银杏毛状根褐化机制的初步研究

该实验依据现有发根农杆菌侵染法,以银杏无菌苗叶片为外植体,对农杆菌诱导银杏毛状根的实验操作进行优化,并观察记录银杏毛状根继代生长情况,测定继代不同天数的毛状根组织内多酚含量、多酚合成相关酶基因和POD基因相对表达量,以探讨银杏毛状根褐化的生理机制。结果显示:(1)银杏农杆菌侵染最适预培养时间为3d、菌种为R1601、共培养基为White、侵染时间为30min,优化侵染操作后的银杏毛状根诱导率可达72.36%;银杏毛状根继代生长的最适IBA浓度为0.5mg/L。(2)银杏毛状根初始发根时颜色为乳白色,多在伤口处簇生,伸长生长时无向地性,不分叶正背面都能在伤口处诱导出根,且在除菌培养20d时毛状根长可达0.4～0.8cm;继代培养25d时毛状根为白色,继代35d时部分毛状根从根部至中部开始转黄,继代45d时大部分毛状根由白转黄,根部至中部开始有褐化出现。(3)继代35d时毛状根的PAL、C4 H、4CL和POD基因表达量分别为25d的68%、44%、51%、52%;继代45d时PAL、C4 H、4CL、POD基因表达量分别为35d的19%、75%、86%、105%。(4)继代35d、45d的银杏毛状根总酚类含量分别为17.14和13.78mg/g。研究发现,PAL基因表达量变化与其下游的两个关键酶基因(C4 H、4CL)表达量变化不一致,且PAL基因表达量的大幅降低并未对下游C4 H、4CL基因表达有明显影响,说明PAL基因表达量变化对其下游合成速率影响不大,可能不是银杏毛状根苯丙氨酸代谢途径的限速酶;而C4 H与4CL基因表达量变化趋势高度一致,说明C4H应是该途径中的限速酶之一;推测继代前期可能是由PPO作为关键酶参与毛状根褐化反应,导致后期毛状根生长受抑制,POD基因表达水平随之上升,POD参与褐化反应。

Preliminary Study on Browning Mechanism of Ginkgo biloba Hairy Roots

According to the existing Agrobacterium rhizogenes infection method, we used the leaves of Ginkgo biloba L. sterile seedlings as explants to optimize the experimental operation of Agrobacterium induced ginkgo hairy roots, and observe the growth of ginkgo hairy roots. The polyphenol content, relative expression of enzyme gene which related to polyphenol synthesis and POD gene in hairy root tissues on different days of subculture were determined to explore the physiological mechanism of browning of ginkgo hairy roots. The results show: (1) the optimal pre incubation time of Agrobacterium rhizogenes infection was 3 d, the strain was R1601, the co culture medium was White, and the infection time was 30 min. The induction rate of Ginkgo biloba hairy root after optimization of infection operation reached 72.36%; the optimal IBA concentration for ginkgo hairy root growth was 0.5 mg/L. (2) The hairy roots of ginkgo have a milky white color when initial rooting and they are mostly clustered at the wound. These hairy roots are non directional when growing and can be induced at the wound both on the front and back of the leaves. The root length can reach 0.4-0.8 cm at 20 days after sterilization. After 25 days of subculture, the hairy roots are white. However, at 35 days of subculture, part of the hairy roots which from bottom to middle turned yellow. At the 45 days of subculture, most of the hairy roots turned from white to yellow, and browning began to appeared from the root to the middle. (3) The relative expression levels of PAL, C4H, 4CL and POD of hairy roots in 35 d after subculture were respectively account for 68%, 44%, 51% and 52% of 25 days, and the relative expression of PAL, C4H, 4CL and POD gene at 45 d after subculture were respectively account for 19%, 75%, 86%, and 105% of 35 days. (4) The total phenolic contents of ginkgo hairy roots at 35 and 45 d after subculture were 17.14 and 13.78 mg/g, respectively. The change of PAL gene expression was inconsistent with the expression of other two key enzyme genes (C4H, 4CL) at downstream, and the significant decrease of PAL gene expression did not affect the expression of C4H and 4CL gene, which indicate the change of PAL gene expression has little effect on the downstream synthesis rate, and it may not be the rate limiting enzyme of the phenylalanine metabolic pathway of ginkgo hairy root; while the trend of C4H and 4CL gene expression is highly consistent, indicating that C4H should be one of the rate limiting enzymes in this pathway; it is speculated that PPO may be involved in the early stage of browning reaction of hairy roots, which leads to the inhibition of hairy root growth and the increase of POD gene expression level, then bring POD participates in browning reaction.