mtlD/gutD双价耐盐基因转化杨树、猕猴桃的研究
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摘要
我国“三北”及沿海地区有大量盐碱地。随着工业迅速发展、人口猛增和可耕地面积的减少，培育抗盐植物材料，开发利用盐碱地显得日益重要。
杨树为“三北”地区主要人工造林树种，栽培数量很大；猕猴桃为一新兴果树，经济价值较高。提高这2个树种抗盐性，扩大其栽培区域是开发利用盐碱地的有效途径，具有重要的生产意义。
本实验计划通过将mtlD/gutD双价耐盐基因导入杨树、猕猴桃，来提高这2个树种的抗盐能力。转化方法以叶盘法为主，辅助使用了基因枪法。所用植物双价双元表达载体为pBIMG，其上含有双价抗盐基因mtlD/gutD、双重增强的35S启动子和TMV“Ω”片段的翻译增强子及NPT-Ⅱ卡那霉素抗性标记基因，农杆菌菌株为LBA4404。杨树选用“三北”地区栽植数量较多的美洲黑杨×青杨杂种，猕猴桃选用经济价值较高的秦美猕猴桃。主要研究内容如下。
1利用正交等实验设计开展了杨树、猕猴桃叶片外植体高效再生系统建立研究。通过系统实验，筛选出杨树叶片外植体不定芽诱导最佳配方为MS+6-BA0.5mg/L+NAA 0.01mg/L，不定芽生根诱导最适配方为1/2MS+NAA 0.01mg/L；猕猴桃叶片外植体不定芽诱导最佳配方为：MS+6-BA 6mg/L+NAA 0.1mg/L，不定芽生根诱导最适配方为：1/2MS+NAA 0.01mg/L+IBA 0.5mg/L+GA 1mg/L。
2通过卡那霉素敏感实验，确定了杨树叶片不定芽诱导卡那霉素临界敏感浓度为50mg/L，不定芽生根诱导卡那霉素临界敏感浓度为60mg/L；秦美猕猴桃叶片不定芽诱导卡那霉素临界敏感浓度为50mg/L，不定芽生根诱导卡那霉素临界敏感浓度为40mg/L。
3利用叶盘法开展了mtlD/gutD双价耐盐基因转化杨树的研究。经叶盘不定芽诱导及不定芽生根诱导阶段卡那霉素连续筛选，获得了38株生根卡那霉素抗性植株。抗性植株经PCR检测，28株呈阳性。对其中7株阳性植株进行southern及Western杂交，有4株二者均呈阳性，确证双价耐盐基因成功整合到杨树基因组中并得到表达；利用叶盘法和基因枪相结合的办法，开展了mtlD/gutD双价耐盐基因转化猕猴桃的研究。获得卡那霉素生根抗性植株20株，其中6株经PCR检测呈阳性，初步证明双价耐盐基因转化获得成功。
4开展了转基因植株耐盐性测定。结果表明，杨树未转基因植株NaCl最高耐受浓度为0.2％，4株转基因植株抗NaCl能力最低为0.4％，最高达0.6％，比对照有较大幅度提高；猕猴桃未转基因植株NaCl最高耐受浓度为0.1％，6株PCR阳性植株中，2株抗NaCl能力与对照一致(为0.1％)，推测为假阳性，另4株NaCl耐受浓度为0.2～0.4％，比对照有不同程度提高。
2 milD／gutD双价耐盐基因转化杨树、猕猴桃的研究
本实验在建立了杨树、猕猴桃叶片外植体高效再生系统及确定了卡那霉素耐受浓
度的基础上，利用叶盘法、叶盘法和基因枪相结合的办法分别开展了milD哈utD双价
耐盐基因转化杨树和猕猴桃的研究。经PCR、Southern及Western杂交检测，确证
milD哈utD双价耐盐基因成功整合到4株杨树基因组中并得到表达。同时，PCR检测
也初步证明部分猕猴桃植株转化获得成功。耐盐测定表明转化植株耐盐性比对照均有
不同程度提高。这在杨树、猕猴桃耐盐基因工程研究中尚属首次。
A large area of the saline-alkali land are mainly sited the northwest, north and northeast parts of China and the east coast region. With development of industry, growth of population and decreasing of arable land, selecting and breading plant material for salt resistance and rationally using saline-alkali land are very significant.
Poplar is the major species in the northwest, north and northeast parts of China and the number of the species in the area is big. Kiwifruit is a new kind of fruit tree with high economic value. Improving salt resistance of the two species and enlarging their planting region are good ways for using saline-alkali land rationally and have an important meaning in practice.
In this experiment, mtlD/gutD divalent genes were transformed into poplar and kiwifruit trees. Leaf-disc method and microprojectile bombardment were adopted for gene transformation. Binary plant expression vecter is pBIMG which contains mtlD/gutD divalent genes, double strength 35S promoter, translation enhancer from TMV " & "fragment and NPT- II kanamycin-resistant marker gene. The strain of Agrobacterium tumefaciens is LBA4404. Populus deltoilds X P. cathayana and Qingmei kiwifruit variety, were used in the experiment as plant materials. The main research works are as follows.
