胡萝卜（Daucus carota）抗冻蛋白基因的克隆及其对番茄（Lycopersicon esculentum）的转化
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摘要
许多农作物，尤其是一些果品和蔬菜，不仅在田间栽培过程中，而且在收获后的
冷藏、冻藏和速冻加工过程中都会遇到冷冻伤害。然而，用常规的育种方法来改良作
物的抗寒性会遇到许多困难。随着分子生物学的发展，基因克隆技术逐步介入植物
抗寒研究，而率先进入该领域的基因即为极区鱼类抗冻蛋白（antifreeze proteins AFPs）
基因（afp）。
自二十世纪80年代，鱼类的afp结构被研究清楚以后，人们先后将其afp转入郁
金香、烟草、油菜、玉米、番茄、马铃薯等作物，力图提高这些作物的抗寒性，取得
了一些有益的结果，但离人们的期望还相差较远。
1998年10月英国York大学的Dawn Worrall等发表了胡萝卜AFP及其基因的论
文，标志着第一个植物afp基因的发现，对于植物抗冻基因工程具有非常重要的意义。
他们不仅测定了胡萝卜AFP的体外热滞值及重结晶抑制活性，而且将其cDNA连接
在表达载体的双CaMV 35S启动子之后导入烟草，获得了正确表达。
本研究用PCR的方法克隆了胡萝卜afp，构建了其植物表达载体，并用农杆菌介
导法将其导入番茄之中，初步获得了转化植株。
以英国胡萝卜变种var. autumn King，中国胡萝卜变种var. sativus Hoffm Deutschl
的3个地方品种——宁夏吴忠胡萝卜、陕西华县胡萝卜和陕西汉中胡萝卜为材料，用
PCR方法克隆了抗冻蛋白基因afp，发现afp在所试材料中普遍存在。用宁夏吴忠胡
萝卜的afp核苷酸序列和英国胡萝卜var. autumn King的afp进行了对比，在所测1004
个核苷酸中，有35个碱基不同，其中无义突变20个，有义突变15个，按有义突变
计，同源性为98.5％。
以克隆到的var. autumn King的afp为目的基因，以pUC_m-T Vector为载体，构建
成胡萝卜afp的克隆载体pTAF。用EcoRI消化重组质粒pTAF使其线性化，再用DNA
聚合酶ⅠKlenow大片段补平末端，然后用XbaⅠ消化，获得一末端粘，一末端平的目
的片段（afp）。植物表达载体pBI121用XbaⅠ和SmaⅠ双酶切，获得一末端粘，一末
端平的线性质粒。将目的片段与线性质粒在T_4DNA连接酶的作用下进行定向连接，
构建成胡萝卜afp的植物表达载体pBAF。用直接转化法将重组子pBAF导入根癌农
杆菌LBA4404。
以番茄‘ZF’的子叶和下胚轴作为外植体，对细胞分裂素与生长素的7个组合
进行了比较试验，确定MS+BA 1.0mg/L+IAA 0.2mg/L为最佳生芽培养基。最佳生根
培养基为MS+IAA 0.05mg/L。
番茄无菌苗出芽后 5——7天，子叶切成 0．scm X 05cm片段，下胚轴切成 Icm节
段，于生芽培养基26℃260o LW预培养24h。农杆菌LBA4404过夜培养，用Ms培
养液稀释 10——20倍，侵染外植体 smin，28 ’C黑暗共培养 48h。然后在 MS＋BA
l刀mg／L＋IAA 0．Zot＋Kan 25mg／L＋cef 200mg／L筛选培养基上诱芽，每两周转接 1
次。芽长至Zcm时，切下转至＊S＋M人0刀smN＋Kan 12．sin叭Ce门伽恤叭培养基
上生根。
到目前为止，877个外植体共获得不定芽221个，已转至生根培养基上80个，
获得生根幼苗 12株，对 IO株生根幼苗进行了 PCR检测，其中 2株呈阳性，初步证
明胡萝卜吻基因已经导入该两株番茄体内。
Cloning of a carrot gene encoding antifreeze protein
and its transformation to tomato
Doctor Yin Mingan
Director Cui Hongwen
Fan Daiming
Abstract
Antifreeze protein gene (afk) in carrot Daucus carota var. autumn King from British
and three native carrot cultivars (Daucus carota var. sat ivus Hoffm Deutschl), Wuzhong
carrot in Ningxia, Huaxian carrot in shaanxi and Hanzhong carrot in shaanxi, was cloned
by PCR (polymerase chain reaction). It was found that af~ existed in all tested materials.
