摘要
枫香(Liquidambar formosana Hance.)作为我国著名的秋色叶树种,主要分布于秦岭淮河以南地区,是我国重要的乡土树种。由于其生长迅速,适应性强,树形美观,叶秋季红艳,具有很强的园林特性,故在我国很多城镇作为行道树和庭院观赏树栽培。但是枫香结实多、个体大且果实硬,严重影响了其应用和推广。对于此种情况,培育不育的无球果枫香成为一种行之有效的解决方法。
本研究通过对根癌农杆菌介导的枫香遗传转化体系的优化和改进,得到了转化GUS的转基因植株。在此基础上,对悬铃木PaFT基因的转化进行了研究。主要研究结果如下：
1.对农杆菌介导的枫香叶片转基因体系中菌液的制备进行了优化,比较了在AB重悬液中分别加入0μM,50μM, 100μM AS后,枫香叶片的GUS瞬间表达和抗性芽的诱导率,得到在菌液中添加100μMAS叶片的GUS瞬间表达可达到100%。共培养后,将叶片放入只含300 mg/L Cef培养基中培养30 d后再转入25 mg/LKan+300 mg/LCef选择培养基筛选,叶片抗性芽的诱导率和每个外植体平均出芽丛数最高分别达到91.6±3.0%和3.12±0.55个。
2.比较了预培养时间的长短对枫香叶片遗传转化的影响,分别对枫香叶片进1 d,3d,5 d和7d的预培养,观察统计其GUS瞬间表达和抗性芽的诱导率,结果表明,在预培养3d和7d时枫香叶片的GUS瞬间表达率都可达到100%。共培养后,将叶片放入只含300 mg/LCef培养基中培养30d后再转入25 mg/L Kan+300 mg/L Cef选择培养基筛选,抗性芽的诱导率分别为92.3±2.7%和92.6±3.4%,且每个外植体平均出芽丛数也较高,分别达到3.23±0.01个和2.98±0.56个。
3.在枫香叶片抗性不定芽筛选策略上,从降低选择培养基中Kan浓度和推迟筛选时间两个因素上进行了研究,其中,Kan浓度设置为0 mg/L,10 mg/L和20 mg/L,推迟时间为0d,10d,20d和30d。实验结果表明,将叶片放入只含300 mg/L Cef培养基培养30d后再转入25 mg/L Kan+300 mg/L Cef选择分化培养基筛选,抗性芽的诱导率和每个外植体平均出芽丛数分别为93.4±7.4%和3.15±0.67个,但阳性率较低；而将叶片先放入10 mg/L Kan+300 mg/L Cef选择分化培养基上培养10d的阳性率最高达到88.9%。
4.在对枫香叶片遗传转化体系进行优化的基础上培育转基因植株,进行GUS组织化学染色和PCR检测,结果表明共有19株转化苗PCR检测呈阳性,2株转化苗表达GUS活性,初步表明外源GUS基因已经整合到枫香转化植物基因组中。
5.以枫香叶片为受体,进行PaFT基因的转化,结果表明：转化PaFT基因的抗性植株经PCR检测,有1株呈阳性。
Formosan sweetgum, mainly distributed in the area South of Qinling Mountain and Huaihe River, is one of the important native tree species in China. Due to its many desirable traits, such as fast growth, wide adaptability, high resistance and red leaves in autumn, formosan sweetgum is becoming popular in urban greenland as street tree in many towns and as ornamental trees in courtyard. However, the spiny fruits of the tree disintegrate very slowly and cause a nuisance on lawns and walks and sterility or fruitless breeding would greatly improve the usefulness of formosan sweetgum.
By optimizing of Agrobacterium-mediated transformation system and genetic transformation, transgenic plantlets were finally obtained successfully with the stable integration of GUS gene in the genome of Liquidambar formosana Hance. Based on the improved systems, genetic transformation with PaFT genes was carried out, and the main results were as follows:
1. The method of culturing bacteria was improved in the Agrobacterium-mediated transformation system of formosan sweetgum. We added 0μM,50μM, 100μM, 200μM AS in the AB suspensions, after comparison of the GUS transient expression and the induction rate of resistance buds, we discovered that the GUS transient expression was 100% in the AB suspensions added 100μM AS. After co-culture, sweetgum was cultured 30 days on the medium of 300mg/L Cef. and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef. The induction rate of resistance buds was up to 91.6±3.0% and the average number of sprouting plexus of each explants was up to 3.12±0.55.
2. The effect of the time of pre-cuture on the transformation system of formosan sweetgum was compared. The sweetgum was pre-cutured respectively 1 d,3 d,5 d and 7 d. The GUS transient expression and the induction rate of resistance buds were observed. As a result, the GUS transient expression was all 100% after pre-cutured 3d and 7d. After co-culture, sweetgum was cultured 30 days on the medium of 300mg/L Cef., and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef. The induction rate of resistance buds was 92.3±2.7% and 92.6±3.4%. And the average number of sprouting plexus of each explants was up to 3.23±0.01 and 2.98±0.56 respectively.
3. The reducing the concentration of Kan in the selection medium and prolonging the time of selection was examed. The concentration of Kan was 0 mg/L,10 mg/L and 20 mg/L. and the time of selection was 0 d,10 d,20 d and 30 d was tested. As a result, sweetgum was cultured 30 days on the medium of 300mg/L Cef. and then selected on the selection medium of 25 mg/L Kan+300 mg/L Cef, the induction rate of resistance buds was 93.4±7.4%, and the average number of sprouting plexus each explants was 3.15±0.67,but the positive rate was very low. When the sweetgum explants were cultured on the selection medium with 10 mg/L Kan+300 mg/L Cef for 10 days, the positive rate was up to 88.9%.
4. Based on the optimized Agrobacterium-mediated transformation system of formosan sweetgum, the transgenic plants were obtained.19 kanamycin resistant plants were positive in the PCR detection, and 2 transgenic plants expressed of GUS activity. It indicated that exogenous GUS gene was integrated into the genome of formosan transgenic plant..
5. PaFT gene was used for transforming formosan sweetgum, PCR detection results show that 1 resistant plant was positive
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