【摘要】：茯苓Wolfiporia cocos是我國傳統中藥材之一,具有極高的藥用價值和膳食營養價值。本課題選取茯苓菌種GIM5.219,對其原生質體的制備與再生條件開展了系統的研究;對PEG介導的原生質體茯苓轉化體系進一步優化;運用現代分子生物學技術及方法研究參與調控菌核形成的相關基因Smk,以達到在茯苓種植過程中減少對松木資源的依賴,精準調控茯苓菌核發育形成條件并提高產量,促進茯苓種植產業化發展的最終目的。實驗結果如下:1、結果表明:GIM5.219菌株在固體和液體培養基上0-2d之間生長緩慢,固體上2-6d內菌絲生長增快,從第7d開始生長趨于穩定;液體中2-7d菌絲球生長較快,8d后菌絲球生長趨于穩定。2、從8個方面對茯苓原生質體制備與再生條件進行篩選研究,得出了最優條件:采取培養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制備原生質體,在馬鈴薯再生培養基上采用雙層涂布法進行再生,原生質體數最高能達到39.20×107/g,再生率最高為32.7%。3、從6個方面對PEG介導的原生質體茯苓轉化體系進行篩選實驗,得到了最優條件:采用液體培養4d的菌絲球所制備的原生質體,用MTC溶液(0.6mol/L甘露醇,50mmol/L Ca Cl2,10mmol/L Tris-HCl、p H7.5)將其濃度調節為1×108個/ml,向原生質體中加入2ug DNA/(100μl原生質體),用45%PEG4000進行轉化,所得的抗性轉化子數最多,為27個。將其進行PCR驗證,所有的轉化子均能擴增出hph特異片段,表明外源DNA已經插入到茯苓基因組中,且能夠隨著茯苓傳代而穩定的遺傳。4、構建了Smk基因沉默重組載體p CIT-Smk,經PEG介導轉入到茯苓原生質體中,成功驗證了進一步優化的PEG介導的茯苓原生質體轉化體系的可行性。5、篩選出了繼代培養5代后仍能穩定沉默Smk基因的轉化菌株,表明轉化子具有遺傳穩定性。相對熒光定量實驗結果表明:未進行轉化的茯苓菌株GIM5.219中Smk基因的表達量是轉化子的28133.5倍,說明轉化子中的Smk基因已被沉默,隨著茯苓傳代而穩定地遺傳。6、沉默Smk基因的轉化子菌株在培養過程中,菌絲的生長速度以及菌落的形態特征都發生了明顯變化,表明Smk基因可能是調控菌絲生長發育、菌核形成的重要基因。本研究進一步完善了目前茯苓原生質體制備與再生的研究工作,優化了茯苓原生質體轉化體系。研究了茯苓菌絲生長發育、菌核形成的相關基因Smk,為茯苓栽培、品種選育工作提供了理論基礎,為指導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，

本文編號：2303789