發(fā)布時(shí)間：2018-12-14 03:26

【摘要】：【目的】在植物中,內(nèi)質(zhì)網(wǎng)脅迫(endoplasmic reticulum stress,ERS)和未折疊蛋白應(yīng)答(unfolded protein response,UPR)參與環(huán)境脅迫響應(yīng)過(guò)程,然而,玉米種子老化過(guò)程中內(nèi)質(zhì)網(wǎng)脅迫相關(guān)基因表達(dá)情況尚未見(jiàn)報(bào)道。文章利用基因數(shù)字表達(dá)譜技術(shù)探究玉米種子老化過(guò)程中內(nèi)質(zhì)網(wǎng)脅迫相關(guān)基因表達(dá)規(guī)律,以期為揭示種子衰老的分子機(jī)制提供理論依據(jù)�！痉椒ā恳杂衩纂s交種鄭單958種子為材料,采用高溫(45℃)高濕(相對(duì)濕度100%)的方法進(jìn)行人工老化處理。分別提取未老化處理(對(duì)照)和老化處理3 d的玉米種胚總RNA,利用Illumina Hi Seq TM 2000平臺(tái)進(jìn)行高通量測(cè)序。去除原始數(shù)據(jù)中的接頭序列、包含模糊堿基的序列以及低質(zhì)量序列,獲得Clean reads,利用短序列比對(duì)軟件SOAPaligner/SOAP2將Clean Reads分別比對(duì)到玉米參考基因組和參考基因序列,采用RPKM(reads per kb per million reads)方法計(jì)算基因的表達(dá)量,根據(jù)FDR(false discovery rate)0.001和|log2 ratio(T/CK)|≥1的標(biāo)準(zhǔn)篩選差異表達(dá)的基因,對(duì)獲得的差異表達(dá)基因(differentially expressed genes,DEGs)進(jìn)行KEGG(kyoto encyclopedia of genes and genomes)數(shù)據(jù)庫(kù)功能注釋分析,篩選出響應(yīng)人工老化的內(nèi)質(zhì)網(wǎng)脅迫相關(guān)差異表達(dá)基因。利用q RT-PCR技術(shù)定量分析內(nèi)質(zhì)網(wǎng)脅迫相關(guān)基因在不同人工老化時(shí)間內(nèi)的表達(dá)特性。【結(jié)果】基因數(shù)字表達(dá)譜鑒定結(jié)果表明,有104個(gè)差異表達(dá)基因在人工老化過(guò)程中參與內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工(protein processing in endoplasmic reticulum)通路,其中內(nèi)質(zhì)網(wǎng)脅迫相關(guān)基因有97個(gè)(81個(gè)上調(diào)表達(dá),16個(gè)下調(diào)表達(dá))。對(duì)差異表達(dá)基因功能注釋分析表明,內(nèi)質(zhì)網(wǎng)脅迫的標(biāo)志性蛋白基因Bi P以及分子伴侶蛋白基因CRT、CNT和GRP94等顯著上調(diào)表達(dá)。參與內(nèi)質(zhì)網(wǎng)相關(guān)性降解(endoplasmic reticulum-associated degradation,ERAD)途徑的有83個(gè)差異表達(dá)基因(70個(gè)上調(diào),13個(gè)下調(diào)),其中啟動(dòng)ERAD途徑的關(guān)鍵酶基因EDEM(ER degradation enhancing mannosidase I-like protein)下調(diào),參與蛋白泛素化的E2泛素結(jié)合酶基因Ubc H5、E3泛素連接酶基因Hrd1和Doa10等也發(fā)生顯著的表達(dá)變化。q RT-PCR結(jié)果表明,內(nèi)質(zhì)網(wǎng)脅迫相關(guān)基因在不同人工老化時(shí)間內(nèi)表現(xiàn)表達(dá)多樣性和復(fù)雜性�！窘Y(jié)論】人工老化處理能造成玉米種胚細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)脅迫。細(xì)胞通過(guò)上調(diào)分子伴侶基因表達(dá)和誘導(dǎo)ERAD途徑響應(yīng)內(nèi)質(zhì)網(wǎng)脅迫,但ERAD途徑受阻可能引起錯(cuò)誤折疊蛋白聚集,從而進(jìn)一步加劇細(xì)胞損傷,最終導(dǎo)致種子活力降低甚至喪失。
[Abstract]:[objective] in plants, endoplasmic reticulum stress (endoplasmic reticulum stress,ERS) and unfolded protein response (unfolded protein response,UPR) are involved in the response to environmental stress. The expression of genes related to endoplasmic reticulum stress during seed aging in maize has not been reported. In this paper, gene expression patterns related to endoplasmic reticulum stress in maize seeds during aging were studied by using the technique of gene digital expression profiling. In order to provide theoretical basis for revealing the molecular mechanism of seed senescence. [methods] the seeds of maize hybrid Zhengdan 958 were used as materials and the method of high temperature (45 鈩，

本文編號(hào)：2377828