农杆菌介导的棉花转化体系优化与抗病基因导入
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摘要
本试验利用根癌农杆菌介导的转基因方法，将双价抗真菌病基因几丁质酶基因(Chitinase)和β-1,3-葡聚糖酶基因(Glucanase)导入棉花中棉所24中。主要研究结果如下:
1.研究了用于遗传转化的外植体的获取时期。剥去种皮的种子要经过0.1%的升汞消毒5min，并用无菌水冲洗3-5次，在苗培养基MS1中生长7天，这时的外植体易于被农杆菌感染和生成愈伤。
2.抗生素筛选压的确定。经过浓度梯度实验，75mg/L作为抗性愈伤组织的筛选压。
3.确定了农杆菌感染外植体的最佳浓度与感染时间。用进入对数生长期的农杆菌（OD600为0.3-0.7）菌液感染下胚轴5min，抗性愈伤的诱导率在40%左右。
4.共培养时间的确定。经过感染的外植体，在不加任何抗生素的MS2培养基上，培养48h后，再接种到加了选择抗生素和抑菌抗生素的MS2培养基上诱导抗性愈伤，这样，既可使农杆菌充分感染受伤的外植体细胞，又不致使农杆菌过度生长而毒害外植体，抑制愈伤的生成。
5.培养基的优化。
① 愈伤诱导培养基：MS1+IAA,KT和2,4-D各0.1 mg/L，pH值6.0；
② 胚性愈伤诱导培养基：MS1+少量IAA和KT，IAA/KT为1/4，pH值6.5；
③ 胚状体和再生苗培养基：MS1+IAA、6-BA各0.5mg/L， Asp、Gln各1g/L ，活性炭1.5g/L，pH值6.2。
6.共获得9棵独立转基因植株，经过PCR检测，外源目的基因已经整合到棉花中棉所24的基因组中。
7.利用上面已经优化的转化体系，将单价抗病葡萄糖氧化酶（GO）基因导入中棉所394，并且获得4棵独立转基因植株，经过PCR检测，外源目的基因已经整合到棉花中棉所394的基因组中。
In this study chitinase and β-1,3-glucanase were transferred into Gossypium hirszrtum CCRI 24 by Agrobacterium tumefaciens .The main results as follows:
1.The time when the explant can be obtained in genetic transformation. Asepsis treated seedlings were wiped off their seed coats,and then sterilized by 0.1%Hg for 5 min . After washed 3 to 5 times by sterilized water ,they had been grown on MS1 culture medium for 7 days. Then they were easily infected by Agrobacterium and induced calli.
2.Ascertain of the screening pressure of antibiotics .Through concentration grading experiment, 75mg/Lwas regarded as the screening pressure of resistance-callus.
3.The aptimal concentration and infection time when Agrobacterium infects explant .When the Agrobacterium that had entered log-growth period was used to infect hypocotyl, the induction rate of resistance-callus was about 40%.
4.Ascertain of the time of co-culture . Infected explant had been cultured for 48h on the MS2 culture medium that had been added selective antibiotics and antibiotic for microbe-inhibition so as to induce resistance-callus .In this way, the explant could be fully infected by Agrobacterium and also the over-growth Agrobacterium would not poison the explant and inhibit the growth of calli .
5.Optimum of culture medium.
① Callus-inducing medium: MS1+IAA,Ktand 2,4-D each 0.1 mg/L，pH=6.0；
② Embryo-callus inducing medium: MS1+ IAA and KT，IAA/KT=1/4，pH=6.5；
③ Embryogenic bud and regenerated seeding inducing medium:
MS1+IAA、6-BA each 0.5mg/L， Asp、Gln each 1g/L ，active carbon 1.5g/L，pH=6.2 .
6.A totel of 9 transgenic plant had been gaind. By PCR detection ,the foreign genes had been integrated in the genome of Gossypium hirsutum CCRI 24.
