【摘要】：茯苓Wolfiporia cocos是我國傳統(tǒng)中藥材之一,具有極高的藥用價(jià)值和膳食營養(yǎng)價(jià)值。本課題選取茯苓菌種GIM5.219,對其原生質(zhì)體的制備與再生條件開展了系統(tǒng)的研究;對PEG介導(dǎo)的原生質(zhì)體茯苓轉(zhuǎn)化體系進(jìn)一步優(yōu)化;運(yùn)用現(xiàn)代分子生物學(xué)技術(shù)及方法研究參與調(diào)控菌核形成的相關(guān)基因Smk,以達(dá)到在茯苓種植過程中減少對松木資源的依賴,精準(zhǔn)調(diào)控茯苓菌核發(fā)育形成條件并提高產(chǎn)量,促進(jìn)茯苓種植產(chǎn)業(yè)化發(fā)展的最終目的。實(shí)驗(yàn)結(jié)果如下:1、結(jié)果表明:GIM5.219菌株在固體和液體培養(yǎng)基上0-2d之間生長緩慢,固體上2-6d內(nèi)菌絲生長增快,從第7d開始生長趨于穩(wěn)定;液體中2-7d菌絲球生長較快,8d后菌絲球生長趨于穩(wěn)定。2、從8個(gè)方面對茯苓原生質(zhì)體制備與再生條件進(jìn)行篩選研究,得出了最優(yōu)條件:采取培養(yǎng)6d的茯苓菌絲,在p H為5.6-6.2的3.5%溶壁酶+1%崩潰酶的混合酶液中,34-36℃靜置酶解2.5-3h制備原生質(zhì)體,在馬鈴薯再生培養(yǎng)基上采用雙層涂布法進(jìn)行再生,原生質(zhì)體數(shù)最高能達(dá)到39.20×107/g,再生率最高為32.7%。3、從6個(gè)方面對PEG介導(dǎo)的原生質(zhì)體茯苓轉(zhuǎn)化體系進(jìn)行篩選實(shí)驗(yàn),得到了最優(yōu)條件:采用液體培養(yǎng)4d的菌絲球所制備的原生質(zhì)體,用MTC溶液(0.6mol/L甘露醇,50mmol/L Ca Cl2,10mmol/L Tris-HCl、p H7.5)將其濃度調(diào)節(jié)為1×108個(gè)/ml,向原生質(zhì)體中加入2ug DNA/(100μl原生質(zhì)體),用45%PEG4000進(jìn)行轉(zhuǎn)化,所得的抗性轉(zhuǎn)化子數(shù)最多,為27個(gè)。將其進(jìn)行PCR驗(yàn)證,所有的轉(zhuǎn)化子均能擴(kuò)增出hph特異片段,表明外源DNA已經(jīng)插入到茯苓基因組中,且能夠隨著茯苓傳代而穩(wěn)定的遺傳。4、構(gòu)建了Smk基因沉默重組載體p CIT-Smk,經(jīng)PEG介導(dǎo)轉(zhuǎn)入到茯苓原生質(zhì)體中,成功驗(yàn)證了進(jìn)一步優(yōu)化的PEG介導(dǎo)的茯苓原生質(zhì)體轉(zhuǎn)化體系的可行性。5、篩選出了繼代培養(yǎng)5代后仍能穩(wěn)定沉默Smk基因的轉(zhuǎn)化菌株,表明轉(zhuǎn)化子具有遺傳穩(wěn)定性。相對熒光定量實(shí)驗(yàn)結(jié)果表明:未進(jìn)行轉(zhuǎn)化的茯苓菌株GIM5.219中Smk基因的表達(dá)量是轉(zhuǎn)化子的28133.5倍,說明轉(zhuǎn)化子中的Smk基因已被沉默,隨著茯苓傳代而穩(wěn)定地遺傳。6、沉默Smk基因的轉(zhuǎn)化子菌株在培養(yǎng)過程中,菌絲的生長速度以及菌落的形態(tài)特征都發(fā)生了明顯變化,表明Smk基因可能是調(diào)控菌絲生長發(fā)育、菌核形成的重要基因。本研究進(jìn)一步完善了目前茯苓原生質(zhì)體制備與再生的研究工作,優(yōu)化了茯苓原生質(zhì)體轉(zhuǎn)化體系。研究了茯苓菌絲生長發(fā)育、菌核形成的相關(guān)基因Smk,為茯苓栽培、品種選育工作提供了理論基礎(chǔ),為指導(dǎo)茯苓遺傳種植工作具有重要意義。
[Abstract]:Poria cocos Wolfiporia cocos is one of the traditional Chinese medicinal materials, which has high medicinal value and nutritional value. The preparation and regeneration conditions of protoplasts of Poria cocos strain GIM5.219, were studied systematically, and the transformation system of protoplast Poria cocos mediated by PEG was further optimized. Modern molecular biological techniques and methods were used to study the related gene Smk, involved in the regulation of sclerotia formation in order to reduce the dependence on pine resources during the cultivation of Poria cocos, to accurately regulate the conditions for the development and formation of the sclerotia of Poria cocos and to increase the yield. The ultimate aim of promoting the industrialization of Poria cocos planting. The results were as follows: 1. The results showed that the growth of GIM5.219 strain was slow between 0 and 2 days on solid and liquid medium, and the mycelium growth increased within 2 to 6 days on solid, and the growth tended to be stable from the 7th day. After 8 days, the mycelium growth tended to be stable. 2. The preparation and regeneration conditions of Poria cocos protoplast were studied from 8 aspects, and the optimum conditions were obtained as follows: culture for 6 days of Poria cocos mycelia. The protoplasts were prepared in the mixture of 3.5% wall-lysing enzymes and 1% crumbling enzymes (pH = 5.6-6.2). The protoplasts were hydrolyzed at 34-36 鈩，

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