侧柏古树组织培养再生及其端粒相关基因克隆研究

发布时间：2018-07-22 12:01

【摘要】：古树是独特的风景园林资源,是城市人文景观和自然景观的综合载体,但是古树面临不同程度衰老甚至死亡的问题,保护古树可以充分发挥其在园林绿化中的重要作用。为了保护古树景观资源,本文以陕西黄帝陵的侧柏古树为研究对象,以古树上当年生枝条茎尖为外植体,通过组织培养对古树进行克隆,完全保留了古树的性状;同时通过克隆端粒基因,在分子水平研究古树衰老,对古树复壮进行初步研究。主要的研究结果如下:1.灭菌方法的选择:通过对外植体灭菌方法的筛选发现,侧柏古树外植体适宜的消毒方法是先用75%酒精处理30 s,无菌水冲洗5次,然后用0.1%Hg Cl2消毒8 min,此时的污染率最低,死亡率也最低,成活率最高,初步解决了侧柏古树的灭菌问题。2.初代培养基筛选:利用MS、1/2MS、B5、WPM与DKW 5种基本培养基对侧柏古树的外植体进行组织培养,经一个月培养后发现,1/2MS培养基对侧柏古树嫩枝的组织培养有良好的效果,B5培养基诱导的效果比1/2MS稍差,诱导率低。MS、WPM与DKW培养基对外植体培养的效果都不理想,可能与培养基本身的营养有关。然后以1/2MS为基本培养基筛选出了NAA和6-BA适宜的使用浓度,最后得出初代培养最适宜培养基为:1/2MS+0.2 mg/L NAA+0.1 mg/L 6-BA。3.增殖培养基筛选:通过对不同基本培养基(MS、1/2MS、WPM、B5、DKW)和不同激素(6-BA、KT、NAA)组合的优化,增殖最适培养基为:1/2MS+0.05 mg/L6-BA+0.2 mg/L NAA+0.1 mg/L KT。4.生根培养基的筛选:内、外源激素的配比关系对不定根的形成起着决定性的作用。试验表明,在生根培养中WPM效果较1/2MS好,这与初代培养、增殖培养时明显不同。说明生根阶段所需要的营养物质有所不同,侧柏古树最适生根培养基为WPM+0.5mg/L NAA+0.5 mg/L IBA。5.三个端粒基因的克隆:应用RACE技术从侧柏中克隆出3个端粒基因的全长:Po TRF1、Po POT1和Po Ku70。Po TRF1基因长度为1464 bp,包括176 bp 5’-非编码区(UTR,untranslated region),和361 bp的3'-UTR,26 bp的poly A尾巴,927 bp的开放阅读框(ORF,open reading frame),编码蛋白质的分子量是119 k Da,等电点是5.04。Po POT1基因长度为1337 bp,包括163 bp 5’-UTR,和556 bp的3'-UTR,28 bp的poly A尾巴,618 bp的ORF,编码蛋白质的分子量是110 k Da,等电点是4.98。Po Ku70基因长度为1010 bp,包括81 bp 5’-UTR,和368 bp的3'-UTR,24 bp的poly A尾巴,561 bp的ORF,编码蛋白质的分子量是82 k Da,等电点是5.02。在NCBI中BLAST比对发现:Po TRF1、Po POT1和Po Ku70编码的蛋白质序列与拟南芥和杨树中的同源性分别为54%、43%;54%、44%;和70%、66%。
[Abstract]:Ancient trees are unique resources of landscape architecture and comprehensive carriers of urban human and natural landscape. However, ancient trees are faced with the problem of aging and even dying to varying degrees, so protecting ancient trees can give full play to their important role in landscape greening. In order to protect the ancient tree landscape resources, this paper takes the ancient Platycladus orientalis in Huangdi Mausoleum of Shaanxi Province as the research object, takes the old branch stem tip on the ancient tree as the explant, carries on the clone through the tissue culture to the ancient tree, completely retains the ancient tree characteristic; At the same time, by cloning telomere gene, the aging of ancient trees was studied at molecular level, and the rejuvenation of ancient trees was preliminarily studied. The main findings are as follows: 1: 1. The selection of sterilization method: through the screening of explant sterilization method, it was found that the suitable disinfection method for explants of Platycladus orientalis was to first treat them with 75% alcohol for 30 s, wash them with sterile water for 5 times, and then sterilize them with 0.1 Hg Cl 2 for 8 min. The pollution rate of the explants was the lowest at this time. The mortality rate was also the lowest and the survival rate was the highest. The problem of sterilizing the ancient trees of Platycladus orientalis was preliminarily solved. 2. 2. Screening of primary culture medium: the explants of ancient Platycladus orientalis were cultured in 5 basic media, MS1 / 2 MSB5 WPM and DKW, respectively. After one month of culture, it was found that 1 / 2 MS medium had a better effect on tissue culture of shoots of ancient Platycladus orientalis than that on 1 / 2 MS medium, and the induction rate was low. Both MSWPM and DKW medium were not effective in explant culture. It may be related to the nutrition of the medium itself. Then the suitable concentration of NAA and 6-BA was screened out on 1 / 2 MS as the basic medium. Finally, the optimal medium for primary culture was: 1 / 2 MS 0.2 mg / L NAA 0.1 mg / L 6-BA.3. Culture medium selection: by optimizing the combination of different basic medium (MS1 / 2MSPPMB5DKW) and different hormone (6-BAKTNAA), the optimal medium for proliferation was: 1 / 2 MS 0.05 mg / L 6-BA 0.2 mg / L NAA 0.1 mg / L KT.4. Selection of rooting medium: endogenous and exogenous hormones play a decisive role in the formation of adventitious roots. The results showed that the effect of WPM in rooting culture was better than that in 1 / 2 MS, which was obviously different from that in primary culture and proliferation culture. The optimum rooting medium of Platycladus orientalis was WPM 0.5 mg / L NAA 0.5 mg / L IBA.5. Cloning of three telomere genes: the full length of three telomere genes were cloned from Platycladus orientalis by race. The full length of the three telomere genes was 1464 BP in length, including the poly A tail of 361bp, the 3- UTR26 BP, and the 1464 BP length of the Po Ku70. Po TRF1 gene, including the 176bp UTRuntranslated region), and the 361bp 3- UTR26bp poly A tail motif of the three telomere genes, which were cloned from Platycladus orientalis by race technique. The molecular weight of ORF open reading frame), protein is 119k Da. the isoelectric point is 5.04.Po POT1 gene length is 1337 BP, including 163bp 5tr -UTRs, and 556bp poly A tail 618bp, the molecular weight of coding protein is 110k. The isoelectric point is 4.98.Po Ku70 gene with a length of 1010 BP, including 81bp 5r-UTR-UTRR, and 368bp 3H-UTR-24bp poly A tail 561bp ORF.The molecular weight of the protein is 82kDaand the isoelectric point is 5.02bp. In the NCBI, blast showed that the homology of the protein sequences encoded by the two proteins were 54443 and 5445, respectively, and 70666666 in Arabidopsis thaliana and poplar, respectively.
【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S791.38

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