Afp seguence of Wuzhong carrat was compared with that of Daucus carota var. autumn
King. There were 35 different bases between two varieties in 1004 seguenced nucleotides,
among which there were 20 monsense mutations and 15 sense mutations. Based on sense
mutations homology was 98.5%.
Cloning vector pTAF of afp from carrot var. autumn King was constructed with
pUCmT vector. pTAF was digested with EcoR I and became linear. Its ends were filled
with DNA Polymerase I Klenow fragment. Then it was digested with Xba I and a
designed fragment( afr )with a cohesive end and a blunt end was released. Plant expression
vector pBI 121 was digested with Xba I and Sma I and a linear plasmid with a cohesive
end and a blunt end was obtained. The linear plasmid and the designed fragment (afp)
were directively ligated with T4 DNA ligase, and the plant expression vector of carrot aTh
was constructed. Then the expression vector was transferred into agrobacterium
tumefacien LBA4404 by direct DNA tranfer.
Cotyledon and hypocotyl from tomato 慫F?were used as explant, and 7 combinations
between cytomin and auxin were tested and compared. It was determined that MS+BA
1 .OmgIL+IAA 0.2mg/L was the best shooting medium. The best rooting medium was
MS+IAA 0.O5mgIL.
When tomato seedings grew 5? days after germination, cotyledons were cut into
0.5cmX 0.5cm discs and hypocotyls into 1cm segments. The discs and segments were
pre-cultured on shooting medium for 24h (26 C, 2600 Lux). Agrobacterium LBA4404
were cultured overnight and diluted 1 0?0 times with MS medium, in which explants
were soaked for 5 minutes. Then the exptants were co-cultured for 48h (28 慍, dark). After
that, the explants were cultured on medium MS+BA 1 .Omg/L 盜AA 0.2mg/L+Kan
25mgIL+cef 200mg/L for selection with fransfer every two weeks. When shoots were 2cm
high, they were cut and transferred on medium MS+IAA 0.O5mglL+Kan 12.5 mg/L+eef
I OOmgIL for rooting.
Up to now, 221 shoots were obtained from 877 explants. 80 shoots were transferred
on rooting medium and 12 plants were transferred in field. 10 plants were assayed by PCR
and 2 of them were positive, which preliminarily proved that carrot atj gene had been
transferred into the tomatoes.
冷藏、冻藏和速冻加工过程中都会遇到冷冻伤害。然而，用常规的育种方法来改良作
物的抗寒性会遇到许多困难。随着分子生物学的发展，基因克隆技术逐步介入植物
抗寒研究，而率先进入该领域的基因即为极区鱼类抗冻蛋白（antifreeze proteins AFPs）
基因（afp）。
自二十世纪80年代，鱼类的afp结构被研究清楚以后，人们先后将其afp转入郁
金香、烟草、油菜、玉米、番茄、马铃薯等作物，力图提高这些作物的抗寒性，取得
了一些有益的结果，但离人们的期望还相差较远。
1998年10月英国York大学的Dawn Worrall等发表了胡萝卜AFP及其基因的论
文，标志着第一个植物afp基因的发现，对于植物抗冻基因工程具有非常重要的意义。
他们不仅测定了胡萝卜AFP的体外热滞值及重结晶抑制活性，而且将其cDNA连接
在表达载体的双CaMV 35S启动子之后导入烟草，获得了正确表达。
本研究用PCR的方法克隆了胡萝卜afp，构建了其植物表达载体，并用农杆菌介
导法将其导入番茄之中，初步获得了转化植株。
以英国胡萝卜变种var. autumn King，中国胡萝卜变种var. sativus Hoffm Deutschl
的3个地方品种——宁夏吴忠胡萝卜、陕西华县胡萝卜和陕西汉中胡萝卜为材料，用
PCR方法克隆了抗冻蛋白基因afp，发现afp在所试材料中普遍存在。用宁夏吴忠胡
萝卜的afp核苷酸序列和英国胡萝卜var. autumn King的afp进行了对比，在所测1004
个核苷酸中，有35个碱基不同，其中无义突变20个，有义突变15个，按有义突变
计，同源性为98.5％。
以克隆到的var. autumn King的afp为目的基因，以pUC_m-T Vector为载体，构建
成胡萝卜afp的克隆载体pTAF。用EcoRI消化重组质粒pTAF使其线性化，再用DNA
聚合酶ⅠKlenow大片段补平末端，然后用XbaⅠ消化，获得一末端粘，一末端平的目
的片段（afp）。植物表达载体pBI121用XbaⅠ和SmaⅠ双酶切，获得一末端粘，一末
端平的线性质粒。将目的片段与线性质粒在T_4DNA连接酶的作用下进行定向连接，
构建成胡萝卜afp的植物表达载体pBAF。用直接转化法将重组子pBAF导入根癌农
杆菌LBA4404。
