喜樹組織培養(yǎng)及遺傳轉(zhuǎn)化的研究.pdf

1、杭州師范大學(xué)碩士學(xué)位論文 喜樹組織培養(yǎng)及遺傳轉(zhuǎn)化的研究 摘要 摘要 喜樹（Camptotheca acuminata Decne.）是我國特有植物，分布于長江流域及西南各省。喜樹不但是一種優(yōu)良的綠化樹種，還是一種藥用價值非常高的藥用植物。喜樹堿（camptothecin, CPT） 是喜樹體內(nèi)發(fā)現(xiàn)的唯一通過抑制拓撲異構(gòu)酶Ⅰ （Topoisomerase Ⅰ） 而具有

2、抗癌活性的天然植物活性成分。 喜樹堿及其衍生物已用于多種癌癥的治療， 在臨床試驗上顯示了廣泛的抗癌活性， 但是喜樹資源有限， 通過基因工程和細胞工程提高喜樹細胞藥用活性成分含量具有潛在的應(yīng)用價值和廣闊的市場。 近年來， 隨著分子生物學(xué)和植物細胞凋亡研究的深入， 人們發(fā)現(xiàn)細胞凋亡不但是植物體正常生長發(fā)育必不可少的組成部分， 而且是植物抵御病原體的重要手段。超敏反應(yīng)（hypersensitive response, HR）是植物細胞為限制病

3、原菌生長的一種細胞凋亡， 它引起的細胞死亡還可以激發(fā)鄰近組織的特異性防御反應(yīng)和植株的系統(tǒng)獲得抗性， 從而使植物的次生代謝產(chǎn)物含量增加。 目前通過基因工程手段提高喜樹細胞藥用活性成分含量的研究未見報道。在本研究中，將促進細胞凋亡基因Bax和抑制細胞凋亡基因PpBi-1、ced-9轉(zhuǎn)化到喜樹細胞中誘導(dǎo)其表達，測定了非轉(zhuǎn)化喜樹細胞和三種轉(zhuǎn)化喜樹細胞喜樹堿和10-羥基喜樹堿的含量，分析了細胞凋亡基因?qū)ο矘浠钚猿煞趾康挠绊憽嶒灚@得以下結(jié)果：

4、1. 喜樹愈傷組織的誘導(dǎo)與抑制褐化：幼葉外植體的最佳誘導(dǎo)培養(yǎng)基為：SH+NAA 2.5mg/L+2,4-D 0.5mg/L+6-BA 0.25mg/L；種胚外植體的最佳誘導(dǎo)培養(yǎng)基為：MS+2,4-D 1.0mg/L+6-BA 0.5mg/L。抑制幼葉外植體褐化的方法為：增加外植體大小，吹干培養(yǎng)基表面水分。抑制愈傷組織繼代培養(yǎng)褐化的方法為：最佳誘導(dǎo)培養(yǎng)基附加30mg/L抗壞血酸和5g/L活性碳；1次繼代后，將質(zhì)地致密的愈傷組織轉(zhuǎn)移到MS

5、培養(yǎng)基上培養(yǎng)。 2. 建立了喜樹再生體系： 種胚的外植體在WPM+NAA 1.0 mg/L +6-BA 5.0 mg/L培養(yǎng)基上的芽誘導(dǎo)率最高，為73.88%；喜樹無菌苗的莖段最適合誘導(dǎo)再生，在SH+NAA 1.0mg/L +6-BA 5.0 mg/L的培養(yǎng)基上可以誘導(dǎo)成芽，誘導(dǎo)率為42%，平均每個外植體產(chǎn)生4.6個芽；誘導(dǎo)根的最佳培養(yǎng)基為1/2MS+IBA 2mg/L。 3. 建立了喜樹細胞懸浮培養(yǎng)體系：挑選疏松愈傷組織接入到WP

