渗透胁迫信号传导关键基因对黑穗醋栗遗传转化的研究
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摘要
干旱、高盐及低温等环境条件，都会构成对植物的渗透胁
迫，使植物缺水受伤害，甚至导致植物死亡，是造成作物减产
的主要原因。生产上主要采用抗逆性强的品种。黑穗醋栗(Ribes
nigrum L. )属于多年生木本果树，世代周期长，杂合性高，许
多重要经济性状属于多基因控制的数量性状，遗传机理不明，
利用常规方法进行品种选育困难重重。利用基因工程手段将外
源基因导入作物中，培育出新的品种已成为现代农业和农作物
育种的发展方向。但是黑穗醋栗属于木本果树，离体培养技术
还不完善，再生有困难，常常无法获得再生植株，并且缺乏高
效的遗传转化方法。
针对以上问题，本研究以黑穗醋栗为试材进行组织培养和
遗传转化的初步研究，本研究构建了 osCDPK7 基因和
osMAPK4 基因的植物表达载体；建立了黑穗醋栗茎尖再生体
系；利用基因枪法和农杆菌介导法将 osCDPK7 基因和
osMAPK4 基因转化黑穗醋栗，以期获得转基因植株，来增强
作物抗逆性。
本研究主要研究结果如下：
1. 载体构建
构建了 2 个植物表达载体 PBC7E12 和 PBME12。载体 PBC7E12 上带有组成型启动
子 E12 调控的 osCDPK7 基因，植物筛选标记为 nptⅡ基因。载体 PBME12 上带有组成型
启动子 E12 调控的 osMAPK4 基因，植物筛选标记为 nptⅡ基因。
2. 黑穗醋栗再生体系的建立
（1） 黑穗醋栗愈伤组织的诱导
利用黑穗醋栗叶片、叶柄和茎段诱导愈伤组织，研究了
不同 PGR 配比对黑穗醋栗愈伤组织诱导的影响，为下
一步通过愈伤组织诱导不定芽奠定了基础。
（2） 黑穗醋栗茎尖培养培养基的确定
V
摘 要
确 定 了 黑 穗 醋 栗 茎 尖 最 佳 分 化 和 继 代 培 养 基 为
MS+1mg/L BA，30g/L 蔗糖，0.8%琼脂，pH5.8。
最佳从生芽生根培养基为 1/2MS，30g/L 蔗糖，0.8%琼
脂，pH5.8。
3. 基因枪法对黑穗醋栗茎尖的遗传转化
确定茎尖分化阶段卡那霉素的筛选压力为 25mg/L。丛生
芽生根阶段卡那霉素筛选压力为 20mg/L。利用基因枪法转化
黑穗醋栗茎尖获得抗性芽，抗性芽率为 8.4％。
4. 农杆菌介导法对黑穗醋栗茎尖的遗传转化
利用农杆菌介导法转化黑穗醋栗茎尖获得抗性芽，抗性芽
率为 10.5％。
5. 转基因植株的分子检测
基因枪法转化获得的抗性植株，经 PCR 检测，获得 osCDPK7 基因阳性的植株 1 株，
获得 osMAPK4，基因阳性的植株 1 株。PCR 阳性率为 9.1%。
目前，利用农杆菌介导法转化黑穗醋栗茎尖已获得大量抗性苗，正在继续对转化植
株进行筛选培养。下一步将对利用农杆菌介导法转化黑穗醋栗茎尖获得的抗性芽进行检
测。
Drought, high salinity and low temperature are common stress condition that adversely
affect plant growth and crop production. Using new cultivars which had high resistance to
these stress is a good way to defense the stress. But blackcurrants (Ribes nigrum L. ) are
perennate woody fruit crops. Because of long life cycles , genetic heterogeneity and most of
economic traits controlled by quantitative trait loci or multiple genes , genetic improvement in
blackcurrant by traditional breeding is very difficult., improvement of source strength by
molecular technology. Transferring extraneous gene to crops by genetic engineering to breed
new varieties has become the research trend in crop breeding. But as a perennate woody fruit
crops, technics in tissue culture of blackcurrant still remain many limitations, It is very
difficult to regeneration and lack of efficient method of transformation.