以番茄‘ZF’的子叶和下胚轴作为外植体，对细胞分裂素与生长素的7个组合
进行了比较试验，确定MS+BA 1.0mg/L+IAA 0.2mg/L为最佳生芽培养基。最佳生根
培养基为MS+IAA 0.05mg/L。
番茄无菌苗出芽后 5——7天，子叶切成 0．scm X 05cm片段，下胚轴切成 Icm节
段，于生芽培养基26℃260o LW预培养24h。农杆菌LBA4404过夜培养，用Ms培
养液稀释 10——20倍，侵染外植体 smin，28 ’C黑暗共培养 48h。然后在 MS＋BA
l刀mg／L＋IAA 0．Zot＋Kan 25mg／L＋cef 200mg／L筛选培养基上诱芽，每两周转接 1
次。芽长至Zcm时，切下转至＊S＋M人0刀smN＋Kan 12．sin叭Ce门伽恤叭培养基
上生根。
到目前为止，877个外植体共获得不定芽221个，已转至生根培养基上80个，
获得生根幼苗 12株，对 IO株生根幼苗进行了 PCR检测，其中 2株呈阳性，初步证
明胡萝卜吻基因已经导入该两株番茄体内。
Cloning of a carrot gene encoding antifreeze protein
and its transformation to tomato
Doctor Yin Mingan
Director Cui Hongwen
Fan Daiming
Abstract
Antifreeze protein gene (afk) in carrot Daucus carota var. autumn King from British
and three native carrot cultivars (Daucus carota var. sat ivus Hoffm Deutschl), Wuzhong
carrot in Ningxia, Huaxian carrot in shaanxi and Hanzhong carrot in shaanxi, was cloned
by PCR (polymerase chain reaction). It was found that af~ existed in all tested materials.
Afp seguence of Wuzhong carrat was compared with that of Daucus carota var. autumn
King. There were 35 different bases between two varieties in 1004 seguenced nucleotides,
among which there were 20 monsense mutations and 15 sense mutations. Based on sense
mutations homology was 98.5%.
Cloning vector pTAF of afp from carrot var. autumn King was constructed with
pUCmT vector. pTAF was digested with EcoR I and became linear. Its ends were filled
with DNA Polymerase I Klenow fragment. Then it was digested with Xba I and a
designed fragment( afr )with a cohesive end and a blunt end was released. Plant expression
vector pBI 121 was digested with Xba I and Sma I and a linear plasmid with a cohesive
end and a blunt end was obtained. The linear plasmid and the designed fragment (afp)
were directively ligated with T4 DNA ligase, and the plant expression vector of carrot aTh
was constructed. Then the expression vector was transferred into agrobacterium
tumefacien LBA4404 by direct DNA tranfer.
Cotyledon and hypocotyl from tomato 慫F?were used as explant, and 7 combinations
between cytomin and auxin were tested and compared. It was determined that MS+BA
1 .OmgIL+IAA 0.2mg/L was the best shooting medium. The best rooting medium was
MS+IAA 0.O5mgIL.
When tomato seedings grew 5? days after germination, cotyledons were cut into
0.5cmX 0.5cm discs and hypocotyls into 1cm segments. The discs and segments were
pre-cultured on shooting medium for 24h (26 C, 2600 Lux). Agrobacterium LBA4404
were cultured overnight and diluted 1 0?0 times with MS medium, in which explants
were soaked for 5 minutes. Then the exptants were co-cultured for 48h (28 慍, dark). After
that, the explants were cultured on medium MS+BA 1 .Omg/L 盜AA 0.2mg/L+Kan
25mgIL+cef 200mg/L for selection with fransfer every two weeks. When shoots were 2cm
high, they were cut and transferred on medium MS+IAA 0.O5mglL+Kan 12.5 mg/L+eef
I OOmgIL for rooting.
Up to now, 221 shoots were obtained from 877 explants. 80 shoots were transferred
on rooting medium and 12 plants were transferred in field. 10 plants were assayed by PCR
and 2 of them were positive, which preliminarily proved that carrot atj gene had been
transferred into the tomatoes.
引文
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