the roots of
S . guianensis under drought st ress. Dehydrat ion and salt str ess st rongly and rapidly induced the expr ession o f
SgN CED1, w hile chill ing also induced the SgN CED 1 expression, though at a low r ate. The accumulat ion of
ABA was correlated with that of S gN CED1 mRNA under dr ought , dehydrat ion, and salt st ress. Chilling
caused ABA accumulat ion, w hich proved to be the r esult of dehydr at ion in the leaves. A ll this demonst rated
that the expression of S gN CED1 r egulates the ABA biosynthesis under environmental str esses.
3. T ransformat ion of tobacco by S gNCED 1 and its functional analysis
Expression vector of pBI-SgN CED 1 w as const ructed and int roduced to tobacco by Agr obacterium-mediat-
ed tr ansformat ion. T he molecular detect ions indicated that SgN CED1 had inserted into the genome o f t rans-
genic tobacco plants and had been expr essed. T he over-expression of SgN CED1 gene resulted in the increase of
endogenous ABA level in t ransgenic tobacco plants, and then enhanced their drought-and salt-tolerance. In ad-
dit ion, w hile the stomatal conductance and transpir at ion rate of t ransgenic plants decr eased, the net pho to syn-
thet ic r ate w as maintained at a no rmal lev el .
4. Responses o f the t ransgenic plants to high light and ox idat ive stress
The over -expression of S gN CED1 gene in t ransgenic tobacco plants increased their high light and oxidat ive
st ress tolerance. The max imal photochemical ef ficiency ( Fv / Fm) , and quantum eff iciency of photosystem Ⅱ
photochem ist ry ( 5 PSⅡ) decr eased in all plants under high l ight , w hereas these eff iciencies w ere maintained at a
299第 3期 杨锦芬等:柱花草 SgN CED1基因的克隆及功能分析
《草地学报》编委会
主　任: 洪绂曾
副主任: 任继周　石元春　陈文新　张子仪　刘更另　张新时
顾　问: 王　培　许　鹏　张自和　李建东　苏大学　苏加楷　陈佐忠　周寿荣
孟昭仪　姜　恕　祝廷成　胡兴宗　胡自治　黄文惠
主　编: 洪绂曾
副主编: 周　禾　云锦凤　韩建国　南志标　龙瑞军　李向林　徐　柱　阿不来提
宗锦耀　才　杰
编　委: 于　卓* 　才　杰*　云锦凤* 　王　涛　王　 *　王宗礼　王明玖* 　王德利*
卢欣石* 龙瑞军* 刘国道 孙启忠 朱进忠 毕玉芬 张新全 李向林
汪诗平* 　沈益新 阿不来提 周　禾* 周道玮 呼天明 孟庆祥 宗锦耀
南志标* 　姚爱兴 施大钊* 洪绂曾* 徐　柱 郭振飞 高洪文* 龚元石
程　序　 程积民　 董宽虎 谢应忠 韩建国* 韩烈保 赖志强* 樊江文
盐见正卫(日本 Japan)　Jerrold L . Dodd(美国USA)
* 常务编委
顾问、编委均以姓氏笔划为序
(接前页)
higher level in the tr ansgenic plants than in the w ild-type plant . Par aquat-induced ox idat iv e st ress resulted in
the decrease of Fv / Fm, 5 PSⅡ, photochemical quenching ( qP) , and non-pho to chemical quenching ( NPQ) in the
w ild type, w hile t ransgenic plants maintained the quenching at a higher lev el than the w ild type contr ols. T he
ion leakage of t ransgenic plants w as maintained at a low er level than that in the w ild-type plant under par aquat
t reatment . T he results indicated that the t ransgenic plants w ere w el l protected against high l ight and par aquat
induced ox idativ e st ress. No obvious increase o f supero xide dismutase o r catalase act iv ity w as measur ed in
t ransgenic plants under non-st ress condit ion. It w as likely that the up-regulated endogenous ABA did not in-
duce ant io xidant enzyme act ivity direct ly.
　　Key words : Sty losanthes guianensis; Abscisic acid; 9-cisepoxycarotenoid diox ygenase( NCED ) ; Environ-
mental st resses; Transgenic tobacco
(责任编辑　才　杰)
300 草　地　学　报 第 14卷