CN1233833C - 一种利用山菠菜badh基因转化培育耐盐番茄的方法 - Google Patents
一种利用山菠菜badh基因转化培育耐盐番茄的方法 Download PDFInfo
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Abstract
一种用转甜菜碱醛脱氢酶基因(BADH)培育耐盐番茄株系的方法，该方法包括如下步骤：a.将含有BADH cDNA的基因片断的双元表达载体pBin438的农杆菌LBA4404感染番茄真叶叶片；b.感染后的番茄叶片置于IM培养基上进行分化培养；c.取分化植株幼嫩叶片提取基因组DNA进行分子检测；d.转基因植株的BADH活性检测和耐盐性鉴定。
Description
1.技术领域
本发明属于植物生物技术领域。通过基因工程抗盐基因转化手段提高植物的耐盐性。
2.技术背景：
植物生长在自然环境中，经常受到不良环境条件的影响。其中，盐害是限制植物的地理分布并在一定程度上影响其产量和质量的一个重要因素(Boyer，1982)。在长期的进化过程中，植物形成了适应和抵御盐胁迫的机制。常见的一种就是合成小分子有机物如氨基酸衍生物(脯氨酸、甜菜碱等)、糖类、醇类等。甜菜碱通过平衡细胞与周围环境的渗透压、稳定蛋白质的四级结构和保护细胞内的酶系统来保护植物细胞，以减少盐胁迫下植物所受的伤害(Bernard等，1988；Papageorgiou等，1995)。
植物细胞中，甜菜碱是在叶绿体内通过两步反应合成。第一步是由胆碱单氧化酶(CMO)催化胆碱形成甜菜碱醛；第二步是在甜菜碱醛脱氢酶(BADH)的作用下由甜菜碱醛氧化生成甜菜碱。编码合成BADH酶的基因已在多种植物中被成功克隆，如菠菜(Weretilnyk等，1990)、山菠菜(肖岗等，1995)、甜菜(McCue等，1992)、高粱(Wood等，1996)等。研究结果表明，许多植物如拟南芥、烟草和番茄属盐敏感植物但自身不能合成和积累甜菜碱(Weretilnyk等，1989；Rhodes等，1993；Nuccio等，1998)。这就引起了人们对导入甜菜碱合成途径合成甜菜碱以提高盐敏感植物耐盐性的兴趣。
番茄是世界上分布广泛的一种蔬菜作物，但其栽培品种对盐比较敏感。土壤和灌溉水的盐碱化在很大程度上降低了番茄的产量，造成很大的经济损失(Foolad，1999；Cuartero等，1999)。在可利用耕地面积日益减少，土质日趋恶化的今天，提高栽培番茄的耐盐性，增强番茄对不良土壤环境的适应能力，不但可以充分利用有限的土地资源，而且具有很大的经济和社会效益。
3.技术内容
番茄是在全世界广为栽培的常规蔬菜种类，栽培番茄品种普遍对盐敏感。本发明通过农杆菌介导的方法，将山菠菜BADH基因导入栽培番茄，提高了栽培番茄品种的耐盐性。实现本发明的具体技术步骤如下：
a，将含有BADH cDNA的基因片断的双元表达载体pBin438的农杆菌LBA4404感染番茄真叶叶片；
b，感染后的番茄叶片置于IM培养基上进行分化培养；
c，取分化植株幼嫩叶片提取基因组DNA进行分子检测；
d，转基因植株的BADH活性检测和耐盐性鉴定。
通过导入甜菜碱合成途径提高植物的耐盐性已有一些报道(Rathinasabapathi等，1994；刘凤华等，1995；郭岩等，1997；Trossat等，1997；李银心等，2000)，但以蔬菜作为研究对象的很少。番茄是一种经济价值较高、市场潜力较大的蔬菜，栽培番茄品种普遍对盐敏感；研究表明，番茄植物中缺乏内源BADH合成途径(McCue和Hanson，1990；Weretilnyk等，1989)，因此，通过将BADH基因导入番茄，建立番茄甜菜碱合成途径，可以提高栽培番茄品种的耐盐性。
尽管植物的耐盐性是一个由多基因控制的复杂数量性状，通过基因工程提高植物的耐盐性仍然存在挑战。本技术发明通过农杆菌介导的山菠菜BADH基因转化番茄栽培品种“百利春”，证明通过该技术手段可以提高番茄的耐盐性。
4.附图说明：
图1、双元表达载体结构简图。
双元表达载体pBin438含有双35S启动子和TMV的Ω片段翻译增强子，山菠菜BADH cDNA片段插入pBin438的BamHI和KpnI位点。
图2A-C、转基因番茄分化。
A.未感染叶片在卡那筛选培养基上死亡；B.转基因愈伤组织；C.转基因植株
图3A-C、转基因番茄分子检测。
A.PCR检测；B.Southern检测；C.Northern检测
图4、转基因番茄中的BADH活性
