本文關(guān)鍵詞： 中間錦雞兒 組織培養(yǎng) CiPP2C8-like基因 CiPP2C27-like基因　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：中間錦雞兒抗旱、耐寒,對(duì)沙地環(huán)境有很強(qiáng)的適應(yīng)能力,具有防風(fēng)固沙,保持水土,改善局部小環(huán)境的作用,對(duì)保護(hù)和恢復(fù)生態(tài)平衡有著重要的意義。近二十年來(lái),我國(guó)西北地區(qū)生態(tài)環(huán)境日趨惡化,錦雞兒屬在沙地植被恢復(fù)進(jìn)程中常作為主要物種。因此,盡快培育出具有優(yōu)良遺傳特性的中間錦雞兒品種迫在眉睫。植物在生長(zhǎng)過(guò)程中,很難逃避各種變化不定的災(zāi)害性環(huán)境因素,如低溫、干旱和高鹽等,為適應(yīng)環(huán)境的脅迫而形成了不同的生理生化機(jī)制。其中蛋白磷酸酶PP2C參與植物的生長(zhǎng)發(fā)育、細(xì)胞周期、信號(hào)轉(zhuǎn)導(dǎo)和滲透脅迫等各種生物學(xué)過(guò)程。本研究以中間錦雞兒莖段為外植體,經(jīng)器官再生途徑長(zhǎng)出植株,探索主要影響因素與培養(yǎng)效果的關(guān)系,初步建立了中間錦雞兒組織培養(yǎng)體系,為中間錦雞兒品種的改良和遺傳轉(zhuǎn)化奠定基礎(chǔ);對(duì)CiPP2C8-like和CiPP2C2 7-like基因進(jìn)行了克隆和生物信息學(xué)分析,通過(guò)qRT-PCR技術(shù)發(fā)現(xiàn)CiPP2C8-like基因受鹽和脫水脅迫誘導(dǎo)。主要研究結(jié)果如下:1.以中間錦雞兒莖尖和莖段分別為外植體進(jìn)行了愈傷誘導(dǎo),結(jié)果顯示莖段比莖尖的愈傷誘導(dǎo)率高。2.利用SPSS19軟件設(shè)計(jì)GA3、6-BA、NAA和活性炭AC的四因素三水平正交試驗(yàn)。以中間錦雞兒的莖段為外植體,在GA3 0.15mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.1g/L的MS培養(yǎng)基中,愈傷誘導(dǎo)率為33.33%;影響愈傷誘導(dǎo)的因素順序依次為GA3、NAA、AC和6-BA;最佳激素配比為GA3 0.2mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.3g/L。3.在6-BA1.0mg/L+NAA0.2mg/LMS培養(yǎng)基中,叢生芽誘導(dǎo)率為13.33%,長(zhǎng)勢(shì)良好,有 3-5 個(gè)叢生芽;在 GA30.05mg/L+6-BA0.05mg/L+KT 1.0mg/L MS 培養(yǎng)基中,叢生芽誘導(dǎo)率為20.00%,但只有1-2個(gè)叢生芽,部分褐化。4.叢生芽長(zhǎng)至2-3cm時(shí),用手術(shù)刀剝離,在GA30.15mg/L+IAA0.5mg/L的MS培養(yǎng)基中生根率最高,生根率為60.00%。將經(jīng)過(guò)生根培養(yǎng)后的小植株從培養(yǎng)瓶中取出,移栽成活率為100%。5.克隆得到了CiPP2C8-lik 和CiPP2C27-like基因cDNA全長(zhǎng)序列。CiPP2C8-like基因的cDNA長(zhǎng)為999 bp,共編碼333個(gè)氨基酸,起始密碼子為ATG,終止密碼子為T(mén)AG;CiPP2C27-like基因的cDNA長(zhǎng)為1149 bp,共編碼383個(gè)氨基酸,起始密碼子為ATG,終止密碼子為T(mén)AG。以上兩個(gè)基因所編碼的蛋白均屬于親水蛋白。6.利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)脅迫處理后CiPP2C8-like和CiPP2C27-like基因表達(dá)量的變化。實(shí)驗(yàn)結(jié)果顯示,鹽脅迫處理后,CiPP2C8-like基因表達(dá)量上調(diào),在48h最高,達(dá)到未處理的33倍;脫水脅迫處理后,CiPP2C8-like基因表達(dá)量上調(diào),在12h最高,達(dá)到未處理的252倍。7.構(gòu)建了表達(dá)載體 CiPP2C8-like-HA。
[Abstract]:Caragana intermedium has the functions of drought resistance, cold tolerance, strong adaptability to sandy land environment, wind and sand prevention, soil and water conservation, and improvement of local environment, which is of great significance to the protection and restoration of ecological balance. The ecological environment of northwest China is deteriorating day by day. Caragana is often used as the main species in the process of vegetation restoration in sandy land. Therefore, it is urgent to cultivate the species of Caragana intermedium with excellent genetic characteristics as soon as possible. It is difficult to escape from various environmental factors, such as low temperature, drought and high salt, which have formed different physiological and biochemical mechanisms to adapt to environmental stress. Protein phosphatase (PP2C) is involved in plant growth and development, cell cycle, etc. In this