7. Using this optimized transformation system, we obtained four transgenic plants into which the sigle GO gene was transferred.and the detection by PCR method showed that the target gene has been inserted into the genome of CCRI 394.
1.研究了用于遗传转化的外植体的获取时期。剥去种皮的种子要经过0.1%的升汞消毒5min，并用无菌水冲洗3-5次，在苗培养基MS1中生长7天，这时的外植体易于被农杆菌感染和生成愈伤。
2.抗生素筛选压的确定。经过浓度梯度实验，75mg/L作为抗性愈伤组织的筛选压。
3.确定了农杆菌感染外植体的最佳浓度与感染时间。用进入对数生长期的农杆菌（OD600为0.3-0.7）菌液感染下胚轴5min，抗性愈伤的诱导率在40%左右。
4.共培养时间的确定。经过感染的外植体，在不加任何抗生素的MS2培养基上，培养48h后，再接种到加了选择抗生素和抑菌抗生素的MS2培养基上诱导抗性愈伤，这样，既可使农杆菌充分感染受伤的外植体细胞，又不致使农杆菌过度生长而毒害外植体，抑制愈伤的生成。
5.培养基的优化。
① 愈伤诱导培养基：MS1+IAA,KT和2,4-D各0.1 mg/L，pH值6.0；
② 胚性愈伤诱导培养基：MS1+少量IAA和KT，IAA/KT为1/4，pH值6.5；
③ 胚状体和再生苗培养基：MS1+IAA、6-BA各0.5mg/L， Asp、Gln各1g/L ，活性炭1.5g/L，pH值6.2。
6.共获得9棵独立转基因植株，经过PCR检测，外源目的基因已经整合到棉花中棉所24的基因组中。
7.利用上面已经优化的转化体系，将单价抗病葡萄糖氧化酶（GO）基因导入中棉所394，并且获得4棵独立转基因植株，经过PCR检测，外源目的基因已经整合到棉花中棉所394的基因组中。
In this study chitinase and β-1,3-glucanase were transferred into Gossypium hirszrtum CCRI 24 by Agrobacterium tumefaciens .The main results as follows:
1.The time when the explant can be obtained in genetic transformation. Asepsis treated seedlings were wiped off their seed coats,and then sterilized by 0.1%Hg for 5 min . After washed 3 to 5 times by sterilized water ,they had been grown on MS1 culture medium for 7 days. Then they were easily infected by Agrobacterium and induced calli.
2.Ascertain of the screening pressure of antibiotics .Through concentration grading experiment, 75mg/Lwas regarded as the screening pressure of resistance-callus.
3.The aptimal concentration and infection time when Agrobacterium infects explant .When the Agrobacterium that had entered log-growth period was used to infect hypocotyl, the induction rate of resistance-callus was about 40%.
4.Ascertain of the time of co-culture . Infected explant had been cultured for 48h on the MS2 culture medium that had been added selective antibiotics and antibiotic for microbe-inhibition so as to induce resistance-callus .In this way, the explant could be fully infected by Agrobacterium and also the over-growth Agrobacterium would not poison the explant and inhibit the growth of calli .
5.Optimum of culture medium.
① Callus-inducing medium: MS1+IAA,Ktand 2,4-D each 0.1 mg/L，pH=6.0；
② Embryo-callus inducing medium: MS1+ IAA and KT，IAA/KT=1/4，pH=6.5；
③ Embryogenic bud and regenerated seeding inducing medium:
MS1+IAA、6-BA each 0.5mg/L， Asp、Gln each 1g/L ，active carbon 1.5g/L，pH=6.2 .
6.A totel of 9 transgenic plant had been gaind. By PCR detection ,the foreign genes had been integrated in the genome of Gossypium hirsutum CCRI 24.
7. Using this optimized transformation system, we obtained four transgenic plants into which the sigle GO gene was transferred.and the detection by PCR method showed that the target gene has been inserted into the genome of CCRI 394.
引文
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