图5、转基因番茄在盐胁迫下叶片电导率的变化
图6、T1植株PCR检测：A.T1-1；B.T1-8
图7、T1植株Northern检测 1，2：T1-1 PCR阳性植株；3：T1-1 PCR阴性植株；4：T1-8 PCR阴性植株；5，6：T1-8 PCR阳性植株
图8、盐胁迫对转BADH基因T1代种子发芽率的影响
图9、转BADH基因T1植株电导率变化
5.实施例
1〕材料
“百利春”番茄(Lycopersicon esculentum Mill)种子由中国农科院购得。种子分别用70％酒精和0.1％升汞进行表面消毒1分钟和15分钟，然后用无菌蒸馏水冲洗三次。播种于不加任何激素的MS基本培养基中(Murashige等，1962)。培养室温度保持在25±2℃，湿度70％-80％，每天光照12小时。
PCR反应所用Taq酶和引物由上海生工公司购买和合成。限制性内切酶和Random Primer Labeling System Kit由Takara公司购买。Trizol由GIBCO公司购买。
植物表达载体由中国科学院遗传研究所陈受宜研究员提供。双元表达载体pBin438含有双35S启动子和TMV的Ω片段翻译增强子，BADH cDNA片段插入pBin438的BamHI和KpnI位点(图1、)。
2)农杆菌转化和转基因番茄的筛选
用液体YEB+卡那霉素50mg/L培养基将含表达载体的LBA4404农杆菌培养至OD560＝0.5后，用无激素的MS液体培养基稀释10倍。将番茄无菌真叶叶片于其中感染5分钟。随后取出叶片，用无菌滤纸吸干表面菌液，置于IM培养基(MS+IAA 0.2mg/L+BA 2.0mg/L+ZT 0.1mg/L)上黑暗中共培养48小时。之后转移至IM+卡那霉素50mg/L+羧苄青霉素500mg/L培养基上进行分化培养，每天光照12小时。
观察结果显示，未经农杆菌感染的叶片在筛选培养基上2周后变黄或变白，叶片死亡，伤口处无愈伤长出。经农杆菌感染的番茄叶片，2-3周后开始叶缘出现绿色芽点，5-6周后分化出小植株。当这些小植株长到2-3cm高时，将其转移至MS+卡那霉素500mg/L+羧苄青霉素500mg/L培养基上生根。这些小植株能顺利生根并且生长基本正常(图2、)。
3)转基因植株的命名
当代转基因株系为TG0加上各自的株系号，如TG0-1，TG0-2。各株系严格自交的后代为TG1加上各自的株系号，如TG1-1，TG1-2等。
4)转基因植株的分子检测
取0.1-0.2g分化植株的幼嫩叶片用CTAB法提取基因组DNA。PCR反应体系按标准方法。所用引物为：5’-AGAATGGCGTTCCCAATTCCTGCTC-3’和5’-TTCAAGGAGACTTGT ACCATCCCCA-3’，反应程序为：95℃变性1分钟，55℃退火1分钟，72℃延伸1.5分钟，共35个循环(肖岗等，1995)。
将基因组DNA(20μg)分别用HindIII和EcoRI酶切，在1.0％琼脂糖凝胶50V电泳5小时，并将DNA转移至Hybond-N+尼龙膜上(Sambrook等，1989)。探针以BADH cDNA为模板，用a-32P-dCTP，Randon Primer LabelingSystem(Takara)试剂盒标记。
RNA提取用Trizol一步法，用1.2％甲醛变性胶电泳，转膜、杂交方法及所用探针同Southern杂交。
PCR和Southern杂交检测结果表明BADH基因已整合到6个株系的基因组中(图3A，B)，但从Southern结果可以看出，不同株系中整合位点和BADH基因拷贝数有较大差异(图3B)。这可能在一定程度上影响了BADH基因在番茄中的表达水平，其中一个株系完全检测不到表达的信号(图3C)。
5〕转基因植株的BADH活性检测
不同的转基因株系的植株移栽到装有土壤和蛭石(1∶1)的花盆中，温室条件下生长(25±2℃，湿度60％-80％，每天光照12小时)。以MS营养液灌溉。40天后，将每个转基因株系植株分为两组。一组以MS营养液灌溉；另一组以MS+NaCl灌溉直至NaCl终浓度达到180mM。BADH活性的测定按郭岩等(1997)的方法进行。一个酶活单位定义为在标准反应体系内每分钟每毫克蛋白质消耗1nmol的NAD。
BADH活性测定结果表明，不论NaCl胁迫存在与否，6个株系的植株中都可以检测到明显的活性。但转基因植株在盐胁迫下的BADH活性却显著高于无盐胁迫下(图4、)。
6〕转基因植株叶片的相对电导率(REc)的测定