study, the stem segments of Caragana intermedia were used as explants to grow plants through organ regeneration pathway, and to explore the relationship between the main factors and the culture effect, and other biological processes such as signal transduction and osmotic stress. The tissue culture system of Caragana intermedia was established preliminarily, which laid the foundation for the improvement and genetic transformation of Caragana intermedia, cloning and bioinformatics analysis of CiPP2C8-like and CiPP2C2 7-like genes. CiPP2C8-like gene was induced by salt and dehydration stress by qRT-PCR technique. The main results were as follows: 1. Callus induction was carried out with stem tip and stem segment of Caragana intermedia, respectively. The results showed that the callus induction rate of stem segment was higher than that of stem tip. Using SPSS19 software to design four factors and three levels orthogonal experiment of GA _ 3N _ 6-BANAA and activated carbon AC. The stem segment of Caragana intermedia was used as explant in MS medium of GA3 0.15mg / L 6-BA 0.2mg / L NAA 0.4mg / L AC 0.1 g / L. The rate of callus induction was 33.33, the order of factors affecting callus induction was GA _ 3N _ NAA _ (AC) and 6-BA, the best hormone ratio was GA3 0.2mg / L 6-BA 0.2mg / L NAA 0.4mg / L AC 0.3g / L. 3.In 6-BA 1.0mg / L NAA0.2mg/LMS medium, the induction rate of cluster buds was 13.3333, the growth rate was good, and there were 3-5 cluster buds. In GA30.05mg/L 6-BA 0.05 mg / L KT 1.0 mg / L MS medium, the induction rate of cluster buds was 20.00%, but there were only 1 or 2 clusters of buds, and partial browning. 4. When the buds grew to 2-3 cm long, the rooting rate was the highest in MS medium of GA30.15mg/L IAA0.5mg/L when the buds grew to 2-3 cm, and the rooting rate was the highest in MS medium of GA30.15mg/L IAA0.5mg/L. The rooting rate was 60. 00. the rooting rate of the small plant was extracted from the culture bottle, and the survival rate of transplanting was 100. 5. The length of cDNA of the cDNA sequence of CiPP2C8-lik and CiPP2C27-like gene was 999bp. encoding 333 amino acids. The initial codon is ATG, and the termination codon is TAGN CiPP2C27-like gene. The cDNA length of the gene is 1149bp. it encodes 383 amino acids. The initial codon is ATG and the termination codon is tag. The proteins encoded by the above two genes belong to hydrophilic protein .6.Real-time fluorescence quantitative PCR technique is used to detect the changes of CiPP2C8-like and CiPP2C27-like gene expression after stress. After salt stress, the expression of CiPP2C8-like gene was up to 33 times at 48h, and the expression of CiPP2C8-like gene was up to 252 times at 12h after dehydration stress. The expression vector CiPP2C8-like-HA was constructed.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2;S793.3

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