According to above-mentioned problems, this study make use of blackcurrant as material to
carry on tissue culture and genetic transformation. In this research construct plant expression
vectors with osCDPK7 gene and osMAPK4 gene. The regeneration system of blackcurrant
has been established. Both of the two genes were transformed into Blackcurrant mediated by
Agrobacterium and particle bombardment, in order to get some transgenic plants to enhance
the stress-tolerant.
The main results were summarized as follows.
1. Vector construction
Plant expression vector PBC7E12 was constructed, on which osCDPK7 gene was regulated
by the constitutive promoter E12, and NptⅡ gene as selectable marker. Plant expression
vector PBME12 was constructed, on which osMACDPK7 gene was regulated by the
constitutive promoter E12, and NptⅡ gene as selectable marker.
2. Establishment of Blackcurrant regeneration system
(1). Callus induction of Blackcurrant
Callus induction with leaf, petiole and stem segment. Study the effect of different
PGR on callus induction, it was the basis of the regeneration of callus for the next step.
(2). Make certain the optimal differentiation medium of blackcurrant shoot tip culture is MS+
1mg/L BA, 30g/L sucrose, 0.8%agar, pH5.8.
The optimal medium of root regeneration from shoots was 1/2MS，30g/L sucrose,
0.8%agar, pH5.8.
3. Transformation of blackcurrant shoot tip by particle bombardment
The concentration of Km selection in phase of shoot differentiation was 25 mg/L , in phase
of root regeneration , this concentration was 20 mg/L. Transformation of blackcurrant shoot
VII
摘 要
tip by particle bombardment,we get some resistant shoots. The percentage of resistant shoot
was 8.4％.
4. Transformation of blackcurrant shoot tip by Agrobacterium mediate
We get some resistant shoots. The percentage of resistant shoot was 10.5％.
5. Identification analysis of transgenic plant
Resistant shoots from particle bombardment were detected. one PCR positive transgenic
plants with gene osCDPK7 and one PCR positive transgenic plants with gene osMAPK4 were
acquired.
迫，使植物缺水受伤害，甚至导致植物死亡，是造成作物减产
的主要原因。生产上主要采用抗逆性强的品种。黑穗醋栗(Ribes
nigrum L. )属于多年生木本果树，世代周期长，杂合性高，许
多重要经济性状属于多基因控制的数量性状，遗传机理不明，
利用常规方法进行品种选育困难重重。利用基因工程手段将外
源基因导入作物中，培育出新的品种已成为现代农业和农作物
育种的发展方向。但是黑穗醋栗属于木本果树，离体培养技术
还不完善，再生有困难，常常无法获得再生植株，并且缺乏高
效的遗传转化方法。
针对以上问题，本研究以黑穗醋栗为试材进行组织培养和
遗传转化的初步研究，本研究构建了 osCDPK7 基因和
osMAPK4 基因的植物表达载体；建立了黑穗醋栗茎尖再生体
系；利用基因枪法和农杆菌介导法将 osCDPK7 基因和
osMAPK4 基因转化黑穗醋栗，以期获得转基因植株，来增强
作物抗逆性。
本研究主要研究结果如下：
1. 载体构建
构建了 2 个植物表达载体 PBC7E12 和 PBME12。载体 PBC7E12 上带有组成型启动
子 E12 调控的 osCDPK7 基因，植物筛选标记为 nptⅡ基因。载体 PBME12 上带有组成型
启动子 E12 调控的 osMAPK4 基因，植物筛选标记为 nptⅡ基因。
2. 黑穗醋栗再生体系的建立
（1） 黑穗醋栗愈伤组织的诱导
利用黑穗醋栗叶片、叶柄和茎段诱导愈伤组织，研究了
不同 PGR 配比对黑穗醋栗愈伤组织诱导的影响，为下
一步通过愈伤组织诱导不定芽奠定了基础。
（2） 黑穗醋栗茎尖培养培养基的确定
V
摘 要
确 定 了 黑 穗 醋 栗 茎 尖 最 佳 分 化 和 继 代 培 养 基 为
MS+1mg/L BA，30g/L 蔗糖，0.8%琼脂，pH5.8。
最佳从生芽生根培养基为 1/2MS，30g/L 蔗糖，0.8%琼
脂，pH5.8。
3. 基因枪法对黑穗醋栗茎尖的遗传转化
确定茎尖分化阶段卡那霉素的筛选压力为 25mg/L。丛生
芽生根阶段卡那霉素筛选压力为 20mg/L。利用基因枪法转化
黑穗醋栗茎尖获得抗性芽，抗性芽率为 8.4％。
4. 农杆菌介导法对黑穗醋栗茎尖的遗传转化
利用农杆菌介导法转化黑穗醋栗茎尖获得抗性芽，抗性芽
率为 10.5％。
5. 转基因植株的分子检测
基因枪法转化获得的抗性植株，经 PCR 检测，获得 osCDPK7 基因阳性的植株 1 株，
获得 osMAPK4，基因阳性的植株 1 株。PCR 阳性率为 9.1%。