6、M+2,4-D 1.0mg/L +6-BA 0.5 mg/L液體生長培養(yǎng)基， 接種量10%、 蔗糖濃度3%、 pH5.5、 搖床轉(zhuǎn)速120rpm、 25 ℃下散光培養(yǎng)，7d繼代一次，為較好的懸浮培養(yǎng)體系，24d時，喜樹細胞增值量達到最大，為接入重量的12.86倍。 4. 建立了遺傳轉(zhuǎn)化體系：取喜樹無菌苗的莖段和葉片為外植體，預(yù)培養(yǎng)2d，用根癌農(nóng)桿菌分別侵染，侵染時間為15min，共培養(yǎng)2d，清洗后轉(zhuǎn)到添加頭孢霉素500mg/L的培養(yǎng)基

7、，14d時加入篩選壓潮霉素10mg/L，1-2個月得到抗性愈傷組織，加入雌二醇10mg/L誘導(dǎo)基因表達，PCR檢測表明目的基因已經(jīng)轉(zhuǎn)化到喜樹基因組中，RT-PCR檢測表明目的基因可以正常表達。 5. 建立了喜樹堿和10-羥基喜樹堿檢測方法：建立了高效液相色譜熒光法測定喜樹組I杭州師范大學(xué)碩士學(xué)位論文 喜樹組織培養(yǎng)及遺傳轉(zhuǎn)化的研究 Abstract Abstrac

8、t Camptotheca acuminata Decne., belonging to nyssaceae family, grows in the southern China. Camptotheca acuminata is not only fine trees, or a very high medicinal value of medicinal plants. Its secondary metabolism produ

9、cts camptothecin is the only body found by inhibiting topoisomerase Ⅰwith the anti-cancer activity of natural plant active ingredients. Camptothecin and its derivatives have been used in the treatment of many types of ca

10、ncer and shown a wide range of anti-cancer activity in clinical trials. Because of tight supply, through genetic engineering and cell engineering increase Camptotheca acuminata cells active ingredients in medicinal have

11、potential applications and the vast market value. In recent years, as molecular biology and plant cell apoptosis in-depth study, it was found that apoptosis is not only the essential component for normal growth and devel

12、opment of plants, and is an important means to resist pathogens. hypersensitive response (HR) is an apoptosis which is for plant cell to limit the growth of pathogen, it can also stimulate plant defense response system o

13、r acquired resistance in nearby organizations, result in that the plant secondary metabolites were increased. Currently it have not been reported that through genetic engineering means to increase the content of medicina

14、l active ingredients in Camptotheca acuminata cell. In this study, we had transformed promote apoptosis gene Bax and inhibition of apoptosis gene PpBi-1, ced-9 into Camptotheca acuminata cells and induced them expression

15、, determined the content of CPT and HCPT in non-transformed cells and three transformed cells, analyzed the apoptosis gene on the content of active ingredients of Camptotheca acuminata. We had obtained following findings

16、: 1. Callus induction of Camptotheca acumianta and darkening inhibition: the optimal culture medium for callus induction in young leaf is SH+NAA 2.5mg/L +2,4-D 0.5 mg/L +6-BA 0.25mg/L, and MS+2,4-D 1.0mg/L+6-BA 0.5mg/L i

17、n mature embryo; The way to inhibit darkening of young leaf is to add the explants sizes and dry the water on the surface of medium; One way to inhibit darkening of callus is to supplement 30mg/L VC and 5g/L AC in the op

18、timal culture medium; the other way is to transfer the close callus to MS medium after the first subculture. 2. Establishment of Camptotheca acuminata regeneration system: The induction rates is highest when embryo expla

19、nts cultured in WPM + NAA 1.0mg / L +6-BA 5.0mg / L medium, bud induction rates is 73.88%. stem segments of Camptotheca acuminata aseptic seedling is most suitable induced regeneration. The explants of stem segments on S

20、H medium supplemented with 6-benzyladenine (6-BA) 5mg/L and α-napthalene acetic acid (NAA) 1mg/L were most effective for shoot induction. The average regenerated shoots is 4.6 per segment, and the shoot regeneration freq