相对电导率的测定按Leopold等(1984)的方法进行。对照及各转基因株系分别用0mM、90mM、180mM和270mM NaCl处理。
番茄叶片的电导率随着盐胁迫强度的增加而逐渐升高，表明细胞受到的伤害越来越严重(图5、)。但可以看出，在正常表达BADH基因的5个株系中，其电导率都有不同程度的降低，这表明转BADH基因番茄在不同程度上提高了耐盐性。
7〕转BADH基因番茄T1代卡那抗性分离比的测定
经分子鉴定BADH基因确已整合入基因组并能正确表达的转基因株系，自交结实。其种子播种在浸有MS+卡那霉素100mg/L溶液的滤纸上，并使溶液在培养皿中没过滤纸，用Parafilm膜封口以减少水分蒸发对抗生素浓度造成的影响。20天后统计对卡那霉素敏感和不敏感的幼苗比率，并进行χ2检验。
本实验对能正常表达BADH基因的两个株系T0-1和T0-8自交获得的种子分别进行了卡那抗性鉴定。χ2检验结果表明这两个后代群体分离均呈3∶1分离，符合孟德尔分离比例(表1)。由此可以推断，这两个转基因株系(T0-1，T0-8)中BADH基因的插入位点是一个或在同一条染色体上，并能遗传给后代。PCR结果也证明了这一点(图6、)。Northern结果表明凡是基因组中存在BADH基因的植株，均能检测到表达信号(图7、)。
表1 转BADH基因番茄T1代卡那抗性分离比
| 群体 | 实测值 | 期望值 | χ2 | 显著性 | ||
| 抗性 | 敏感 | 抗性 | 敏感 | |||
| T1-1 | 147 | 53 | 150 | 50 | 0.624 | 0.25＜P＜0.5 |
| T1-8 | 145 | 55 | 150 | 50 | 0.414 | 0.5＜P＜0.75 |
8〕转基因番茄T1代植株分子鉴定
PCR扩增条件及Northern杂交方法同T0代。
9〕转BADH基因番茄T1植株耐盐性检测
将灭菌的T1种子置于试管中的MS培养基上，对其发芽粒数、苗高、根长及须根数进行逐日记录。并应用SPSS统计软件进行方差分析。同时对60日龄幼苗在0mM、180mM NaCl胁迫下的叶片电导率进行测定。
T1-1和T1-8的种子发芽率在90mM和140mM NaCl胁迫下显著高于对照(图8、)。而幼苗的苗高、主根长和须根数量也比对照有很大提高(表2)。对NaCl胁迫下叶片的电导率测定发现，转基因植株的电导率明显低于对照(图9、)。
表2 T1代植株生长指标Ducan分析
| 群体 | 指 标 | ||
| 苗高(cm)0mM 90mM 140mM | 根长(cm)0mM 90m 140mMM | 须根数(条)0mM 90mM 140mM | |
| WTT1-1T1-8 | 6.60a 3.25c 0.60d6.52a 5.04b 2.91c6.65a 4.64b 3.10c | 9.68a 4.40d 0.62e9.72a 6.86b 5.84cd9.83a 6.12 6.06bc bc | 8.4a 0.0e 0.0e8.3a 3.7b 1.6d8.5a 4.2b 2.8c |
数字后标同一字母的表示差异不显著。
参考文献
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Claims (5)
1.一种用转甜菜碱醛脱氢酶基因(BADH)培育耐盐“百利春”番茄株系的方法，该方法包括如下步骤：
a.将含有BADH cDNA的基因片断的双元表达载体pBin438的农杆菌LBA4404感染“百利春”番茄真叶叶片；
b.感染后的“百利春”番茄叶片置于IM培养基上进行分化培养；
c.取分化植株幼嫩叶片提取基因组DNA进行分子检测；
d.转基因植株的BADH活性检测和耐盐性鉴定。
2.根据权利要求1所述的方法，其中农杆菌LBA4404菌液浓度OD560值等于0.5，用无激素的MS液体培养基稀释10倍，感染番茄真叶叶片。
3.根据权利要求1所述的方法，其中感染后的番茄叶片置于IM培养基上分化培养，IM培养基由MS、IAA 0.2mg/L、BA 0.2mg/L、ZT 0.1mg/L所组成。
4.根据权利要求1所述的方法，其中所述的转基因植株的基因组DNA的分子检测，是以α-32P-dCTP标记的BADH cDNA模板为探针进行检测。
5.根据权利要求1所述的方法，其中所述的转基因植株的BADH活性检测是指MS培养基加盐NaCl直至NaCl终浓度达到180mM，测定BADH酶活性和叶片的相对电导率。
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