目前，利用农杆菌介导法转化黑穗醋栗茎尖已获得大量抗性苗，正在继续对转化植
株进行筛选培养。下一步将对利用农杆菌介导法转化黑穗醋栗茎尖获得的抗性芽进行检
测。
Drought, high salinity and low temperature are common stress condition that adversely
affect plant growth and crop production. Using new cultivars which had high resistance to
these stress is a good way to defense the stress. But blackcurrants (Ribes nigrum L. ) are
perennate woody fruit crops. Because of long life cycles , genetic heterogeneity and most of
economic traits controlled by quantitative trait loci or multiple genes , genetic improvement in
blackcurrant by traditional breeding is very difficult., improvement of source strength by
molecular technology. Transferring extraneous gene to crops by genetic engineering to breed
new varieties has become the research trend in crop breeding. But as a perennate woody fruit
crops, technics in tissue culture of blackcurrant still remain many limitations, It is very
difficult to regeneration and lack of efficient method of transformation.
According to above-mentioned problems, this study make use of blackcurrant as material to
carry on tissue culture and genetic transformation. In this research construct plant expression
vectors with osCDPK7 gene and osMAPK4 gene. The regeneration system of blackcurrant
has been established. Both of the two genes were transformed into Blackcurrant mediated by
Agrobacterium and particle bombardment, in order to get some transgenic plants to enhance
the stress-tolerant.
The main results were summarized as follows.
1. Vector construction
Plant expression vector PBC7E12 was constructed, on which osCDPK7 gene was regulated
by the constitutive promoter E12, and NptⅡ gene as selectable marker. Plant expression
vector PBME12 was constructed, on which osMACDPK7 gene was regulated by the
constitutive promoter E12, and NptⅡ gene as selectable marker.
2. Establishment of Blackcurrant regeneration system
(1). Callus induction of Blackcurrant
Callus induction with leaf, petiole and stem segment. Study the effect of different
PGR on callus induction, it was the basis of the regeneration of callus for the next step.
(2). Make certain the optimal differentiation medium of blackcurrant shoot tip culture is MS+
1mg/L BA, 30g/L sucrose, 0.8%agar, pH5.8.
The optimal medium of root regeneration from shoots was 1/2MS，30g/L sucrose,
0.8%agar, pH5.8.
3. Transformation of blackcurrant shoot tip by particle bombardment
The concentration of Km selection in phase of shoot differentiation was 25 mg/L , in phase
of root regeneration , this concentration was 20 mg/L. Transformation of blackcurrant shoot
VII
摘 要
tip by particle bombardment,we get some resistant shoots. The percentage of resistant shoot
was 8.4％.
4. Transformation of blackcurrant shoot tip by Agrobacterium mediate
We get some resistant shoots. The percentage of resistant shoot was 10.5％.
5. Identification analysis of transgenic plant
Resistant shoots from particle bombardment were detected. one PCR positive transgenic
plants with gene osCDPK7 and one PCR positive transgenic plants with gene osMAPK4 were
acquired.
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