本文選題：單核體菌絲 + 農(nóng)桿菌��； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：香菇(Lentinula edodes)是世界上最著名的食用菌之一,是世界第二大食藥用真菌,產(chǎn)量?jī)H次于雙孢蘑菇(Agaricus bisporus),在中國(guó)已有近千年的栽培歷史[1]。由于其突出的食藥作用和價(jià)值,人們對(duì)香菇的需求量明顯上升,從而促進(jìn)了香菇產(chǎn)業(yè)迅猛發(fā)展,自1987年香菇總產(chǎn)量第一次超過(guò)日本以來(lái),中國(guó)一直保持著世界第一香菇大國(guó)的地位[2]。不過(guò)與發(fā)展迅猛的香菇產(chǎn)業(yè)相比,香菇的遺傳轉(zhuǎn)化研究較為落后。隨著兩個(gè)香菇全基因組測(cè)序的完成和公布[3-4],香菇的遺傳及功能基因研究將成為熱點(diǎn)。遺傳轉(zhuǎn)化技術(shù)是運(yùn)用分子生物學(xué)相關(guān)方法和基因工程相關(guān)方法將本來(lái)不屬于受體菌株基因組的基因片段插入到受體基因組并整合,再通過(guò)復(fù)制、轉(zhuǎn)錄、翻譯等一系列過(guò)程,最終可以達(dá)到定向的改變受體菌株或者細(xì)胞生理生化特性的目的。因此,穩(wěn)定、高效、便捷的遺傳轉(zhuǎn)化體系的構(gòu)建將具有重要意義和價(jià)值。本文將探索以農(nóng)桿菌為介導(dǎo)的遺傳轉(zhuǎn)化體系在香菇原生質(zhì)體單核體菌株中進(jìn)行隨機(jī)插入突變的研究。通過(guò)香菇菌絲對(duì)潮霉素的抗性試驗(yàn),確定了農(nóng)桿菌轉(zhuǎn)化初篩濃度為10mg/L,復(fù)篩濃度為12mg/L。通過(guò)對(duì)比在不同頭孢噻肟鈉Cef濃度下香菇菌絲的生長(zhǎng)情況,確定了Cef的最佳抑制農(nóng)桿菌的濃度為600mg/L。構(gòu)建的質(zhì)粒pYN6982和GPiE以潮霉素抗性基因hyg作為篩選標(biāo)記基因,以增強(qiáng)型熒光蛋白基因egfp作為報(bào)告基因,以農(nóng)桿菌EHA105和LBA4404為介導(dǎo),同時(shí)轉(zhuǎn)化了3個(gè)香菇單核體菌株。香菇受體菌絲創(chuàng)新性地采用小米粒培養(yǎng)基進(jìn)行培養(yǎng)和轉(zhuǎn)化,采用小米粒培養(yǎng)基培養(yǎng)菌絲不需要用打孔器選取直徑相同的菌塊,每粒小米粒都是大小均勻的個(gè)體,可以直接挑取使用,也無(wú)需再接種于鋪有無(wú)菌玻璃紙的MYG固定培養(yǎng)基中,不易污染雜菌。農(nóng)桿菌侵染菌絲后,小米粒方便挑取,相較于常用的PDA菌塊或菌絲球的方法,這種方法可更快捷得到更多的易于分散的受體材料,并可更加方便、快捷的統(tǒng)計(jì)轉(zhuǎn)化效率。受體菌絲經(jīng)過(guò)農(nóng)桿菌侵染,共培養(yǎng)階段培養(yǎng),通過(guò)潮霉素抗性篩選,經(jīng)PCR以及測(cè)序驗(yàn)證,轉(zhuǎn)化子中含有潮霉素抗性基因的片段。經(jīng)過(guò)熒光顯微觀察,經(jīng)轉(zhuǎn)化的香菇菌絲體內(nèi)可以觀測(cè)到綠色熒光,而未經(jīng)轉(zhuǎn)化的對(duì)照菌株則沒(méi)有熒光信號(hào)的產(chǎn)生,表明增強(qiáng)型綠色熒光蛋白在轉(zhuǎn)化香菇菌絲中得到了表達(dá),再經(jīng)過(guò)轉(zhuǎn)化子的有絲分裂穩(wěn)定性試驗(yàn)得到穩(wěn)定遺傳的轉(zhuǎn)化子。對(duì)部分香菇轉(zhuǎn)化子進(jìn)行了在PDA培養(yǎng)基上菌落形態(tài)觀察試驗(yàn),與未轉(zhuǎn)化的對(duì)照香菇菌絲相比,發(fā)現(xiàn)小部分轉(zhuǎn)化子菌落的形態(tài)和生長(zhǎng)速度有所差異,可能是T-DNA的插入破壞了轉(zhuǎn)化子基因組中與菌落形態(tài)相關(guān)的功能基因,從而導(dǎo)致了轉(zhuǎn)化子和對(duì)照菌絲的差異。本研究探索出一種采用小米粒培養(yǎng)基培養(yǎng)菌絲并進(jìn)行轉(zhuǎn)化的新方法,并且從菌絲在小米粒培養(yǎng)基上生長(zhǎng)時(shí)間、農(nóng)桿菌類型、質(zhì)粒上啟動(dòng)子類型、農(nóng)桿菌侵染過(guò)程中超聲時(shí)間、共培養(yǎng)時(shí)間和溫度等方面進(jìn)行探索和優(yōu)化。同時(shí)比較了含有不同啟動(dòng)子的質(zhì)粒在相同農(nóng)桿菌中轉(zhuǎn)化效率以及同一質(zhì)粒在不同農(nóng)桿菌中的轉(zhuǎn)化效率。結(jié)果表明菌絲在小米粒培養(yǎng)基上培養(yǎng)15~20天為最佳的農(nóng)桿菌侵染轉(zhuǎn)化時(shí)間;不同的農(nóng)桿菌EHA105和LBA4404侵染菌絲后的轉(zhuǎn)化效率沒(méi)有太大的差異;含有雙孢蘑菇Agpd的轉(zhuǎn)化效率要高于含有靈芝Gl-gpd啟動(dòng)子的轉(zhuǎn)化效率;農(nóng)桿菌侵染過(guò)程中超聲震蕩2分鐘為最佳時(shí)間;共培養(yǎng)階段加入乙酰丁香酮能夠提高轉(zhuǎn)化效率;共培養(yǎng)時(shí)間5天為最佳培養(yǎng)時(shí)間,共培養(yǎng)最佳溫度為25℃。建立了較穩(wěn)定的農(nóng)桿菌介導(dǎo)的香菇遺傳轉(zhuǎn)化的體系,并獲得可穩(wěn)定遺傳和表達(dá)的香菇轉(zhuǎn)化子菌株。
[Abstract]:Letinous edodes (Lentinula edodes) is one of the most famous edible fungi in the world. It is the world's second largest edible medicinal fungus. The yield is second only to Agaricus bisporus (Agaricus bisporus). In China, there has been nearly a thousand years of cultivation history in China. Because of its prominent food and drug effect and value, the demand for letinous edodes has risen obviously, thus promoting the rapid development of letinous edodes industry. Rapid development, since the first letinous edodes output in 1987 has exceeded Japan for the first time, China has kept the position of the world's largest letinous edodes country [2]., but compared with the rapid development of letinous edodes industry, the genetic transformation of letinous edodes is relatively backward. With the completion of the complete genome sequencing of two letinous edodes and the publication of [3-4], the genetic and functional gene research of letinous edodes. It will be a hot spot. Genetic transformation technology is the use of molecular biology related methods and genetic engineering related methods to insert the gene fragments originally not belong to the receptor strains into the receptor genome and integrate, and then through a series of processes such as replicating, transcription, translation and so on, in order to achieve directional change of the receptor strain or cell physiology. Therefore, the construction of a stable, efficient and convenient