1 ? Leaf explant regeneration systems of the poplar and kiwifruit were established by orthogonal design, the experiment results indicated that the optimum medium compositions of poplar were MS+6-BA 0.5 mg/L+NAA O.Olmg/L for inducing shoot from leaf explant, and 1/2 MS+NAA O.Olmg/L for inducing root from shoot. The optimum medium compositions of kiwifruit were MS+6-BA6mg/L+NAA 0.01 mg/L for inducing shoot, and 1/2MS+NAA O.Olmg/1+IBA 0.5mg/L +GA Img/L for inducing roots.
2> kanamycin sensitive experiment showed that the critical kanamycin sensitive concentrations for inducing shoots and roots of the poplar were 50 mg/L and 60mg/L.The critical kanamycin sensitive concentrations for inducing shoots and roots of the Qingmei kiwifruit were 50 mg/L and 40 mg/L.
3 > On the basis of experiments above, a study on transforming mtlD/gutD divalent salt resistance genes into poplar by leaf disc method was carried out. Through kanamycin continuously screening in the shoots and roots induction stage, 38 kanamycin-resistant rooted poplar shoots were obtained. PCR analysis showed that 28 of these kanamycin-resistant rooted plants were positive. 7of these 28 PCR positive plants were
taken out for Southern blotting and Western blotting , the result indicated that 4 of the 7 plantlets were positive for both. It means that mtlD/gutD divalent genes were successfully integrated into the genome of these 4 plantlets, and the proteins expressed by mtlD/gutD divalent genes were synthesized. Combining leaf disc method and microprojectile bombardment, a study on transforming mtlD/gutD divalent salt tolerance genes into kiwifruit was conducted. Through successive selection in the period of shoot and root induction under high lever kanamycin pressure, 20 kanamycin-resistant rooted plants was obtained, out of which, 6 was positive through PCR analysis.
4. Results from salt tolerance tests indicated that the NaCl tolerance concentration of 4 transgenic poplar plantlets can reach to 0.4~0.6% while the highest NaCl tolerance concentration for normal poplar (control) was 0.2%. The highest NaCl tolerance concentration for normal kiwifruit (control) was 0.1%. In 6 PCR positive kiwifruit plantlets, the salt resistant ability of 2 plantlets was the same as that of control, but the sodium chloride tolerance concentrations of another 4 kiwifruit plantlets can reach to 0.2%-0.4%, which was higher than that of control plantlets.
On the basis of establishment of leaf explant regeneration system for poplar and kiwifruit species and the kanamycin tolerance concentration , studies on transforming mtlD/gutD divalent salt tolerance genes into poplar and kiwifruit species by leaf-dish method ,and combination of leaf disc and microprojectile bombardment method were developed. Re
杨树为“三北”地区主要人工造林树种，栽培数量很大；猕猴桃为一新兴果树，经济价值较高。提高这2个树种抗盐性，扩大其栽培区域是开发利用盐碱地的有效途径，具有重要的生产意义。
本实验计划通过将mtlD/gutD双价耐盐基因导入杨树、猕猴桃，来提高这2个树种的抗盐能力。转化方法以叶盘法为主，辅助使用了基因枪法。所用植物双价双元表达载体为pBIMG，其上含有双价抗盐基因mtlD/gutD、双重增强的35S启动子和TMV“Ω”片段的翻译增强子及NPT-Ⅱ卡那霉素抗性标记基因，农杆菌菌株为LBA4404。杨树选用“三北”地区栽植数量较多的美洲黑杨×青杨杂种，猕猴桃选用经济价值较高的秦美猕猴桃。主要研究内容如下。
1利用正交等实验设计开展了杨树、猕猴桃叶片外植体高效再生系统建立研究。通过系统实验，筛选出杨树叶片外植体不定芽诱导最佳配方为MS+6-BA0.5mg/L+NAA 0.01mg/L，不定芽生根诱导最适配方为1/2MS+NAA 0.01mg/L；猕猴桃叶片外植体不定芽诱导最佳配方为：MS+6-BA 6mg/L+NAA 0.1mg/L，不定芽生根诱导最适配方为：1/2MS+NAA 0.01mg/L+IBA 0.5mg/L+GA 1mg/L。
2通过卡那霉素敏感实验，确定了杨树叶片不定芽诱导卡那霉素临界敏感浓度为50mg/L，不定芽生根诱导卡那霉素临界敏感浓度为60mg/L；秦美猕猴桃叶片不定芽诱导卡那霉素临界敏感浓度为50mg/L，不定芽生根诱导卡那霉素临界敏感浓度为40mg/L。
3利用叶盘法开展了mtlD/gutD双价耐盐基因转化杨树的研究。经叶盘不定芽诱导及不定芽生根诱导阶段卡那霉素连续筛选，获得了38株生根卡那霉素抗性植株。抗性植株经PCR检测，28株呈阳性。对其中7株阳性植株进行southern及Western杂交，有4株二者均呈阳性，确证双价耐盐基因成功整合到杨树基因组中并得到表达；利用叶盘法和基因枪相结合的办法，开展了mtlD/gutD双价耐盐基因转化猕猴桃的研究。获得卡那霉素生根抗性植株20株，其中6株经PCR检测呈阳性，初步证明双价耐盐基因转化获得成功。
4开展了转基因植株耐盐性测定。结果表明，杨树未转基因植株NaCl最高耐受浓度为0.2％，4株转基因植株抗NaCl能力最低为0.4％，最高达0.6％，比对照有较大幅度提高；猕猴桃未转基因植株NaCl最高耐受浓度为0.1％，6株PCR阳性植株中，2株抗NaCl能力与对照一致(为0.1％)，推测为假阳性，另4株NaCl耐受浓度为0.2～0.4％，比对照有不同程度提高。
2 milD／gutD双价耐盐基因转化杨树、猕猴桃的研究
本实验在建立了杨树、猕猴桃叶片外植体高效再生系统及确定了卡那霉素耐受浓
度的基础上，利用叶盘法、叶盘法和基因枪相结合的办法分别开展了milD哈utD双价
耐盐基因转化杨树和猕猴桃的研究。经PCR、Southern及Western杂交检测，确证
milD哈utD双价耐盐基因成功整合到4株杨树基因组中并得到表达。同时，PCR检测
也初步证明部分猕猴桃植株转化获得成功。耐盐测定表明转化植株耐盐性比对照均有
不同程度提高。这在杨树、猕猴桃耐盐基因工程研究中尚属首次。
