本文選題：金針菇免疫調(diào)節(jié)蛋白 + RNAi載體��； 參考：《分子植物育種》2017年11期

【摘要】：為了探究金針菇FIP-fve基因?qū)疳樄奖旧淼纳飳W(xué)功能,本研究克隆FIP-fve基因正反片段并構(gòu)建其RNAi載體,并將其轉(zhuǎn)化農(nóng)桿菌LBA4404,建立以農(nóng)桿菌介導(dǎo)的金針菇遺傳轉(zhuǎn)化體系。雙酶切瓊脂糖電泳檢測(cè)結(jié)果表明成功構(gòu)建了FIP-fve基因的RNAi載體:p TCK303-fve(F)-fve(R),并用液氮轉(zhuǎn)化法獲得重組農(nóng)桿菌轉(zhuǎn)化子。金針菇遺傳轉(zhuǎn)化以菌絲體為外植體,潮霉素B(Hygromycin B)和頭孢霉素(Cephalosporins)使用濃度分別為9μg/m L和600μg/m L。重組農(nóng)桿菌濃度OD600為0.5,侵染金針菇菌絲體30 min,共培養(yǎng)培養(yǎng)基AS使用濃度為200μg/m L,共培養(yǎng)25℃3 d,抗性篩選10 d。最終通過潮霉素抗性篩選和PCR檢測(cè)從60個(gè)外植體中篩選得到5個(gè)金針菇遺傳轉(zhuǎn)化子,轉(zhuǎn)化率為8.33%。經(jīng)過連續(xù)培養(yǎng),對(duì)比觀測(cè)轉(zhuǎn)化子和野生型金針菇,FIP-fve基因沉默的金針菇轉(zhuǎn)化子菌絲的生長(zhǎng)在第3天、第6天和第10天明顯慢于野生型金針菇菌絲。初步說明FIP-fve基因?qū)疳樄骄z體時(shí)期的生長(zhǎng)發(fā)育具有一定調(diào)節(jié)作用,這為研究金針菇FIP-fve以及FIP對(duì)真菌本身生物學(xué)功能提供一定研究基礎(chǔ)和直接證據(jù)。
[Abstract]:In order to investigate the biological function of Flammulina velutipes (Flammulina velutipes) FIP-fve gene on Flammulina velutipes, the positive and negative fragments of FIP-fve gene were cloned and its RNAi vector was constructed, and the Agrobacterium tumefaciens LBA4404 was transformed into Flammulina velutipes genetic transformation system mediated by Agrobacterium tumefaciens. The results of double enzyme digestion agarose electrophoresis showed that the RNAi vector of FIP-fve gene was successfully constructed, and the recombinant Agrobacterium tumefaciens transformant was obtained by liquid nitrogen transformation. The genetic transformation of Flammulina velutipes (Flammulina velutipes) with hygromycin (B(Hygromycin B) and ceftomycin Cephalosporins (Cephalosporins) concentration was 9 渭 g / mL and 600 渭 g / mL, respectively. The concentration of recombinant Agrobacterium tumefaciens was 0.5, infecting the mycelium of Flammulina velutipes for 30 min, the concentration of co-culture medium was 200 渭 g / mL, co-cultured at 25 鈩，

本文編號(hào)：1985138