genetic transformation system will be of great significance and value. This paper will explore the random insertion mutation of a genetic transformation system based on Agrobacterium tumefaciens in the single nucleosome of letinous edodes protoplast. The resistance test of hypha to hygromycin by letinous edodes mycelium has determined agriculture. The initial screening concentration of bacilli was 10mg/L, and the concentration of rescreening was 12mg/L. by comparing the growth of letinous edodes mycelium under the concentration of different cefotaxime sodium (Cef) in different cefotaxime sodium. The best inhibition of the concentration of Agrobacterium tumefaciens was determined by 600mg/L. constructed plasmid pYN6982 and GPiE with the hygromycin resistance gene hyg as a screening marker gene, and the enhanced fluorescent protein gene was enhanced. As a reporter gene, EGFP is mediated by Agrobacterium tumefaciens EHA105 and LBA4404, and 3 single nucleosome strains of letinous edodes are transformed. The mycelium of letinous edodes receptor is innovatively used for culture and transformation of small rice medium. The individual, which can be used directly, and does not need to be inoculated in the MYG fixed medium with aseptic glass paper, is not easy to pollute the heterozygous bacteria. After Agrobacterium tumefaciens infect the mycelium, the small rice grains are convenient to pick up. Compared to the commonly used PDA bacteria block or the method of bacterial silk ball, this method can get more easily dispersed receptor materials and can be more convenient. The receptor mycelium was infected by Agrobacterium tumefaciens, co culture stage and screened by hygromycin resistance. The hygromycin resistance gene fragment contained in the transformant was verified by PCR and sequencing. After fluorescence microscopy, the transformed letinous edodes mycelium could observe green fluorescence and unconverted control bacteria. No fluorescence signal was produced, indicating that the enhanced green fluorescent protein was expressed in the transformed letinous edodes mycelium, and then a stable hereditary transformant was obtained through the mitotic stability test of the transformant. The colony morphology of some letinous edodes transformants was observed on the PDA medium and the unconverted control of the control of the mycelium of letinous edodes. It was found that the morphology and growth speed of the small portion of the colonies were different. It was possible that the insertion of T-DNA destroyed the functional genes related to the colony morphology in the transformant genome, which led to the difference between the transformants and the control mycelium. The growth time of mycelia on the medium of rice grain, Agrobacterium type, promoter type, ultrasonic time, co culture time and temperature during Agrobacterium infection process were explored and optimized. Meanwhile, the conversion efficiency of plasmids containing different promoters in the same Agrobacterium and the same plasmid in different Agrobacterium tumefaciens were compared. The results showed that mycelia was the best Agrobacterium infection transformation time in the culture of 15~20 days on the medium of small rice grain, and the conversion efficiency of Agrobacterium tumefaciens EHA105 and LBA4404 was not very different, and the conversion efficiency of Agpd containing Agaricus bisporus was higher than that of the Gl-gpd promoter with Ganoderma lucidum. In the process of infection, the ultrasonic vibration of 2 minutes is the best time, and the conversion efficiency can be improved by adding acetyl syringone in the co culture stage. The optimum incubation time is 5 days, and the optimum temperature is 25. A stable Agrobacterium mediated transformation system of letinous edodes is established, and the transformation of letinous edodes to stable inheritance and expression is obtained. Substrains.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S646.12

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