A large area of the saline-alkali land are mainly sited the northwest, north and northeast parts of China and the east coast region. With development of industry, growth of population and decreasing of arable land, selecting and breading plant material for salt resistance and rationally using saline-alkali land are very significant.
Poplar is the major species in the northwest, north and northeast parts of China and the number of the species in the area is big. Kiwifruit is a new kind of fruit tree with high economic value. Improving salt resistance of the two species and enlarging their planting region are good ways for using saline-alkali land rationally and have an important meaning in practice.
In this experiment, mtlD/gutD divalent genes were transformed into poplar and kiwifruit trees. Leaf-disc method and microprojectile bombardment were adopted for gene transformation. Binary plant expression vecter is pBIMG which contains mtlD/gutD divalent genes, double strength 35S promoter, translation enhancer from TMV " & "fragment and NPT- II kanamycin-resistant marker gene. The strain of Agrobacterium tumefaciens is LBA4404. Populus deltoilds X P. cathayana and Qingmei kiwifruit variety, were used in the experiment as plant materials. The main research works are as follows.
1 ? Leaf explant regeneration systems of the poplar and kiwifruit were established by orthogonal design, the experiment results indicated that the optimum medium compositions of poplar were MS+6-BA 0.5 mg/L+NAA O.Olmg/L for inducing shoot from leaf explant, and 1/2 MS+NAA O.Olmg/L for inducing root from shoot. The optimum medium compositions of kiwifruit were MS+6-BA6mg/L+NAA 0.01 mg/L for inducing shoot, and 1/2MS+NAA O.Olmg/1+IBA 0.5mg/L +GA Img/L for inducing roots.
2> kanamycin sensitive experiment showed that the critical kanamycin sensitive concentrations for inducing shoots and roots of the poplar were 50 mg/L and 60mg/L.The critical kanamycin sensitive concentrations for inducing shoots and roots of the Qingmei kiwifruit were 50 mg/L and 40 mg/L.
3 > On the basis of experiments above, a study on transforming mtlD/gutD divalent salt resistance genes into poplar by leaf disc method was carried out. Through kanamycin continuously screening in the shoots and roots induction stage, 38 kanamycin-resistant rooted poplar shoots were obtained. PCR analysis showed that 28 of these kanamycin-resistant rooted plants were positive. 7of these 28 PCR positive plants were
taken out for Southern blotting and Western blotting , the result indicated that 4 of the 7 plantlets were positive for both. It means that mtlD/gutD divalent genes were successfully integrated into the genome of these 4 plantlets, and the proteins expressed by mtlD/gutD divalent genes were synthesized. Combining leaf disc method and microprojectile bombardment, a study on transforming mtlD/gutD divalent salt tolerance genes into kiwifruit was conducted. Through successive selection in the period of shoot and root induction under high lever kanamycin pressure, 20 kanamycin-resistant rooted plants was obtained, out of which, 6 was positive through PCR analysis.
4. Results from salt tolerance tests indicated that the NaCl tolerance concentration of 4 transgenic poplar plantlets can reach to 0.4~0.6% while the highest NaCl tolerance concentration for normal poplar (control) was 0.2%. The highest NaCl tolerance concentration for normal kiwifruit (control) was 0.1%. In 6 PCR positive kiwifruit plantlets, the salt resistant ability of 2 plantlets was the same as that of control, but the sodium chloride tolerance concentrations of another 4 kiwifruit plantlets can reach to 0.2%-0.4%, which was higher than that of control plantlets.
On the basis of establishment of leaf explant regeneration system for poplar and kiwifruit species and the kanamycin tolerance concentration , studies on transforming mtlD/gutD divalent salt tolerance genes into poplar and kiwifruit species by leaf-dish method ,and combination of leaf disc and microprojectile bombardment method were developed. Re
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